CN1017652B - Invivo cellular tracking - Google Patents

Abstract

A fixture for use with a pull tool to form the parts of a grommet about an opening in material to reinforce the material opening.

Description

Invivo Cellular tracking

The present invention relates to the method for Invivo Cellular tracking and measure the cells in vivo method of life.

Invivo Cellular tracking and life-span determination need come labeled cell with a sign at cell inner stablity, that is, this sign can not lost from cell also influences cell function indistinctively.Existing available sign can not provide these essential characteristics.For example, fluorescence antibody dissociates inapplicable because of it easily from cell.Other possible sign can not use because of its interference cell function.

Cyanine dye has been used for various biological applications.The dioxy carbonyl cyanine dye has been used for carrying out Arneth's count.Gunter Valet, Max Planck Ges Wissensch; Patent registration No. 84-102307/17, " by selectively staining and measurement volumes and fluorescence quantitative measurement simultaneously haemocyte ".But the dyestuff used is short chain carbonyl cyanine dye (being less than 10 carbon) and the variation of film potential is reacted in these researchs.And the short chain carbonyl cyanine dye enters the mitochondria of cell, is cytotoxic, and when washed cell, no matter whether cell membrane potential changes, these dyestuffs all leak out cell easily.In many other biological tests, lack the aliphatic chain cyanine dye with other.But short chain molecule can react and passes cell membrane film potential, enters mitochondria.H.M.Shapiro, U.S. Patent number 4,343,782, August 10 nineteen eighty-two.Short chain dyestuff also pair cell is toxic and can not be used for Invivo Cellular tracking.

Tricarbocyanine dye (Fox, I.J., et al., proc.Mayo Clinic, 32:478-484,1957) and Evans blue (Evans-Blue) dyestuff (SChad, H., et al., Pfluegers Arch.Eur.J.Physiol., 370(2): 139-144,1977) be used for measuring in the body cardiac output by dilution method.Dow（Dow，P.，Physiol。Rev., 36:77-102,1956) the method is described as, inject certain blood vessel internal indicator of known quantity in the venous side of lung, measure the time course of the artery concentration of this indicator, to determine capacity at injection and sampling point-to-point transmission.These dyestuffs are not used for staining cell.

The present invention is the spike cell and the new method in mensuration cells in vivo life-span in the body.According to Cellular tracking new method of the present invention, at first cell is used cyanine class dye marker, give the experimenter with its injection then, cyanine class dyestuff is used for determining the position of cell.In another embodiment, determine cell survival by the speed of measurement markers vanished cell.

In body of the present invention, in spike cell and the mensuration cells in vivo method of life, use cyanine class dye marker cell.At this, the compound with following array structure is called cyanine class dyestuff:

Wherein:

Y is the methylene that oxygen, sulphur, methylene or alkyl replace;

M is 0-3;

N is 12-22.

The methylene that alkyl used herein replaces means list or the disubstituted methylene that methyl, ethyl or the propyl group with any combination replaces.

The compound of said structure can be represented with following general abbreviated formula:

DiYCn（2m+1）

Sims，P.J.，et al.，Biochem，13：3315（1974）。Like this, be sulphur as Y in this compound, there are three carbon to connect two rings, and the aliphatic chain of two 14 carbon is arranged, then be expressed as DiSC ₁₄(3).Similarly, DiIC ₁₄(5) represent that Y is an isopropyl in this compound, have five carbon to connect two rings, and the aliphatic chain of two 14 carbon is arranged.

Be included in the compound that is called cyanine class dyestuff herein, it is compound with said structure of one or more replacements, suppose that such substituted compound is dissolved in the cell marking medium at mark in the required time at least, and have sufficiently high film partition factor to link to each other with the cell membrane of mark with maintenance.Such compound also must be under the required concentration of mark not appreciable impact cell activity.The solubleness of mensuration dyestuff as described below in the cell marking medium: by cyanine class dyestuff is dispersed in the mark medium, over time with conventional fluorescence spectrophotometry commercial measurement fluorescence intensity.Fluorescence intensity weakens and shows dye precipitated and be attached on the chamber wall.For example, inject the fluorescence intensity of the red blood cell of donor animal behind the available known flow cytometer method monitoring mark, measure dyestuff and whether keep linking to each other, the fluorescence intensity of the substantially invariable labeled cell in injection back, the stability of susceptible of proof dyestuff in cell membrane with cell membrane.

Mix the compound of said structure of the atom of an available magnetic resonance imaging technology for detection, be also included within the compound that is called cyanine class dyestuff herein.Such compound can prepare with known technology, for example, fluorine atom is mixed in the methyl of aliphatic chain.With gamma radiation body as ¹²⁵The compound of the said structure of I mark also is included into cyanine class dyestuff at this.

Used cyanine class dyestuff can be from various sources such as Molecular Probe among the present invention, Inc., and Eugene, Oregon has bought, and also available known synthetic method prepares from existing initial substance.Hamer，F.M。, " cyanine dye and allied compound ", Interscience Publishers(1964).

