CN107144695B - Application of the Arl13b albumen in cancer diagnosis - Google Patents
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- CN107144695B CN107144695B CN201710255066.9A CN201710255066A CN107144695B CN 107144695 B CN107144695 B CN 107144695B CN 201710255066 A CN201710255066 A CN 201710255066A CN 107144695 B CN107144695 B CN 107144695B
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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Abstract
Application of the Arl13b albumen in cancer diagnosis, Arl13b albumen or its nucleic acid sequence can be used for the reagent or kit of preparation diagnosis cancer.It is reacted using anti-Arl13b protein specific antibody or Arl13b protein-specific nucleic acid probe as cancer diagnosing agent with cell sample, the binding capacity of antibody or probe is obtained with the detectable group of probe or antibody coupling by detecting, it is compared with the binding capacity of normal cell, it is convenient to which tentative diagnosis is made to conditions of patients.Marker of the Arl13b as tumour in the present invention can be used for the condition-inference and Index for diagnosis of tumour；Arl13b is alternatively arranged as the potential therapy target of tumour.
Description
Technical field
The invention belongs to technical field of molecular biology, in particular to gene diagnosis.
Background technique
With the variation of human habitat, living standard and life style, malignant tumour becomes increasingly common and serious
Threaten one of human life and the principal disease of quality of life.Global annual new cancer more than 1,400 ten thousand, wherein relatively conventional
Be lung cancer, breast cancer；The annual whole world has 8,200,000 people to die of cancer, and the most common cause of the death is lung cancer.Pathogenesis of cancer map is aobvious
Show, although China's cancer morbidity is 2.8%, the death rate is in medium level on the upper side, is for global medium level
27%.The incidence of China's cancer has certain features: onset of liver cancer 50.5%, the death rate 51.4%, equal Zhan Quanqiu
Onset of liver cancer, dead half；The cancer of the esophagus, gastric cancer equally account for whole world morbidity, dead nearly 50%；Lung cancer ratio is higher, accounts for about
Global new cases 1/3.Globocan predicts global pathogenesis of cancer, death, arrives the year two thousand twenty, it is contemplated that about 17,140,000 people will
Cancer is suffered from, 10,050,000 people will die of cancer.
Although cancer patient is continuously increased in recent years, the five year survival rate of cancer is significantly improved.With nineteen ninety-five -1999
Year compare, 5 years survival rates of -2009 years 2005 major cancers in China have obtained obvious raising, such as: gastric cancer by
15.3% increases to 31.3%, and colon cancer increases to 54.6% by 33.5%, and the carcinoma of the rectum increases to 53.2% by 28.9%, liver cancer
12.5% is increased to by 2.4%, lung cancer increases to 17.5% by 7.5%, and breast cancer increases to 80.9% by 53.8%, cervical carcinoma
59.9% is increased to by 40.1%.The raising for the treatment of technology progress such as surgical technic level；Advanced chemotherapeutics and radiotherapy skill
The application of art and the discovery etc. of more and more efficiently targeted drugs are the major reasons that cancer cure rate improves.But people
Be more attributed to the fact that early discovery, early diagnosis and early treatment to cancer lesion.
Arl13b is a small molecule GTP enzyme, is specifically assembled in the primary cilium of a variety of organs.Arl13b pairs
The formation of primary cilium and the performance of function play very crucial effect, participate in vesicle transport, cell differentiation, cell movement and
Cytoskeleton such as forms at the processes.In the case where Arl13b protein delation, cilium is short and small and structural damage.In the mankind,
The mutation of Arl13b gene will lead to Joubert syndrome.The mouse of Arl13b gene delection have ciliary structures impaired and
Phenomena such as Hedgehog signal path activity is abnormal, and show symptom similar with Joubert syndrome patients.In the recent period, have
The growth of document report Arl13b adjusting tumour cell.
Therefore, research, which is invented, has urgent demand for the accurate and specific diagnostic reagent or kit of cancer.
Summary of the invention
The object of the present invention is to provide index, diagnostic reagent or kit, the Arl13b albumen of being capable of Accurate Diagnosis cancer
The purposes and vitro detection of diagnostic kit its expression quantity method.
It is an object of the present invention to provide a kind of indexs of cancer diagnosis.
Second object of the present invention has been to provide a kind of cancer diagnosing kit.In a preferred example of this aspect,
Anti- Arl13b antibody coupling has detectable group, can detect group and is selected from chromophore, chemiluminescent groups, fluorogen or same position
Element.
