CN106754680A - A kind of separation method of placenta derived stem cells and its application - Google Patents
A kind of separation method of placenta derived stem cells and its application Download PDFInfo
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- CN106754680A CN106754680A CN201611240190.XA CN201611240190A CN106754680A CN 106754680 A CN106754680 A CN 106754680A CN 201611240190 A CN201611240190 A CN 201611240190A CN 106754680 A CN106754680 A CN 106754680A
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Abstract
A kind of separation method the present invention relates to new placenta derived stem cells and its application in ischemic disease is treated.Specifically, in the present invention, using a kind of new complex enzyme fluid composition, placenta derived stem cells are efficiently separated.Placenta derived stem cells by after outer-gene modification, can induction of vascular is newborn in animal model, by improving offshoot circulation, play the effect for the treatment of ischemic.
Description
Technical Field
The present invention relates to the field of methods for the isolation and genetic modification of placental derived stem cells and the treatment of ischemic diseases. Specifically, the invention relates to a novel method for separating placenta-derived stem cells and application of gene modified cells thereof in treating ischemic diseases.
Technical Field
The ischemic diseases include coronary heart disease, lower limb arterial ischemia, diabetic arteriosclerosis, etc. The major arterial lesions of ischemic diseases can be treated by adopting vascular bypass, endovascular intervention and the like, but patients with peripheral vascular occlusion and intolerance to surgery have poor treatment effect by using a single medicament. The combination of stem cells and gene therapy can effectively improve the ischemic tissues of the body.
The stem cell transplantation provides a new treatment method for limb ischemic diseases. The placenta contains various cell components such as hematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells, and the like. Placental derived stem cells (PSCs) have been the focus of research because they are easily available, abundant and have no adverse effect on donors. The number of cells and biological properties obtained by various isolation and culture methods vary. For example, the method of separating and collecting the placental blood mononuclear stem cells can enrich hematopoietic cells, and thus can be used for treating leukemia, various immunological diseases and the like through hematopoietic stem cell transplantation. Most of the cells obtained by cutting, digesting, culturing and the like are mesenchymal stem cells, and can be used for treating osteogenesis and immune diseases.
The placenta-derived stem cells PSCs are expected to become an alternative source of bone marrow mesenchymal stem cells (BMSCs). The current research shows that PSCs have the capacity of secreting pro-vascular growth factors and inducing differentiation to other tissue cells [ Lu GH, Yong WS, Xu ZM, et al human plant specific tissue samples-derived sensory stem cells, differential expression and so on, and do not have any adverse reaction to long-term culture in video. neuron report.2012,23(8):512-518 ]. Therefore, the PSCs also have good application prospect in treating ischemic diseases.
Hepatocyte Growth Factor (HGF) is a multifunctional growth factor with angiogenic activity. HGF gene therapy has good effect in early animal experiments and clinical researches of ischemic diseases.
The stem cell modified by HGF gene is a regenerative medical means with clinical application prospect for promoting angiogenesis, and has more superiority compared with simple stem cell or gene therapy. The invention evaluates the treatment effect of transplanting the Ad-HGF modified placenta-derived stem cells (PSCs) in a rabbit limb ischemia model, and provides experimental basis for future application and clinic.
Disclosure of Invention
The invention provides a method for separating placenta-derived stem cells and application of HGF modified placenta-derived stem cells in treating ischemic diseases. Specifically, in the invention, the placenta-derived stem cells are effectively separated by using a novel compound enzyme liquid composition. The placenta-derived stem cells can induce angiogenesis in an animal model after in vitro gene modification, and play a role in treating ischemia by improving collateral circulation.
In order to achieve the above object, the present invention relates to the following aspects.
In a first aspect, the present invention relates to a complex enzyme liquid composition for isolating placental derived stem cells, comprising trypsin, collagenase and dispase.