Use the method for being invented, all available cyanine class of any living cells dye marker.Term cell used herein includes the eukaryotic of nuclear such as white blood corpuscle, various tumour cell, other mammalian cell (as: Chinese hamster ovary cell of tissue culture), yeast; Seedless cell such as red blood cell and blood platelet.Karyocyte is grown or functionating desired as resembling basically the cell of this type, lives so; If akaryote can show the function of its expection and live, as, the red blood cell of a work can transport the carbon dioxide of oxygen; For example the blood platelet of Huoing in aggegation and release test, resemble expection show its function.

Cell marking is to carry out in nonlethal at pair cell, that cell marking repeatably can the be provided medium.For making medium have indispensable characteristic, use the perviousness correctives, at least at mark in the required time, cyanine dye forms stabilizing solution in the perviousness correctives.Available perviousness correctives comprises and resembles sugar, monose such as glucose, fructose, sorbose, wood sugar, ribose, and disaccharide such as sucrose, sugar alcohol such as mannitol, glycerine, inositol, xylitol, adonitol, amino acid such as glycocoll and arginine also have some Gu Shi buffering agent (Good ' s buffers) as N-three (methylol)-methyl-3-aminopropanesulfonicacid acid.Good，N.E.，et al.，Biochem.15，467-477（1966），Good，N.F.and S.Izawa，Methods Enzymol.，24，Part B，53（1968），Feguson，W.J.，et al.，Anal.Biochem.104：301-310（1980）。Yet some clone may be to one or more perviousness correctives sensitivities, particularly to sugar alcohol.Therefore, will carry out code test before mark can survive in the perviousness correctives that will use to determine cell.In addition, a small amount of buffering agent can be added the mark medium to regulate pH.

Contact the influence of various perviousness correctives pair cell activity, determine by the doubling time of measuring the Yac cell at the various perviousness correctivess of cells contacting after 30 minutes.The Yac cell is that a kind of mouse lymph lymphoma tissue culture cells is, can be from American Type Culture Collection, Rockville, Maryland obtains, and at European Journal of Immunology 5:112-117(1975) in be described.Data shown in the table 1 show, and compare with phosphate buffer, and the not influence of contact sucrose, glucose, Gu Shi buffering agent (TAPS, CAPS, EPPS, HEPPSO, DIPSO) pair cell doubling time shows not exist and contact relevant cytotoxicity.

Table 1

The perviousness correctives doubling time (hour)

Phosphate buffer 31.0

Sucrose 41.0

Glucose 34.5

TAPS 32.7

CAPS 45.8

EPPS 32.2

HEPPSO 23.4

DIPSO 36.7

3-amino-1-propane sulfonic acid 99.6

The 3-(N-morpholinyl) propane sulfonic acid sodium (MOPS) A

2-amino-2-methyl-1, ammediol B

2-amino-2-methyl-1-propanol B

N-three (methylol) methyl taurine (TES) B

N, two (2-hydroxyethyl)-2-tarine (BES) A of N-

3-(hexamethylene amino)-2-hydroxyl-1-propane sulfonic acid (CAPSO) A

Triethanolamine B

Three (methylol) aminomethane (TRIZMA) B

Bis-tris propane B

The 2-(N-morpholinyl) ethyl sulfonic acid (MES) B

3-(dimethyl (methylol) methylamino)-2-hydroxyl

Propane sulfonic acid (AMPSO) A

Table 1(is continuous)

The perviousness correctives doubling time (hour)

N, two (2-hydroxyethyl) glycocoll (BICINE) 57.7 of N-

3-((3-courage amidopropyl) Dimethyl Ammonium)-1-B

Propane sulfonic acid salt (CHAPS) B

3-(N-three (methylol) methylamino) 2-

Hydroxy-propanesulfonic acid (TAPSO) 63.6

The 3-(N-morpholinyl)-2-hydroxy-propanesulfonic acid (MOPSO) 178.4

2-((2-amino-2-oxoethyl) amino)

Ethyl sulfonic acid (ACES) 1038.4

Two (2-hydroxyethyl) imino group-three-(methylol)

Methane (BIS-TRIS) A

2-(N-hexamethylene amino) ethane sulfonic acid (CHES) 51.5

N-three-(methylol) methylglycine (TRICINE) A

Gucosamine 288.4

Imidazoles B

Glycylglycine 66.9

A-does not grow or the parts of fine cellular toxicity

The B-acute cytotoxicity

Table 2 has provided the various perviousness correctivess that are used for checking cyanine class colorant dissolubility.Remove post precipitation with centrifuge method, get a certain amount of perviousness correctives that contains cyanine class dyestuff and be dissolved in the ethanol, survey concentration with fluorescence spectrophotometry.Used dyestuff is DiSC ₁₄(5) and DiOC ₁₄(3), the perviousness correctives is an isotonic concentration.The reduction of fluorescence intensity that with the ethanolic solution is standard is directly relevant with the reduction of cyanine class colorant dissolubility.