Third object of the present invention has been to provide a kind of cancer diagnosing kit.In a preferred example of this aspect,
Nucleic acid probe coupling containing Arl13b protein-specific has detectable group, can detect group and is selected from chromophore, chemiluminescence
Group, fluorogen or isotope.
Fourth object of the present invention is that Arl13b albumen or its nucleic acid sequence are in the use for preparing diagnostic reagent or kit
On the way.In a preferred example of this aspect, cancer diagnosing agent is that anti-Arl13b protein specific antibody or Arl13b albumen are special
Specific nuclease probe.
A kind of method that 5th purpose of the invention has been to provide vitro detection specificity Arl13b protein expression, packet
It includes: being reacted with anti-Arl13b protein specific antibody or Arl13b specific dna probe with cell sample, be with normal cell
Control；Compare the binding capacity of antibody or probe, wherein the amount for being higher than control shows that the cell is cancer cell, less than or equal to control
Amount show the cell be normal cell.In a preferred example of this aspect, binding capacity is by detection and probe or antibody
What the detectable group of coupling measured.
The invention has the following advantages that marker of (1) Arl13b as tumour, it can be used for the condition-inference of tumour and pre-
After judge；(2) Arl13b can be used as the potential therapy target of tumour.
Detailed description of the invention
Fig. 1 is influence of the silencing Arl13b to tumor cell proliferation ability.
Fig. 2 is the influence for being overexpressed Arl13b to tumor cell proliferation ability.
Fig. 3 is influence of the silencing Arl13b to tumor cell invasion ability.
Fig. 4 is the influence for being overexpressed Arl13b to tumor cell invasion ability.
Fig. 5 is influence of the silencing Arl13b to tumor cell migration ability.
Fig. 6 is the influence for being overexpressed Arl13b to tumor cell migration ability.
Fig. 7 is that Arl13b silencing inhibits stomach cancer cell in the intracorporal growth of nude mice.
Fig. 8 is that Arl13b is overexpressed promotion stomach cancer cell in the intracorporal growth of nude mice.
Fig. 9 is the expression quantity that Western Bolt method detects Arl13b in stomach organization and cancer beside organism.
The expression and Overall survival and survive without disease that Figure 10 is the different degrees of Arl13b of Kaplan-Meier survival analysis
The relationship of phase.
In the above figure, N-Shh:N-Shh stimulates liquid；Control: control medium；Vector: control stomach cancer cell；
Arl13b-KD: stablize Gastric cancer cell MKN45；GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
Specific embodiment
Embodiment is provided below embodiment of the present invention is described in detail, the following example is only used for illustrating
The bright present invention, and be not construed as limiting the scope of the invention.The person that is not specified actual conditions in embodiment, according to normal conditions into
Row.
Embodiment 1: plate clone method detects influence of the Arl13b to tumor cell proliferation ability.
The stabilization Gastric cancer cell MKN45 (Arl13b-KD) and control stomach cancer cell of the Arl13b silencing of logarithmic growth phase
(Vector) it or is overexpressed the stabilization stomach cancer cell MKN28 (Arl13b) of Arl13b and compares stomach cancer cell (Vector), removal
Culture medium, be added after adding pancreatin sufficiently to digest 2-3min 500 μ l containing 10% serum DMEM high glucose medium in and pancreatin, piping and druming
Cell is mixed, is separated into cell unicellular.It takes 1000 cells/wells to be inoculated into 6 orifice plates, N-Shh is added to stimulate liquid (N-Shh)
Or control medium (Control), and keep cell evenly dispersed.37 DEG C are placed in, 5%CO2It is cultivated 14-20 days in incubator.
When observing macroscopic clone, culture is terminated.Culture medium is removed, 37 DEG C of 4% paraformaldehyde solution fixed 10min are added.
After removing fixer, after PBS is washed 3 times, 0.2% violet staining 20-30min is added.Wash away dyeing liquor extremely PBS more times
PBS clarification, which is placed on, to be air-dried.6 orifice plates are inverted in professional scanner to be scanned.Background is removed using ImageJ software
And to unit area (1cm2) violet staining clone number calculated, * * indicate p < 0.01.The result shows that Arl13b promotes
The proliferation (Fig. 1, Fig. 2) of tumour cell.
Influence of the embodiment 2:Transwell experiment detection Arl13b to tumor cell invasion ability.