In one embodiment of the invention, the complex enzyme liquid composition comprises 0.01-0.1 mg/mL of trypsin, 0.1-5 mg/mL of collagenase and 0.1-5 mg/mL of dispase; preferably, it comprises: 0.03-0.1 mg/mL of trypsin, 0.5-3 mg/mL of collagenase and 0.5-3 mg/mL of dispase; preferably, it comprises 0.05mg/mL trypsin, 1mg/mL collagenase and 1mg/mL dispase.
In one embodiment of the invention, the complex enzyme liquid composition comprises 0.5-3 wt% of trypsin, 0.5-3 wt% of collagenase and 0.5-3 wt% of dispase; preferably, the complex enzyme liquid composition comprises 1-3 wt% trypsin, 1-3 wt% collagenase and 1-3 wt% dispase.
In one embodiment of the invention, the collagenase is collagenase type I;
in one embodiment of the invention, the dispase is a neutral protease, such as neutral protease I.
In a preferred embodiment, the placental derived stem cells are placental mesenchymal stem cells.
In a second aspect, the present invention relates to a method for isolating placental-derived stem cells, comprising the step of pumping the complex enzyme liquid composition of the present invention into the placenta to isolate placental-derived stem cells.
In a preferred embodiment, the complex enzyme liquid composition is pumped through the placenta by umbilical vein and arterial closure.
In a preferred embodiment, the placental derived stem cells are placental mesenchymal stem cells.
A third aspect of the invention relates to a placental derived stem cell produced by the isolation method according to any one of the claims of the present invention. In one embodiment of the present invention, the placental derived stem cells are HGF-modified placental derived stem cells.
In a fourth aspect, the present invention relates to a method of increasing the expression level of angiogenic growth factors comprising infecting placental derived stem cells with Ad-HGF.
In a preferred embodiment, the vascular growth factor comprises HGF, VEGF and bFGF. In a further preferred embodiment, the vascular growth factor is HGF.
In a fifth aspect, the present invention relates to the use of HGF-modified placental derived stem cells for the preparation of a medicament for the treatment of an ischemic disease. Preferably, the ischemic disease is selected from any one or more of coronary heart disease, lower limb arterial ischemia and diabetic arteriosclerosis; preferably, the HGF-modified placental-derived stem cells are the HGF-modified placental-derived stem cells described above.
In a preferred embodiment, the placental derived stem cells are placental mesenchymal stem cells.
In a sixth aspect, the present invention relates to a method of treating ischemic diseases comprising the step of administering a therapeutically effective amount of HGF-modified placental-derived stem cells to a subject. Preferably, the ischemic disease is selected from any one or more of coronary heart disease, lower limb arterial ischemia and diabetic arteriosclerosis; preferably, the HGF-modified placental-derived stem cells are the HGF-modified placental-derived stem cells described above.
In a preferred embodiment, the placental derived stem cells are placental mesenchymal stem cells.
The dosage to be administered will depend on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the condition and past medical history of the patient being treated. It is common practice in the art to start doses at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved.
In a seventh aspect, the present invention relates to isolated, genetically modified, placental derived stem cells for use in the treatment of an ischemic disease.
In a preferred embodiment, the placental derived stem cells are placental mesenchymal stem cells.
In the present invention,
the term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or amelioration of a disease and/or disorder described herein in a subject.
The term "disease and/or disorder" refers to a physical condition of the subject that is associated with the disease and/or disorder of the present invention.
The term "subject" can refer to a patient or other animal, particularly a mammal, e.g., a human, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the invention to treat, prevent, ameliorate, and/or alleviate a disease or disorder described herein.
Drawings
FIG. 1 cell morphology of placental stem cells after isolation and culture.
FIG. 2 detection of cell surface marker expression by flow cytometry. Wherein,
2A, flow cytometry detects the expression of the cell surface marker CD 31.
And 2B, detecting the expression of the cell surface marker HLA-DR by a flow cytometer.
2C, flow cytometry detects the expression of the cell surface marker CD 34.
2D, flow cytometry detects the expression of the cell surface marker CD 45.