Table 2

Perviousness correctives relative intensity of fluorescence (CONC)

DiSC ₁₄（5） DiOC ₁₄（3）

Ethanol 100 100

Glucose 31 100

Fructose 35 100

Sorbose 40 100

Sucrose 41 100

Wood sugar 36 19-52

Ribose 24 100

Lyxose 0.12 1.8

Glycocoll 31 93

Arginine 17 17.2

Glycerine 39 99.5

Inositol 42 92

Xylitol 34 76.4

Sweet mellow wine 29 *

Adonitol 34 ND

Three (methylol)-methylaminos

Propane sulfonic acid (TAPS) 18 ND

3-(hexamethylene amino)-1-

Propane sulfonic acid (CAPS) 40 ND

The N-(2-hydroxyethyl) piperazine-N '-3-

Propane sulfonic acid (EPPS) 18 ND

Table 2(is continuous)

Perviousness correctives relative intensity of fluorescence (CONC)

DiSC ₁₄（5） DiOC ₁₄（3）

N-2-hydroxyethyl piperazine-N '-2-

Hydroxy-propanesulfonic acid (HEPPSO) 20 ND

3-(two (2-hydroxyethyl) amino of N-N-)

-2-hydroxy-propanesulfonic acid (DIPSO) 43 ^{* *}ND

NaCl 6 1.7

Phosphate buffer 2.1 6.5

Na ₂SO ₄7.4 1.6

NaI 1.1 0.14

Choline Chloride 11 ^*6.3

Choline iodide 0.16 2.3

^*In ethanol, precipitate, do not obtain data.

^*Because big crystallization does not precipitate the illusion that causes.

^{* *}Precipitation (suspicious data) in ethanol

ND does not survey

By table 2 as seen, the dissolubility of cyanine class dyestuff when having general salt to exist is than much lower when having sugar (except the lyxose), sugar alcohol, amino acid, Gu Shi buffering agent TAPS, HEPPSO, DIPSO, CAPS and EPPS to exist.In addition, measured DiSC ₁₄(5) stability in following solution: sugared as glucose, fructose, ribose, sorbose, sucrose, wood sugar, sugar alcohol such as glycerine, inositol, xylitol, adonitol, amino acid such as glycocoll and arginine.Cyanine class dyestuff was stablized in the solution of testing 20 minutes at least, and this time is enough carried out repeatably mark, and in many reagent, the amount of cyanine class dyestuff did not significantly reduce in the time of 60 minutes in the solution.

Further, measured the solubleness of cyanine class dyestuff in the medium of the perviousness correctives that contains general salt and this dyestuff of solubilized.Not appreciable impact of distilled water diluting DiSC ₁₄(5) waiting the solubleness of oozing in the glucose solution.But, with only about 20% isotonic sodium chlorrde solution dilution, DiSC ₁₄(5) solubleness in waiting glucose solution that oozes just significantly reduces.Therefore, can in the medium that only contains a small amount of general salt, carry out repeatably cell marking with cyanine dye, these salts such as sodium chloride, potassium chloride, lime chloride, sodium acetate, potassium acetate, sodium sulphate, sodium iodide, Choline Chloride, or choline iodide.Being preferably in generally salt regulates in the infiltrative medium and carries out.

The cell of cyanine class dyestuff that analyze to have adopted mark of the present invention is to determine the influence of labeling process pair cell activity.Can be from American Type Culture Collection, the V79 cell that Rockville, Maryland obtain is 10 with containing concentration ^-5Or 4 * 10 ^-5MDiOC ₁₄(3) solution carries out mark, and the growth kinetics of staining cell is compared with the equal amount of mixture of be unstained cell and dyeing and the cell that is unstained.The V79 cell is by Prescott, D.M., and Ann.New York Acad.Sci. 397:101-109(1982) describes.Cyanine class dye marker does not influence cell doubling time.So the mark cell growth is influence not.And several other conventional cytoactive tests have confirmed also that as trypanblue exclusion method (Trypan Blue Exclusion) and Propidium Iodide exclusive method the active nothing of the cyanine class dye marker pair cell that is undertaken by described method influences.

The influence of cyanine class dye marker pair cell activity also can be determined by measuring erythrocyte fragility.By changing salinity, being suspended in the sodium chloride medium of various osmotic strength of mark with unlabelled red blood cell.The volume distributed median of cell is measured with the Coulter-counter (Coulter Counter) that is connected with a conduit fittings.Measure average external volume and to each salinity mapping, volume increases with the decline of sodium chloride concentration, when salinity was about 0.5 gram/100ml, the volume appearance was fallen suddenly.Erythrocytolysis on this aspect.And no matter whether cell uses cyanine class dye marker, volume change is all identical.In a similar manner, in parallel sample, haemolysis is monitored as the function of sodium chloride concentration.After red blood cell put into the about 2-3 of sodium chloride minute, centrifugal solution was with any undissolved cell of sedimentation.Supernatant solution carries out spectrophotometric analysis to measure hemoglobin concentration.The hemoglobin concentration of each sample is compared with the control sample of all dissolvings, determine dissolving number percent.The concentration of free hemoglobin is quite low, reaches about 0.5 gram/100ml until sodium chloride concentration, and after this, haemoglobin discharges rapidly.The result compares with erythrocyte fragility test, and volume change is directly relevant with the release of haemoglobin.And, haemoglobin to be released in mark and the unlabelled cell be identical.

Press the body internal stability of method of the present invention for check, take out rabbit erythrocyte, use DiSC with the cell of cyanine class dye marker ₁₄(5) mark re-enters again.After this regular percentage of the blood sampling evaluation of markers cell fluorescence intensity of labeled cell when.The erythrocytic number of round-robin descends as the function of time is linear, and is 52 days life-spans of measured labeled cell, very approaching with the mean lifetime of the rabbit erythrocyte of reporting 40 to 60 days.Therefore, cyanine class dye marker does not influence the clearance rate of red blood cell.