Tumour cell is through Nature enemy 12h.The cell Transwell bottom is coated with 50mg/LMatrigel 1:8 dilution
The upper chamber face of film, 4 DEG C air-dry.Residual liquid in culture plate is sucked out, serum-free medium of the 50ul containing 10g/LBSA is added in every hole,
37 DEG C, 30min.The cell Transwell is put into 24 orifice plates, the serum free medium of 300 μ l pre-temperatures, room temperature is added in upper chamber
Lower standing 15-30min, makes matrigel rehydration.Remaining culture liq is sucked again.Digest the stabilization stomach cancer cell of Arl13b silencing
MKN45 (Arl13b-KD) and control stomach cancer cell (Vector) or the stabilization stomach cancer cell MKN28 for being overexpressed Arl13b
(Arl13b) with control stomach cancer cell (Vector), termination is centrifuged after digesting and discards culture solution, washed 1-2 times with PBS, with containing BSA
Serum free medium be resuspended, adjustment cell density is to 1-10 × 105.Take 200 μ l of cell suspension that the cell Transwell is added,
500 culture mediums of the μ l containing 20%FBS are added in room under 24 orifice plates, add N-Shh stimulation liquid (N-Shh) or control medium
(Control), it cultivates for 24 hours.Cell is transferred in new culture hole, is washed to appropriate color, goes to small interior with cotton swab
Portion does not pass through the cell of upper chamber film surface.Toward 100 μ l PBS of small indoor addition, the cell that inverted microscope observation is gone through, 200 ×
The invasion cell number that 10 visuals field are counted under light microscopic, takes its average value, indicates tumour cell with the relative number of invasion cell
Invasive ability, * * indicate p < 0.01.The results show that Arl13b promotes the invasion (Fig. 3, Fig. 4) of tumour cell.
Influence of the embodiment 3:Migration experiment detection Arl13b to tumor cell migration ability.
Compared with ruler in 6 orifice plates behind with marker, uniformly draw horizontal line, it is together per every about 0.5~1cm, horizontal
Across hole.Every hole is at least across 5 lines.About 5 × 10 are added in hole5The stabilization Gastric cancer cell MKN45 of a Arl13b silencing
(Arl13b-KD) and control stomach cancer cell (Vector) or be overexpressed Arl13b stabilization stomach cancer cell MKN28 (Arl13b) and
It compares stomach cancer cell (Vector), inoculation principle is that rear fusion rate reaches 100% overnight.Compare ruler with pipette tips within second day, to the greatest extent
The horizontal line scratch perpendicular to behind is measured, pipette tips are vertical, cannot tilt and (same pipette tips be preferred between different holes).PBS
It washes cell 3 times, the cell under removal stroke, N-Shh stimulation liquid (N-Shh) or serum-free control medium of serum-free is added
(Control).37 DEG C of 5%CO2 incubators are put into, are cultivated.Can be by 0,12, point in time sampling for 24 hours, taking pictures, (the specific time is according to reality
It tests depending on needs).After opening picture using Image J software, random draw takes 6 to 8 horizontal lines, calculates the equal of iuntercellular distance
Value, * * indicate p < 0.01.The result shows that Arl13b promotes the migration (Fig. 5, Fig. 6) of tumour cell.
Embodiment 4:Arl13b promotes the growth of stomach cancer cell in vivo.
Buy 24 BALB/c-nu Female nude mices, 4-6 week old, weight 18-22g；By table of random number by 24 nude mices
It is divided into two groups: 1. slow virus silencing control group (Vector)+Arl13b silencing group (Arl13b-KD)；2. slow virus overexpression pair
According to group (Vector)+Arl13b overexpression group (Arl13b)；And weigh the weight of every nude mice.With 0.25% trypsin digestion
The stabilization Gastric cancer cell MKN45 (Arl13b-KD) of Arl13b silencing and control stomach cancer cell (Vector) are overexpressed Arl13b
Stabilization stomach cancer cell MKN28 (Arl13b) and control stomach cancer cell (Vector), 1000r/min centrifugation, PBS wash 2 times, use
PBS is with restriction I × 108Cell/ml.0.2ml, which is extracted, with the syringe of No. 6 syringe needles of band contains 2 × 107The cell suspension of a living cells,
It is inoculated in the medial thigh skin of every nude mice (left and right is respectively control group, interference or overexpression group).After injection at the 7th day,
It is come into effect when control group and roughly equal processing group knurl and Doxycycline (2mg/ml) inducing expression is added in reference water, every 2
It changes that water is primary, is administered 3-4 week: every three days with the volume (V) of transplantable tumor of vernier caliper measurement: V=a × b2/ 2 (a be length
Diameter, b are minor axis), and observe the variation of the physical signs such as nude mice spirit, activity condition.After 3-4 weeks, euthanasia nude mice is anaesthetized,
Transplantable tumor is cut, and weighs its weight.Compared with control group (Vector), stablizes and lower Arl13b group (Arl13b-KD) mouse
Knurl it is smaller, knurl weight is lighter (Fig. 7).However, being overexpressed Arl13b group stablizing compared with control group (Vector)
(Arl13b) in experiment, it is observed that knurl is bigger after being overexpressed Arl13b, knurl weight is heavier (Fig. 8).