2E, flow cytometry detects the expression of the cell surface marker CD11 b.
2F, flow cytometry detection of the expression of the cell surface marker CD 90.
2G, flow cytometry detects the expression of the cell surface marker CD 105.
2H, flow cytometry detects the expression of the cell surface marker CD 44.
The 2I flow cytometer detected the expression of the cell surface marker CD 73.
FIG. 3 shows HGF gene-modified PMSC transplanted ischemic tissue pro-angiogenic growth factor expression. Wherein,
3A, Q-PCR assay for intracellular HGF expression.
3B, Q-PCR assay of ischemic tissue local VEGF, bFGF and HGF expression.
Fig. 4 evaluates angiogenesis and improvement of blood circulation. Wherein,
4A, arteriography was performed to evaluate angiogenesis and improvement of blood circulation.
4B, HE staining to evaluate angiogenesis and improved blood circulation in the left treatment group.
4C, HE staining right control groups were evaluated for angiogenesis and improved blood circulation.
Detailed Description
In order to further understand the present invention, the technical solutions mentioned above are further illustrated and described below with reference to examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
Example 1: placenta mesenchymaIsolation culture of cells
A new complex enzyme culture system comprising 0.05mg/mL trypsin, 1mg/mL collagenase and 1mg/mL dispase (neutral enzyme) is utilized, and the digestive enzyme combined solution is hermetically pumped into the placenta through the umbilical vein and the artery at the speed of 50 mL/hour, so as to achieve the effects of digesting and separating cells. Digestion was carried out at 37 ℃ for 2 hours. The washed out cells were collected, washed and cultured. The culture conditions were that DMEM containing 15% FBS was isolated and placental mesenchymal stem cells PSCs were cultured in the form of fibroblasts, and the cell state was good (fig. 1).
1. Reagent preparation
a) Storage solution (5mg/mL) of dispase (neutral protease I)
The dispase was dissolved in ultrapure water, the lyophilized enzyme was stored at 2-8 degrees until shelf life, the aqueous solution was stored at-15 to-25 degrees, and the lid was opened to allow the powder to be ejected (if stored under vacuum) and the correct concentration (including the cap attached) was achieved by trying to dissolve it.
b) Storage liquid of collagenase type I (5mg/mL)
100mL of PBS was added 0.5g of collagenase powder, filtered through a 0.2 μm filter and dispensed into 50mL tubes. Can be preserved for several months at-20 ℃ without repeated freezing and thawing.
c) DNase stock solution 1:100(0.1mg/mL)
DNase I was dissolved in 1mL of ultrapure water, transferred to a 50mL tube, and 20mL of ultrapure water was added and mixed well. Stored at-20 degrees.
d) Pancreatin stock solution (2.5% w/v, about 2.5mg/mL)
To 500mL of sterile phosphate buffer was added 2.5g of PVP powder to prepare a PVP solution. The powder was completely dissolved by vigorously shaking the tube to obtain a clear solution. 5g of pancreatin powder was added to 200mL of 0.5% PVP solution, and a sterile magnetic stirrer was placed and stirred at 4 ℃ for 30 min. After 30min, the solution remained cloudy. Subpackaging 1.5mL of the solution into 1.5mL of tubes, centrifuging at 4 ℃ and 15000g for 30 min. The supernatant was collected in a 50mL tube and the precipitate was discarded. The aqueous solution was stored at-20 ℃.
e)PBS
NaCl 8g，KCl 0.2g,，Na2HPO4.12H2O 1.44g，KH2PO40.24g, adding 800mL of distilled water, adjusting the pH to 7.4 by hydrochloric acid, and adding water to a constant volume of 1000 mL. Filtering and sterilizing with 0.22 μm microporous filter membrane.
f)PBS/EDTA
EDTA, penicillin (100U/mL), streptomycin (100mg/mL) was added to PBS, and the mixture was stored at 4 degrees for 1 month. 1.5mg EDTA was added per mL of cord blood or placental cell suspension. The solution is used in placenta and blood collection and cleaning media.