In five rabbits being tested, except that one, the fluorescence intensity of labeled cell is at mark and inject again after 60 days and all remain unchanged substantially.In the 5th animal, be no more than 20% cyanine class dyestuff after circulation 60 days, from labeled cell, discharge.These data add the data that get from tissue culture, and showing does not have dyestuff from the transfer of labeled cell to unlabeled cells, illustrate that cell is by the dyestuff stable labelling.

The living cells of cyanine class dye marker is used for mammal in the present invention, comprises people's Invivo Cellular tracking and cells in vivo durability analysis.Invivo Cellular tracking used herein, comprise measure cell in subject the location and measure the speed of cell by point of fixity, cross the speed of blood vessel as stream of cells.

The red blood cell of importing a part of cyanine class dye marker is measured the life-span of the red blood cell of input.After the input, measure the percent of mark red blood cell in the systemic circulation immediately with routine techniques.Subsequently, the number percent of labeled cell is used to calculate the life-span of input cell.Further, by the relatively variation of labeled cell number percent and the variation of hematocrit, will import hemorrhage difference the in back and come with immune response.Input rate is identical with labeled cell number percent reduced rate, shows by hemorrhage losing blood of causing the higher immune response that then shows the input cell of labeled cell counting reduced rate.Be autofluorescence and self cancellation avoid haemoglobin to cause, preferably be used in the cyanine dye of being excited in the red light wavelength scope and launching the light longer and come tagged blood cells than the excitation wavelength of haemoglobin.The example of this class dyestuff comprises DiC ₁₄(5) and DiSC ₁₄(3).

Use with to the similar technology of the used technology of red blood cell, can draw hematoblastic volume lifetime and distinguish decrease of platelet hemorrhage and that immune response causes.But, blood platelet absorbs or radiative molecule because lacking in used wavelength, various cyanine class dyestuffs are used for hematoblastic mensuration, utilize the above-mentioned method that is used for red blood cell, such life-span determination of blood platelet can be used to distinguish the hemorrhage and immune response after the blood transfusion, also can be used for diagnosing atherosclerotic or other disease, in these diseases, be subjected to patient's the platelet life span of sickness influence different with the patient's who is not subjected to sickness influence platelet life span.In addition, above-mentioned method can be used to carry out the mensuration of the volume lifetime of other cell, comprises white blood corpuscle.

During Cellular tracking was analyzed in vivo, cell was with carrying out mark from the cyanine class dyestuff of external detection.This can comprise from the dyestuff of external detection cyanine class dyestuff as ¹²⁵The DiOC of I mark ₁₄(3).The also available cyanine class dyestuff that has the nuclear magnetic resonance probe carries out Cellular tracking, determines the position of labeled cell like this with regard to the available core magnetic resonance imaging.In addition, the fluorescence of cyanine class dyestuff can be used to the spike cell in the observation in vitro body.Modally be that fluorescence is used for spike cell in the spot of eyes, retina and blood vessel.As described in the following examples, the purposes of Invivo Cellular tracking comprises the detection of primary carcinoma disease and metastasis cancer cell, the contract mensuration of narrow imaging and graft-rejection of the detection of infection site, artery.

The present invention will be described for the following example, and do not mean that top definition of restriction reaches following desired scope of invention.

The colouring method of embodiment 1 tissue culture cells

I. the preparation of cell

The tissue culture cells that uses logarithmic phase is to obtain best result.Suspension culture is taken out and puts into polypropylene centrifuge tube from culture vessel.

When using monolayer cultivation, the cell that must remove supernatant and adhere to the phosphate buffer washing of no calcium ions and magnesium ions is to remove haemocyanin from bottle.(New York #610-5300) covers the bottle end and at room temperature cultivating for Cibco Laboratories, Grand Island, and is loosening and spread out until cell monolayer to add trypsase-EDTA solution.The gained cell suspending liquid is transferred in the polypropylene centrifuge tube, adds isopyknic 10% hyclone (FBS) culture medium (Hazelton) that contains to stop tryptic effect.Cell at room temperature centrifugal 10 minutes with 400 * g.The sucking-off supernatant replaces the sweet mellow wine that isopyknic grade is oozed, with the precipitation of suspension cell again.Current sweet mellow wine washing is in order to remove the cell that plasma proteins and preparation are used to dye from cell suspending liquid.Cell at room temperature centrifugal 10 minutes once more with 400 * g.Sucking-off supernatant, gained cell precipitation in mannitol solution with 2 * 10 ⁶The concentration of individual cell/ml suspends again and is used for dyeing.But some clone is to the use sensitivity of sugar alcohol (sweet mellow wine); In this case, (MW 180.16,54.05g/l) for the glucose solution that oozes of available grade.

II. the preparation of stock staining solution

With absolute ethyl alcohol preparation 2 * 10 ^-3M stoste is as follows:

DiO-C ₁₄（3） MW800（1.600mg/ml）

DiS-C ₁₄（5） MW814（1.628mg/ml）

DiO-C ₁₈（3） MW936（1.872mg/ml）

DiI-C ₁₄（5） MW850（1.700mg/ml）

All dyestuffs are all from Molecular Probes, Eugene, Oregon.

