CN106434747A - Method for inhibiting hepatoma carcinoma nude mouse transplantation tumor capacity through silent secretory clusterin - Google Patents
Method for inhibiting hepatoma carcinoma nude mouse transplantation tumor capacity through silent secretory clusterin Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a method for inhibiting hepatoma carcinoma nude mouse transplantation tumor capacity through silent secretory clusterin. The method includes the two steps of interference and detection. The method has the advantages that the method is simple and efficient, and it is clearly shown that the silent secretory clusterin can obviously inhibit the human hepatoma carcinoma nude mouse subcutaneous tumor formation capacity. It is effectively shown that the transcription of the silent secretory clusterin prolongs the incubation period of a human hepatoma carcinoma cell tumor-bearing nude mouse model. It is effectively shown that the silent secretory clusterin reduces the hepatoma carcinoma nude mouse transplantation tumor growth speed. Through protein and gene analysis, it is effectively shown that the silent secretory clusterin enables the expression level of the silent secretory clusterin in a hepatoma carcinoma nude mouse transplantation tumor and mRNA of the silent secretory clusterin to be smaller than those of a blank control group and a negative control group.
Description
Technical field
The present invention relates to Golgi body secreting type clusterin correlative technology field is and in particular to a kind of silence secreting type clump
The method that raw albumen suppresses hepatocellular carcinoma in nude mice ability.
Background technology
Hepatocarcinoma (Hepatocellular carcinoma, HCC) is one of global most common malignant tumor, it prevent with
Treatment is still a difficult problem for medical circle.Hepatocarcinoma early diagnosis are difficult, poor prognosis, find accurate, special diagnosis and prognostic indicator will
Contribute to HCC preventing and treating.There is the complex process that development is that multi-pathogenesis, polygenes, multi-step and multiple-factor are worked in coordination with HCC, be related to
To oncogene activation, multi signal path and the gene regulation such as antioncogene inactivation and some oncogenes of period of embryo are brought back to life again.Mesh
Before, excision or liver transplantation are still treatment prefered method, but because hepatocellular carcinoma quickly grows, Most patients
Often lose the chance of operative treatment when making a definite diagnosis, Radiotherapy chemotherapy and local treatment can only have been carried out, but currently for advanced liver cancer
Still lack effectively treatment method.Therefore, find Effective target site suppression hepatocarcinoma progress further, to the treatment level tool improving hepatocarcinoma
Significant.
New discovery has the Golgi body clusterin (Clusterin, CLU) of various biological activity, and it is in the nature sulfur
Change heterodimeric glycoprotein, in chromosome 8p 21-p12, molecular structure is highly conserved between kind for gene mapping, participate in tissue weight
Build, lipid transport, complement are adjusted and apoptosis etc..By two hypotypes：Caryogram clusterin (nuclear Clusterin,
NCLU) form with secreting type clusterin (secretory Clusterin, sCLU).Accumulation data display sCLU expression with
Hepatocarcinoma drug resistance, propagation and transfer etc. are closely related.SCLU albumen has cytoprotection, can check irritability apoptosis；Normally
Hepatic tissue low expression, liver cancer tissue overexpression is closely related with drug resistance.
Content of the invention
Present invention aims to the deficiencies in the prior art, now provide a kind of silence secreting type clusterin suppression liver
The method of cancer transplanted tumor in nude mice ability.
For solving above-mentioned technical problem, the technical solution used in the present invention is：A kind of silence secreting type clusterin suppression
The method of hepatocellular carcinoma in nude mice ability, its innovative point is：Including intervention and two steps of detection；
Described intervention concretely comprises the following steps：
（1）For people's secreting type clusterin sequence NCBI:NM_001831.3 designs interference sequence:SCLU-shRNA, 5 '-
GTAAGTACGTCAATAAGGA-3 ', negative control sequence NC-shRNA, 5 '-TTCTCCGAACGTGTCACGT-3 ', and build
Plasmid vector pRNAT-U6.1/Neo- shRNA1, sets up blank control group simultaneously；
（2）By above-mentioned Transfected Recombinant Plasmid to human hepatoma HepG2 cell, and processed using Geneticin, screening obtains steady
The HepG2 cell of fixed transfection；
（3）The HepG2 cell that aforementioned stable is transfected is inoculated nude mice by subcutaneous and is built human liver cancer cell Transplanted tumor model；
（4）The HepG2 cell measuring stable transfection becomes tumor situation and tumor endocrine type clusterin mRNA and egg in nude mice by subcutaneous
White expression；
Described detection includes RT-PCR to the detection of sCLU-mRNA level, PBCA algoscopy in transplanted tumor
To the detection of total protein levels, enzyme-linked immunosorbent assay in transplanted tumor to the detection of sCLU protein level and HE in transplanted tumor
Dyeing and immunohistochemical analysis.