g)HBSS(withCa2+、Mg2+)
NaCl 8g，KCl 0.4g，Na2HPO4.12H2O 1.26g，KH2PO40.06g，MgSO40.098g，CaCl20.14g，D-Glucose 1g，NaHCO30.35g, constant volume to 1000mL, and filtering and sterilizing by a 0.22 mu m microporous filter membrane.
h)PBS/FBS
PBS was added with 2% FBS, penicillin (100U/mL), streptomycin (100mg/mL), and stored at 4 degrees for 1 month. The solution is used in placenta and blood collection and cleaning media.
i) Albumin (0.3% w/v, about 0.3mg/mL)
j) Double antibody
10 ten thousand units of penicillin powder and 10 ten thousand units of streptomycin powder were dissolved in 1mL of PBS buffer solution and diluted 1:1000 at the time of use.
k) RPMI1640 medium
RPMI1640 dry powder (Gibco)1 bag, NaHCO32g, HEPES 2.38g/1L, pH 7.2, 0.45 μm, 0.22 μm microporous membrane twice filtration.
l) Mix digestive juice
Neutral enzymes (also known as dispase) were purchased from roche. 1mg/mL of dispase, 1mg/mL of collagenase type I, 0.05mg/mL of pancreatin and 0.3mg/mL of albumin.
2. Method and step
And 2.1, obtaining the Ad-HGF modified placenta mesenchymal stem cells, culturing in vitro and preparing cell suspension.
The method comprises the following steps of (1) separating and culturing mesenchymal stem cells, clinically taking placenta tissues of full-term lying-in women (agreed by the lying-in women and family members), preparing a placenta collecting and storing device: sterile or disposable sterile canisters. A25000 ten thousand units heparin sodium PBS solution (50 mL) was prepared, and injected into the placenta, and then the umbilical cord was ligated to prevent coagulation of the placenta. Placenta protection fluid: RPMI1640 contains 1% human serum albumin, 1% double-resistant gentamicin and amphotericin B, and 1% heparin sodium. Storing at 4 deg.C for use. The placenta is collected aseptically and placed in a reservoir containing a placental protective solution. Timely transporting to a laboratory for separation.
2.1.1. Washing placenta blood
The umbilical cord is loosened, the placenta is kept down, two venous catheters are respectively inserted into two umbilical arteries, the suture is fixed, the vein is inserted into the other catheter, and the two catheters are also fixed by suturing. Connecting one umbilical artery with a peristaltic pump, cutting off the other umbilical artery, slowly flushing FBS/PBS through an umbilical artery tube, flushing 1000mL, and collecting placental blood through a venous tube. The other umbilical artery was similarly flushed and placental blood was collected via the venous port. Approximately 4000mL were flushed until the placental vasculature appeared white.
2.1.2 placental Stem cell Collection
Connecting a peristaltic pump with an umbilical artery, pumping a mix digestive fluid into the umbilical artery by peristalsis, stopping flow and standing for digestion for 2 hours after pouring 200mL, connecting the peristaltic pump with PBS, pumping the PBS into the umbilical artery, and flushing about 500mL to thoroughly collect stem cells attached to the placenta in the blood vessel. The injected volume is to fill the entire vessel. Filtering with filter screen to collect single cells, suspending the collected cells in MSC culture medium, placing at 37 deg.C and 5% CO2And (5) culturing in a humidity incubator.
The experimental results are as follows: when the placenta mesenchymal stem cells are observed under a microscope, the PMSCs are in a fibroblast shape, and the cell morphology and the growth state are good, as shown in figure 1.