Stock staining solution carries out sonication and dissolves fully with the assurance dyestuff, and makes it reduce to minimum to adhering to of pipe.Prepare stoste with polystyrene tube, just can be observed the dissolving of dyestuff.But come staining cell with polypropylene tube because cyanine class dyestuff in aqueous environments to polyacrylic adhere to the comparison polystyrene adhere to much smaller.

III. cell dyeing

In waiting sweet mellow wine that oozes, cell concentration is transferred to 2 * 10 ⁶Individual cell/ml.Be staining cell, with 2 * 10 ^-3The stock staining solution of M adds in the dyeing liquor, and every 1ml cell suspending liquid adds the 5ul dyestuff.Suction or stir the sample be used to dye so that sample fully mixes.Cell is cultivated 10 minutes in dyestuff after, take out sub-fraction and under fluorescent microscope, check, to guarantee reaching strong and dyeing uniformly.The dyestuff of DiO series uses the exciting light to 488nm to have optionally microscope optical filter, and the dyestuff of DiS and DiI series excites near 575nm just and can observe fluorescence.

After the cultivation, isopyknic PBS is added in the suspending liquid of staining cell.Cell 20 ℃ with 400 * g centrifugal 10 minutes.The sucking-off supernatant, precipitation suspends again with PBS.The repeated centrifugation step, observation post gets the dyestuff that exists in the supernatant.If dyestuff obviously is present in the supernatant, repeated washing does not detect free dyestuff until supernatant with fluorescence spectrophotometry.The last time after the washing, remove supernatant and suspend again and be precipitated to required concentration with suitable culture medium.Institute all carries out under aseptic condition in steps.

The dyeing of embodiment 2 red blood cells

I. the reagent preparation

A. citrate anticoagulation agent

1.66g sodium citrate

0.206g citric acid

0.140g NaH ₂PO ₄

1.61g glucose

Listed each component is dissolved in the 63ml distilled water, and solution PH transfers to 5.6.Last solution is sterilized by 0.22 micron filter membrane.

B. wait and ooze glucose solution

Glucose (54.05g) is dissolved in 1 liter of distilled water.Check osmolarity with Fick (Fiske) osmometer,, transfer to 320mOsm as needs.

II. the preparation of stock staining solution

The dyestuff of dissolving 1.628mg/ml is made into 2 * 10 in absolute ethyl alcohol ^-3The DiIC of M ₁₄(5) stoste.May need sonication to come complete dissolving dye.

III. staining procedure

The bloodletting tube of sodium citrate is equipped with in use, or the syringe of the citrate anticoagulation agent of being prepared is housed, and the amount of anti-coagulants is 1/10th of a syringe volume, aseptic collection whole blood.Reserve sub-fraction as flow cytometer or function test.Blood at room temperature with centrifugal 10 minutes of 100 * g with the precipitation red blood cell.Taking-up contains hematoblastic blood plasma and preserves, and adds the grade that is five times in packed cell volume and oozes the glucose Washed Red Blood Cells.Should be once more at room temperature with 100 * g centrifuge cell 10 minutes, the sucking-off supernatant.Remove haemocyanin and make more than the stronger and uniform washing of dyeing repeats once.After the centrifugal the last time and sucking-off supernatant, Red Blood Cells Suspension again in waiting glucose that oozes make its concentration reach 4 * 10 ⁸Individual cell/ml.

Add before the dyestuff, suction or stirred sample are to guarantee not take place sedimentation.The red blood cell suspension of each milliliter adds the DiSC of 15 microlitres ₁₄(5) stoste (concentration is 2mM in the ethanol).Biased sample is to guarantee that dyestuff is distributed in the solution quickly and evenly immediately.After about 5 minutes, take out sub-fraction and do microscopic examination.On microslide, draw a circle with wax crayon, the small amounts of cells sample in the dyeing liquor is put in the circle.On slide, put a cover plate, observation sample.Can reduce the discal cell-spine-like cell that causes by slide and cover plate with the wax circle and transform (discocyte-echinocyte transformation).Also can prevent this conversion with plastic slide and cover plate.Like this, just can guarantee in whole staining procedure that generation in the dyeing of homogeneous, keeps erythrocytic structure strongly.After 5 minutes, cell should be by level dyeing, and the contact dyestuff time needn't be longer than 10 minutes.

Determine cell by after the level dyeing, isopyknic phosphate buffer is added in the dyeing suspending liquid.Cell at room temperature with 400 * g centrifugal 10 minutes is removed supernatant.After centrifugal, supernatant regular meeting sees the vestige that free dye exists, therefore must be with the phosphate buffer repeated washing of calcic and magnesium, up to not detecting free dye with fluorescence spectrophotometry in supernatant.

At this, cell suspension can be used for experiment in suitable solution, also can be suspended in the no hematoblastic blood plasma and be used for injecting to animal subject again.Concerning injection again, general method is that the red blood cell that will dye is suspended in a certain amount of blood plasma again, and this blood plasma obtains and the centrifugal blood platelet of having removed under 400 * g from collected whole blood centrifugal first.The asptic technique of all using is in steps carried out.