Embodiment 5: the expression quantity of Arl13b in detection stomach organization and cancer beside organism.
Trash ice is got out, 5ml centrifuge tube is weighed and marked, is pre-chilled on ice together with PBS.8 gastric cancers are taken at random
The cancerous tissue of people and its effective vacuum cup transfer equipped with liquid nitrogen of corresponding cancer beside organism's tissue freezing, clip portion of tissue are put
Enter in the centrifuge tube of pre-cooling, remaining tissue is put back in cryopreservation tube, is replaced；Weighing tissue, record；It is washed with the PBS of pre-cooling
Tissue is washed, 2-3 times, washes away the blood of adherency；It shreds tissue, is added containing 1:100 protease inhibitors and 1mM PMSF
RIPA buffer, additional proportion tissue: buffer=1:10 (g/ml)；The homogenised tissue on historrhexis's instrument, 13000g,
10s, until tissue is more broken；Tissue wants the moment to keep on ice；On gyroscope, 4 DEG C of rotation 30min crack tissue sufficiently；Cracking
Liquid is transferred to EP pipe, is centrifuged, 4 DEG C, 12000g, 15min；Take supernatant to get required histone lysate；Take 100 μ l albumen fixed
Western Blot is measured and is, if remaining does not have to temporarily put -80 DEG C of preservations.BCA protein quantification, it is quantitative according to resulting numerical value
Matched curve, the protein concentration after obtaining linear equation and dilution, calculates actual protein concentration.Take suitable albumen
The protein content of Arl13b in Western sample detection normal tissue and cancerous tissue.The result shows that Arl13b table in cancerous tissue
Up to amount (Fig. 9) higher than normal tissue.
The expression quantity of embodiment 6:Arl13b is related to the state of an illness of Patients with Gastric Cancer and prognosis.
Pathology preserves paraffin embedding sample in 154 gastric cancer cases chosen, and organization type is through 1 experienced pathology
Section doctor is determined the histotomy HE dyeing in selected wax stone source.To 154 gastric cancer samples, (including 154 corresponding
Cancer beside organism) carry out immunohistochemical analysis.Immunohistochemistry scoring is scored according to " German sxemiquantitative points-scoring system ", according to 0
~12 points of standards of grading delimit 0~6 point as negative and low expression, and > 6 points are middle high expression.It will be each according to Arl13b points of height
Index scoring for two groups and with clinical pathologic characteristic progressive correlation analysis.Analysis the results are shown in Table 1, as a result prompt: height expression
Arl13b and tumor size, invasive depth are related.
Influence of the high expression to patient's prognosis in stomach organization for clear Arl13b, we to be included in the patient of research into
Row follow-up.Observing time is set as Overall survival (overall surviva), and without disease life cycle (Disease-free
survival).Analyze with Kaplan-Meier and carry out the discovery of single factor test survival analysis: Arl13b high expression can significantly affect trouble
Person OS and DFS, the higher patient survival that scores are shorter (Figure 10).Show that Arl13b has significant correlation with clinical prognosis.
Relationship between table 1Arl13b and clinical pathologic characteristic
In summary specific embodiment measurement result, Arl13b tumor tissues height express, promote tumour cell proliferation,
Migration and invasion.
Therefore, Arl13b albumen or its nucleic acid sequence can be used for the reagent or kit of preparation diagnosis cancer.It will resist
Arl13b protein specific antibody or Arl13b protein-specific nucleic acid probe are reacted as cancer diagnosing agent with cell sample,
The binding capacity for obtaining antibody or probe with the detectable group of probe or antibody coupling by detecting, the binding capacity with normal cell
It is compared, can be used for the diagnosis of cancer.
Claims (2)
- Application of the 1.Arl13b albumen in preparation cancer diagnosis reagent box.
- Application of the 2.Arl13b specific dna probe in preparation cancer diagnosis reagent box.
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| CN111269982B (en) * | 2020-02-20 | 2022-09-23 | 南昌大学 | Application of SNEP1 protein in diagnosis of colorectal cancer |
| CN112294947A (en) * | 2020-11-10 | 2021-02-02 | 南昌大学 | Application of polypeptide in preparing medicine for preventing and/or treating tumor |
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