2.2 flow assay of cell surface markers
The material and the method are as follows:
CD34/FITC, CD73/PE, CD105/PE, CD90/PE, HLA-DR/PE, CD44/PE, CD11b/PE, CD45/PE, CD31/FITC monoclonal antibodies were purchased from BD corporation, FACS flow cytometer (BD Calibur USA),
method and step
1) PSC was cultured for 4 passages, trypsinized to collect cells, washed 2 times with PBS (pre-warmed to room temperature) to make 5X1050.1mL of cell suspension;
2) adding 1 μ l of different antibodies, mouse FITC and PE labeled isotype antibody labeled cells as negative control, wherein the antibodies comprise CD105, CD90, CD73, CD44, CD45, CD11b, CD34, CD31, HLA-DR are incubated for 20min at 37 deg.C in dark;
3) adding 4 to 5 times volume of DMEM medium containing 2% FBS at low temperature to resuspend and wash the cells;
4)400 u l PBS heavy suspension cells, flow cytometry collection, analysis of data.
Flow cytometry measurement results: the PSCs positive cell surface molecules comprise CD105, CD90 and CD73, and the positive rate of CD44 is more than 95%. The hematopoietic cell related molecules CD45, CD11b, CD34, CD31, HLA-DR and the like are less than 1 percent and meet the identification standard of Mesenchymal Stem Cells (MSCs), and the identification standard is shown in figure 2 (2A-2I).
Example 2: Ad-HGF modified PSCs promote HGF expression
Ad-HGF at multiplicity of infection (MOI) was used to infect PSCs, 48H after infection, the cells were harvested, and the expression level of HGF was determined by the QPCR method. After the completion of the imaging, the rabbit limb muscle tissue after the ischemia was collected, and then the expression of VEGF, bFGF and HGF in the limb muscle tissue was measured by the QPCR method.
Materials: DMEM medium and Ad-HGF were prepared from the same house, and VEGF, bFGF, and HGF primers were synthesized by Olympic. QPCR apparatus 7500(ABI, USA),green Real-time PCR Master Mix (TAKARA, Japan).
Method and step
1) Extraction of tissue RNA: after the radiography is finished, taking rabbit limb muscle tissues without blood, adding liquid nitrogen for freezing and grinding, adding TRIZOL for cracking and extracting total RNA of the tissues, and comprising the following steps:
cutting rat quadriceps tissue blocks frozen in a refrigerator with the size of 0.3 x 0.3cm to 80 ℃, adding liquid nitrogen into the rat quadriceps tissue blocks to grind the rat quadriceps tissue blocks in a mortar, adding 0.5ml of a pyrolysis solution to crack, sucking the solution into an EP (EP) tube, and standing the solution for 10min at 4 ℃.
② adding 200 mul of trichloromethane, mixing uniformly, standing for 2-3min at 4 ℃.
③ 12000rpm, centrifuging at 4 ℃ for 15 minutes, taking the supernatant and adding the supernatant into another EP tube.
Fourthly, adding isopropanol with the same volume (about 500 mu L) into an EP tube, standing for 10min at 4 ℃, rotating at 12000rpm, centrifuging for 15 min at 4 ℃, and then absorbing and removing the supernatant.
Fifthly, adding 1mL of precooled 75% ethanol (prepared by 1 per thousand DEPC water) into the precipitate, and centrifuging the precipitate for 10 minutes at 4 ℃ at 10000 rpm. Carefully removing the supernatant by suction, and air-drying for about 10min until the ethanol is completely volatilized.
Sixthly, the supernatant is carefully decanted and the tube is allowed to air dry for 5-10 minutes, taking care not to allow the RNA pellet to dry completely (otherwise its solubility is greatly reduced). Then 30. mu.L of DEPC water was added to the tube to dissolve. 1. mu.L of each extracted RNA sample was assayed for its RNA content using a nucleic acid analyzer.
2) Synthesis of first strand cDNA:
taking 2 mu gRNA, adding DEPC water to make volume reach 11 mu L, adding oligo (dT) primer 1 mu L into nucleic acid-free EP tube
② heating at 65 ℃ for 5 minutes and then rapidly placing on ice for operation, and adding the following components in the nucleic acid-free EP tube: 5 × reaction buffer 4 μ L, Ribockrnase Inhibitor (20u/μ L)1 μ L, dNTP mix 2 μ L, and RevertAId M-MuLV Reverse transfer (200u/μ L)1 μ L. The total reaction volume was 20. mu.L;
mixing the mixture lightly;
and fourthly, the reaction is terminated after the temperature bath is carried out for 60 minutes at 42 ℃.