The dyeing of embodiment 3 blood platelets

I. the preparation of cell

The bloodletting tube of sodium citrate is equipped with in use, or the syringe of the sodium citrate anticoagulant of being prepared is housed, and the amount of anti-coagulants is 1/10th of a syringe cumulative volume, collects whole blood.Cell at room temperature with centrifugal 10 minutes of 100 * g to obtain the abundant blood plasma of blood platelet.All to use in steps plastic suction pipe and polypropylene centrifuge tube to activate preventing relating to hematoblastic institute.

The blood plasma that the sucking-off blood platelet is abundant is also transferred to it in another centrifuge tube.Under 20 ℃, blood platelet was precipitated in centrifugal 10 minutes from blood plasma then with 1000 * g.Collect blood plasma then and give over to function test usefulness, the suspending medium that also can do to inject is again used.This blood plasma should be under 4000 * g centrifugal 10 minutes, and to guarantee removing any residual blood platelet before as the medium that suspends again, this blood plasma is called as PFP (PPP).

The centrifugal blood platelet precipitation that obtains under 1000 * g, care should be used to ground is suspended in the agent of a spot of citrate anticoagulation blood again, with the suspending liquid of the homogeneous that obtains concentrating.After this, can add wherein as thinning agent oozing glucose with the grade of initial blood plasma equivalent.Blood platelet is under 300 * g centrifugal 5 minutes then, the sucking-off supernatant.This step glucose washing is removed remaining plasma proteins and is made dyeing homogeneous and stronger.Again the blood platelet that suspends in glucose solution precipitation can dye.

PC is transferred to 4 * 10 ⁸Individual cell/ml adds 15ul DiOC in every milliliter of platelet suspension ₁₄(3) stoste (concentration is 2mM in absolute ethyl alcohol).Abundant carefully immediately mixing suspension evenly distributes to guarantee dyestuff.Use the fluorescence microscope blood platelet, guarantee to reach level dyeing,, just can carry out separating of free dyestuff in blood platelet and the suspending liquid if like this.

II. use the blood platelet of Sephadex G-100 post separation marking

From the blood plasma that blood platelet enriches, separate blood platelet with Sepharose 2B traditionally.We find that Sephadex G-100 is also very effective in hematoblastic separation.Present technique is used with staining technique, and operates by following method.With blood platelet-dye suspensions upper prop.The dyestuff of small-molecular weight is trapped in the particle, and big blood platelet then directly passes through pillar.Sephadex G-100 can be used for the separating of free dye of fluorescently-labeled blood platelet and suspending liquid by this way.

A. the preparation of post

The operational manual of pressing factory (Pharmacia Laboratories, Piscataway, New Jersey) is with Sephadex G-100 swelling, and washing in acetone (100%), separates hematoblastic medium with preparation.Washing step is following to carry out: Sephadex at room temperature with 300 * g centrifugal 10 minutes is suspended in the Hanks balanced salt solution after removing supernatant again.Use Hanks balanced salt solution repeated washing Sephadex, until no longer smelling the acetone flavor.Gained solution is placed boiling water bath or places vacuum to carry out degasification.

Irritate post with the Sephadex slurry.。Do not make pillar with one with the syringe of the 10cc of connector.Silicone tubing is linked on the head of syringe, regulated the liquid stream of post with an adjustable tubule folder.The holder of doing the syringe bottom with the nylon wire of 47 micron pore size blocks the Sephadex pearl.Should avoid in order to band sintered glass filter disc is the conventional glass column of holder, because these posts may activate blood platelet.Pour in the post with Hanks solution, a small amount of Sephadex slurry is added in the post.Clip is left even as big as making slowly and continuously stream of liquid stream.This operation compresses Sephadex equably and prevents to form duct or air gap.Repeat to add the step of HBSS and Sephadex slurry, until the column volume that obtains required size.Use HBSS(2 void volume before using) with the post cleaning down.

B. hematoblastic separation

Blood platelet dye well suspending liquid is layered on the Sephadex carefully.The clip of column bottom should be opened and the liquid level in the post is descended, until the top that reaches Sephadex liquid.Continuing to flow makes suspending liquid pass Sephadex.When liquid level was in the Sephadex top, it was mobile to slow down once more.Carefully at the top of post add enough HBSS and form cushion, so that do not upset Sephadex pearl face when adding HBSS again.Recover flowing of post once more.Can make platelet suspension in gel, form compact zone in this way, and move through whole pillar with unusual even velocity.Can obtain concentrated blood platelet effluent in this way.Continuing to flow through pillar, is that a pipe is collected with 0.5 to 1.0ml.Containing hematoblastic each pipe can be observed and they are combined by its opacity.Each component that will merge then centrifugal 10 minutes with 300 * g.Sucking-off supernatant, available suitable medium suspend again to precipitate and are used for experiment or analysis, or the PFP supernatant is used for functional examination or injection again.

Embodiment 4 difference the input hemorrhage and immune responses in back

Before the input, a small amount of red blood cell in the blood of every part of desire input of labeled molecule mark of usefulness fluorescence form.After operation and blood input, take out a small amount of venous blood immediately, with the number percent (fluorescence number percent) of conventional flow cytometer or fluorescence spectrophotometry labeled cell.Behind the vein haemospasia, get a certain amount of blood with regular interval and similarly analyze first.If the generation internal haemorrhage, even blood volume descends, staining cell does not change with the ratio of the cell that is unstained yet.If patient is just standing a blood transfusion afterreaction, then Ran Se cell (they are cells of input) is at first destroyed, and staining cell descends with the ratio of the cell that is unstained.This method makes people might distinguish hemorrhage and blood transfusion back immune response.