Q-PCR
The primers were designed and synthesized by Beijing Oakco Biotechnology Ltd.
The primer sequences are as follows:
VEGF genes
Sense primer 5'TAT TCA AGC CTT CCT GCG TG 3' (SEQ ID NO:1)
Antisense primer 5'TCA TCT CCC CTA TGT GCT GG 3' (SEQ ID NO:2)
bFGF gene
The upstream primer is 5'CAA ACC GTT ACC TTG CTA TG 3' (SEQ ID NO:3)
The downstream primer is 5'TTC GTT TCA GTG CCA CAT AC 3' (SEQ ID NO:4)
Hepatocyte Growth Factor (HGF) primers
Upstream primer 5'GGA ACC AGA TGC TAG TAA GC 3' (SEQ ID NO:5)
Downstream primer 5'CGA GCA AGA GGA CAA TAA TC 3' (SEQ ID NO:6)
Internal reference β -actin:
the upstream primer is as follows: 5 'TGTTGTCCCTGTATGCCTCTG 3' (SEQ ID NO:7)
The downstream primer is: 5 'ACCGCTCATTGCCGATAGTG 3' (SEQ ID NO:8)
And (3) PCR amplification:
PCR reaction system:
setting PCR reaction circulation: real-time PCR was performed on the ABI prism 7500 System
The reaction parameters are as follows: 5min at 95 ℃ (pre-denaturation), 1 min at 94 ℃ (denaturation), 1 min at 55 ℃ (annealing), 1 min at 72 ℃ (extension), 30 cycles, and finally 10min at 72 ℃ (extension). After the reaction is completed, the detection of the dissolution curve is carried out to ensure the specificity of the PCR reaction. And carrying out data analysis according to the finally obtained CT value result. Statistical treatment SPSS 19.0 statistical software was used for t-test analysis, and P <0.05 was statistically significant for differences.
The experimental results are as follows: PSCs were infected with Ad-HGF at multiplicity of infection (MOI), and the expression level of HGF was determined by Q-PCR. As shown in FIG. 3A, the expression level of HGF in cells is obviously improved after Ad-HGF gene modification. The Q-PCR detects the expressions of VEGF, bFGF and HGF in the muscle tissue of hind limbs, and the result shows that the level of various blood vessel growth factors is obviously improved in a rabbit animal model. The expression of VEGF \ bFGF \ HGF in the treated group is obviously higher than that in the control group, and the figure is shown in figure 3B.
Example 3: HGF gene-modified PSC transplantation for promoting angiogenesis
3.1 preparation of animal model and Stem cell transplantation:
materials: 10 New Zealand white rabbits were ordered by the laboratory animal center of the military medical academy of sciences (animal license number: SCXK (Jing) 2015) 0005) with a weight of 2-3kg, half female and half male, and an age of 6 weeks.
Method and step
3.1.1 rabbits were anesthetized by 3% sodium pentobarbital (50mg/kg) via ear vein injection, and experimental rabbits were molded on the left and right lower limbs.
3.1.2 after the left and right hind limbs are unhaired and sterilized, a long incision is made from the midpoint of the inguinal ligament to the knee, the femoral artery and branch thereof are respectively ligated and separated from the inguinal ligaments on the two sides to the knee joint and the upper and lower longitudinal incisions, 1mL of Ad-HGF modified stem cell suspension is injected into the thigh muscle group around the femoral artery by 10 points by a 1mL injector for the left lower limb, and the normal saline is injected for the right control group.
3.2 after the transplantation and injection of abdominal aorta angiography, the rabbits are taken from the ear margin and injected with pentobarbital sodium (50mg/kg) for general anesthesia, the abdominal cavity is opened, the abdominal aorta is exposed, 70% diatrizoate meglumine is injected for continuous radiography after the abdominal aorta puncture operation, the double lower limb arteriography is carried out, and the formation condition of collateral circulation is observed. The photographing speed was 4 frames/s.
3.3 HE staining
Method and step
3.3.1 after the contrast of the experimental animal is finished, the muscles of the middle thigh of the lower limbs at both sides are taken and fixed in 10 percent neutral formaldehyde for 24 hours.
3.3.2 dyeing, the whole dyeing process comprises five contents: dewaxing, dyeing, dewatering, clearing and sealing.
(1) Dewaxing:
1. continuously slicing the tissue wax block to a thickness of 3 μm;
2. putting slices into xylene immediately for dewaxing for 10min after baking the slices in a warm box;
3. transferring into anhydrous alcohol for about 2minx2 times;
4. transferring into 90% alcohol for about 2minx2 times;
5. transferring into 80% alcohol for about 2minx2 times;
6. transferring into 70% ethanol for about 2 min;
7. transferring into water, and washing off alcohol for about 2-3 min;
8. transferring into distilled water for about 2 min.
(2) Dyeing:
1. transferring into hematoxylin, and dip-dyeing for 10min, preferably slightly deep-dyeing;
2. moving the mixture into water, and washing out hematoxylin and loose color for about 1-2 min;
3. transferring into differentiation solution (1% hydrochloric acid alcohol), and differentiating for several seconds to 30 seconds to fade the slice to light blue red;
4. moving into running water, washing for 30-60 min to make the tissue appear bright blue or sky blue;
5. transferring the fabric into the illite solution, and dip-dyeing for 2-5 min;
6. the slides were then transferred to water, and the eosin supernatant was washed off and excess dye wiped off with gauze.
(3) And (3) dehydrating:
1. after water on the slide is sucked, the slide is dehydrated in 80 percent alcohol for about 2minx2 times;
2. transferring into 90% alcohol for dehydration for about 4minx2 times;
3. the mixture was completely dehydrated by transferring into absolute alcohol (100% alcohol) for about 5minx2 times.
(4) Is transparent
1. Transferring into xylene I, and transparent for 3-5 min;
2. transferring into xylene II, and transparent for 5-10 min;
(5) sealing and fixing
And (3) sealing with gum, taking out the slice from the xylene II, quickly wiping off xylene on the periphery of the tissue, dripping a drop of gum on the tissue, taking a clean cover glass, carefully adding the cover glass on the sealing agent, and slowly flattening to ensure that the position of the cover glass is moderate. After the slices are sealed, the slices are placed in an incubator and are dried for observation.
The experimental results are as follows: an animal model of rabbit limb ischemia is established by a ligation method, and PSCs modified by Ad-HGF are transplanted at the ligation position. DSA (digital angiography) contrast examination is carried out on abdominal aorta puncture on the 14 th day after treatment, and the number of the generated capillaries after the left limb ischemia treatment is obviously more than that of the capillaries without the right limb ischemia treatment, so that the angiogenesis capacity is obviously enhanced. The results show that: the formation number of collateral blood vessels of the Ad-HGF-PSCs treated group is obviously higher than that of the control group, the blood vessels of the left lower limb (marked 1) of the rabbit are obviously compensated and thickened compared with those of the right limb (marked 2), and the peripheral branches are obviously increased (figure 4A). And (4) carrying out conventional HE staining, and observing the distribution density of blood vessels of lower limbs at two sides. Randomly selecting 10 visual fields with 400 times, counting the number of capillaries and the number of muscle fibers of the rectus femoris, and calculating the capillary density according to the ratio of the two numbers. The results show that the capillary number and muscle fiber number of the rectus femoris muscle was significantly greater in the left treatment group (fig. 4B) than in the right control group (fig. 4C), with statistically significant differences (P < 0.05).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