The diagnosis of embodiment 5 spontaneous thrombopenias

Produce the speed except measuring platelet life span and blood platelet, this method is identical with embodiment 4.Therefore, want mark patient's blood platelet (seeing embodiment 3) itself before the input.Behind the input blood platelet, the venous blood that takes a morsel immediately is with the hematoblastic number of mark (fluorescence number percent) in every milliliter of conventional flow cytometer or the fluorescence spectrophotometry.Behind the vein haemospasia, get the percent of other a certain amount of blood analysis fluorescencepositive cell with certain interval first.The blood platelet of dyeing and undyed hematoblastic number percent as the function construction of time, for providing mark blood platelet extinction speed and unmarked blood platelet, the clinician are produced speed.Blood platelet produces or the speed of extinction is the dynamic yardstick of thrombopenia process.In this way, the clinician can determine whether the symptom that is similar to spontaneous decrease of platelet disease is in the level of blood platelet generation or platelet life span.

The blood platelet of input is also used these dye markers.And, the mensuration of blood donor's platelet life span also with described to be used for self hematoblastic mode identical.

Embodiment 6 atherosclerotic diagnosis

Use this method diagnosing atherosclerotic to be based on such hypothesis, promptly atherosclerotic's the hematoblastic life-span is shorter than the life-span of orthoplastocyte.Patient's sub-fraction blood platelet is carried out mark (pressing the scheme of embodiment 3) with the cell marking dyestuff of fluorescence, NMR or radiolabeled form.

Again the blood platelet of the fluorescence formal notation of this molecule of injection (method is seen embodiment 3), the hematoblastic life-span is measured with continuous venipuncture method, and the remaining hematoblastic quantity of mark is measured (seeing embodiment 5) as the function of time.The mensuration of platelet life span has been indicated the reduction of platelet life span, can be used for atherosclerotic diagnosis.

Another diagnostic method is a radioactive isotope form (gamma emitter) of utilizing this molecule, at external mark patient's blood platelet (with the method for embodiment 3).Re-enter the blood platelet of mark, measure the hematoblastic life-span with conventional isotope method of counting, also available conventional gamma camera is taken a picture to determine whether the mark blood platelet adheres on the vascular wall to the patient.

The detection of embodiment 7 primary tumo(u)rs or metastasis

Combine with the gamma emitter of this molecule or the form of NMR sensitivity with two kinds of different cytology methods.In two kinds of methods, all used the cell (tumor cell-seeking cells) of close tumour cell.A kind of method activation or non-activated lymphocyte, another kind of method monocyte, neutrocyte or blood platelet spike.

Lymphocyte or NK cell adopt interleukin 2 or equivalent molecule to activate with method in vivo.Utilize the method for embodiment 1 then,, and re-enter in patient's body with radioactivity or these cells of NMR formal notation of this molecule.Then the patient is placed under the suitable display, monitor leukocytic " going back to the nest " to determine the position of the unknown tumour that originates from.

A kind of similar methods uses the monocyte that is tried the patient to replace lymphocyte.In the method, monocyte is used special people of tumor type or the monoclonal antibody of mouse is carried out mark, to keep the identification to tumour cell.Foris，G.，et al.，Cell.Immuno。78：276-284（1983）。Then these cells are come mark with the radioactive isotope or the NMR imaging form of labeled molecule, and the patient is given in injection again.Continuous imaging of time discloses the monocytic playback of target.

In the method for another location primary tumo(u)r or metastasis, monocyte, neutrocyte or blood platelet carry out mark and are used for seeking tumour or metastasis as described in embodiment 3.Because these cells occur early at tumor locus, the show tags cell can positioning tumor.

The detection of embodiment 8 infection sites

Spike neutrocyte in this application.Get the neutrocyte of waiting to try the patient, come mark (with the method mark of embodiment 1 and 2) with the radioactive isotope or the NMR imaging form of labeled molecule.Feed back these cells then, do neutrocyte with gamma camera or nuclear-magnetism imaging technique, the imaging of playback is to identify infection site.Imaging can be used for identifying the dynamic change in the neutrocyte aggregation process continuously, and monitoring is by the variation due to the treatment.

Embodiment 9 detects diabetic's retinopathy by checking macular degeneration

With a small amount of red blood cell (O type or self cell) of a kind of labeled molecule mark (method is seen embodiment 2) of form, the excitation wavelength of this labeled molecule is (greater than 700nm) outside the range of human sight.Then cell is injected to the patient again by vein.With the fluorescence retinography of routine, can photograph the photo of continuous retinal vessel.Excite the cell that carries the fluorescent tracing compound with suitable wavelength, on photographic negative, catch institute's emitted fluorescence.Since the red blood cell absorbing dye, and red blood cell can not flow to outside the vascular system, so have only retinal vessel that fluorescent image is just arranged.

In another embodiment, use conventional double light beam laser energizer to measure blood flow rate, this energizer excites identical cell on endovascular two differences.But the distance between two shot points is fixed.Survey fluorescence at each shot point, the length of required time between two shot points is a yardstick of cell flow rate in blood vessel.

Check retinosis with vascular integrity and blood flow rate.A kind of like this detection is used for determining the degree of impaired vision and to the successful property of diabetic therapy.

Embodiment 10 is right after last time, and morbidity detects the morbidity next time that the patient is about to generation

Examine patient's blood platelet is carried out mark (method is seen embodiment 3) with a kind of labeled molecule of form, and the excitation wavelength of this molecule is (greater than 700nm) outside the range of human sight.Give the patient with labeled cell by the vein re-injection then.With a single blood vessel in a conventional condenser (in excitation wavelength) the aligning retina bed.Measure fluorescence with suitable conventional focusing lens and a photomultiplier.The output of photomultiplier shown by numeral and deposit computing machine in.Fluorescence intensity can be used as a yardstick, the blood platelet of indication in the vein be single, two, three, or the like.The danger that the platelet aggregation thing increases expression morbidity for the second time is very big.

The developing of embodiment 11 arteriarctias

With blood platelet (with the method for embodiment 3) or the red blood cell (with the method for embodiment 2) of the radioactive isotope form of this molecule (gamma radiation body) external mark patient self.The input marking cell is taken a picture to the patient with conventional gamma camera, determines whether constriction of lumen of vessels.The red blood cell mark can with self or any blood donor's O type cell do.

Embodiment 12 arthritic diagnosis and by stages

Lymphocyte, monocyte or neutrocyte with the radioactive isotope of this molecule or NMR imaging formal notation (embodiment 1 or 2 method) patient to be tested.Then these labeled cells are fed back to the patient, observe its gathering with conventional image device.When arthritis, the spike of expection monocyte is more useful, and when degenerative joint disease (Degenerative Joint Disease), the spike of expection lymphocyte is more useful.This makes us can measure number and definite result of treatment to conditions of patients in hot joint.

The fast measuring of embodiment 13 graft-rejections

Each plays a significant role lymphocyte, neutrocyte or blood platelet in transplanting or organ rejection response.Take out patient's cell, carry out mark (with the method for embodiment 1-3) with the radioactive isotope or the NMR imaging form of dye molecule.The re-injection labeled cell is done continuous video picture to the patient.Before just repelling, the cells accumulation of injection increases available this method detection in the amount of graft part.

The diagnosis of embodiment 14 multiple sclerosiss

Except will measuring cell spinal cord district or other are imbued with position in the myelinic zone, this application has the method identical with embodiment 13.

Embodiment 15 measures the volume of phase in red blood cell longevity, red blood cell and blood

For distinguishing the reaction after hemorrhage and the blood transfusion, with the method same as described above mensuration phase in red blood cell longevity (embodiment 4).Unique is not both, mark and re-injection patient's self red blood cell.After the injection, with the quantity of every milliliter of tagged blood cells function (using flow cytometer or fluorescence spectrophotometry), to measure the phase in longevity of these cells as the time.With the red blood cell of the dyestuff of fluorescence form dyeing dose known amounts (self or O type) to measure the volume of red blood cell mass and blood.Dyeing red blood cell (10 with dose known amounts ⁹) when zero, inject to individuality, take out sub-fraction blood after 5 minutes.Percent with flow cytometer or fluorescence spectrophotometry labeled cell.By the total cellular score in this dilution gfactor and every cubic millimeter, can determine the volume of red blood cell mass and blood.

More than set forth embodiment preferably more of the present invention, but the invention is not restricted to explanation disclosed herein, we keep all rights of making amendment in the scope of following claim.

Claims (17)

1, a kind of preparation contains being used for Invivo Cellular tracking and measuring the method for the blood sample of cell survival of cell of cyanine class dye marker.

2, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is close tumour cell, so can detect former or the tumour cell that shifts.

3, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is lymphocyte or natural killer cell.

4, a kind of method according to claim 2, lymphocyte wherein or natural killer cell were activated before introducing the experimenter.

5, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is a monocyte.

6, a kind of method according to claim 2, wherein close tumour cell is a monocyte, so kept identification to tumour cell because it links to each other with TS monoclonal antibody.

7, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is little blood plate.

8, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is a neutrocyte.

9, the cyanine class dyestuff that the process of claim 1 wherein of a kind of basis is DISC ₁₄(5) or DiOC ₁₄(3).

10, the cyanine class dyestuff that the process of claim 1 wherein of a kind of basis is with a gamma radiation body tag.

11, the cyanine class dyestuff that the process of claim 1 wherein of a kind of basis is with a nuclear resounce probe mark.

12, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is close infector cell, so but bacterial detection or fungi.

13, a kind of basis the process of claim 1 wherein that the cell of cyanine class dye marker is a red blood cell.

14, a kind of method according to claim 13, the speed that the red blood cell of wherein measuring cyanine class dye marker flows in blood vessel.

15, a kind of basis the process of claim 1 wherein that the excitation wavelength of cyanine class dyestuff is outside human visible spectrum.

16, a kind of method according to claim 13, wherein the red blood cell of cyanine class dye marker is prepared by mark experimenter's cell.

17, a kind of method according to claim 13, wherein the red blood cell of cyanine class dye marker is prepared by any donor 0 type red blood cell of mark.