SEQUENCE LISTING
<110> institute of radiology and radiology medical science institute of military medical science institute of people's liberation force of China
Zaimei JIA
<120> separation method of placenta-derived stem cells and application thereof
<130>IDC150239
<150>201511021411.X
<151>2015-12-31
<160>8
<170>PatentIn version 3.2
<210>1
<211>20
<212>DNA
<213>Artificial
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<223> VEGF Gene sense primers
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tattcaagcc ttcctgcgtg 20
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<212>DNA
<213>Artificial
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<223> VEGF Gene antisense primer
<400>2
tcatctcccc tatgtgctgg 20
<210>3
<211>20
<212>DNA
<213>Artificial
<220>
<223> bFGF Gene upstream primer
<400>3
caaaccgtta ccttgctatg 20
<210>4
<211>20
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<213>Artificial
<220>
<223> bFGF gene downstream primer
<400>4
ttcgtttcag tgccacatac 20
<210>5
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<220>
<223> HGF upstream primer
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ggaaccagat gctagtaagc 20
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<223> HGF downstream primer
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<213>Artificial
<220>
<223> beta-actin upstream primer
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<400>8
accgctcatt gccgatagtg 20
Claims (10)
1.A complex enzyme liquid composition for isolating placental derived stem cells, comprising trypsin, collagenase and dispase.
2. The complex enzyme liquid composition of claim 1, comprising: 0.01-0.1 mg/mL of trypsin, 0.1-5 mg/mL of collagenase and 0.1-5 mg/mL of dispase;
preferably, it comprises: 0.03-0.1 mg/mL of trypsin, 0.5-3 mg/mL of collagenase and 0.5-3 mg/mL of dispase;
preferably, it comprises trypsin 0.05mg/mL, collagenase 1mg/mL and dispase 1 mg/mL;
preferably, the collagenase is collagenase type I;
preferably, the dispase is a neutral protease such as neutral protease I.
3. The complex enzyme liquid composition of claim 1 or 2, wherein the placenta-derived stem cells are placenta mesenchymal stem cells.
4. A method for isolating placental derived stem cells, comprising the step of pumping the complex enzyme liquid composition according to any one of claims 1 to 3 into the placenta, to isolate placental derived stem cells.
5. The method of claim 4, wherein the complex enzyme liquid composition is pumped through the placenta by umbilical vein and arterial occlusion.
6. The method of claim 4 or 5, wherein said placental-derived stem cells are placental mesenchymal stem cells.
7. A placental derived stem cell produced by the isolation method of any one of claims 4 to 6.
8. The placental derived stem cells according to claim 7, which are HGF modified placental derived stem cells.
9. A method for increasing the expression level of an angiogenic growth factor, comprising the step of infecting placental derived stem cells with Ad-HGF;
preferably, the vascular growth factor comprises HGF, VEGF, and bFGF;
preferably, the vascular growth factor is HGF.
The use of HGF-modified placental derived stem cells in the preparation of a medicament for the treatment of an ischemic disease; preferably, the ischemic disease is selected from any one or more of coronary heart disease, lower limb arterial ischemia and diabetic arteriosclerosis; preferably, the HGF-modified placental-derived stem cell is the placental-derived stem cell of claim 8.
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| CN110592005A (en) * | 2019-08-14 | 2019-12-20 | 广州乾晖生物科技有限公司 | Method for separating mesenchymal stem cells |
| CN110974847A (en) * | 2019-12-25 | 2020-04-10 | 博雅干细胞科技有限公司 | Method for treating lower limb ischemic diseases by using stem cells |
| CN115418351A (en) * | 2022-10-18 | 2022-12-02 | 上海君益禾生物医学科技有限公司 | Preparation method of platelet lysate in placenta |
| CN116144583A (en) * | 2023-04-19 | 2023-05-23 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
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| CN116144583A (en) * | 2023-04-19 | 2023-05-23 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
| CN116144583B (en) * | 2023-04-19 | 2023-08-25 | 北京雪颜博润生物科技有限公司 | Method for obtaining human placenta embryo-derived stem cells and application |
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