Further, described RT-PCR is to the concretely comprising the following steps of the detection of sCLU-mRNA level in transplanted tumor：Will be right for blank
Trizol is used to extract total serum IgE according to, negative control and intervention group transplanted tumor tissue homogenate, ultraviolet spectrophotometer measures RNA and contains
Amount and absorbance ratio A260/A280；Synthesis the first chain is illustrated according to cDNA synthetic agent box；Using SYBPremixEx
TaqTM II test kit carries out RT-PCR, and using glyceraldehyde-3-phosphate dehydrogenase as internal reference, described RT-PCR reaction condition is：95
DEG C 30s, 60 DEG C of 45s, 40 circulations；Result uses 2-ΔΔCtMethod is analyzed；MRNA relative to change calculations formula is：Ratio=2-ΔΔCt, Δ Ct=CtsCLU-CtGAPDH, Δ Δ Ct=Δ CtSample-ΔCtBlank, relative expression quantity=2-ΔΔCt；Primer sequence:SCLU,
F:ATCACTGTGACGGTCCCTGTA, R: TCACTCCTCCC GGTGCTT；GAPDH, F:
CAAGGTCATCCATGACAACTTTG, R: GT CCACCACCCTGTTGC TGTAG.
Further, the tool to the detection of total protein levels in transplanted tumor for the described PBCA algoscopy
Body step is：Tumor tissue is shredded, and makes tissue homogenate using tissue homogenate device；Follow PBCA to measure
PBCA working solution is prepared in test kit explanation, fully mixes；Then 10 μ l standard substance phosphate-buffered salts are taken
Solution is diluted to 100 μ l so that ultimate density is 0.5mg/ml；Standard substance are pressed 0 μ l, 1 μ l, 2 μ l, 4 μ l, 8 μ l, 12 μ
L, 16 μ l and 20 μ l are added in the protein standard sample wells of 96 orifice plates, plus phosphate buffered saline(PBS) supplies 20 μ l；By dilute sample
Add in 96 orifice plate sample wells, each hole adds 200 μ l BCA working solutions, places 30 min under 37 DEG C of environment；Then it is cooled to
Room temperature, measures A with microplate reader562, total protein concentration in transplanted tumor is calculated according to standard curve.
Further, the concrete steps to the detection of sCLU protein level in transplanted tumor for the described enzyme-linked immunosorbent assay
For：Follow test kit operation instruction by 1000pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml,
The each 0.1ml of standard substance of 31.3 pg/ml and 15.6 pg/ml sequentially adds in ELISA Plate one row 7 hole, and 1 hole only adds diluted sample
Conduct zero hole of liquid；The test serum homogenised sample having been diluted with sample diluting liquid is added ELISA Plate to remain by every hole 0.1 ml
Yu Kongzhong；Using shrouding membrane closure ELISA Plate, put into 37 DEG C of calorstats and be incubated 90 min, firmly have the final say, discard liquid；To resist
People's secreting type clusterin antibody working solution is sequentially added by every hole 0.1 ml, reacts 60 min under 37 DEG C of environment；Question response
Wash plate 3 times with Tris buffer salt solution after end, soak 1min every time；Biotin-avidin working solution is pressed every hole 0.1ml
Sequentially add, react 30 min under 37 DEG C of environment, wash plate 5 times with Tris buffer salt solution, wash after hardened bundle by every hole 90 μ l
Sequentially add TMB nitrite ion, lucifuge reacts 10 min under 37 DEG C of environment, then sequentially add TMB by every hole 0.1ml and terminate
Liquid, now in the hole blueness is vertical turns yellow；Finally use microplate reader to measure OD value in absorbance 450nm, calculated according to standard curve
SCLU protein concentration in surveyed tissue homogenate.
Further, the concretely comprising the following steps of described HE dyeing and immunohistochemical analysis：HE staining：60 DEG C of paraffin section is baked
Piece 4 hours, is dewaxed 3 times using dimethylbenzene, each 10min, is subsequently placed in 100% and 95% ethanol respectively dehydration 2 times, every time
5min；Using Harris haematoxylin dyeing 5min, using 0.5% eosin stains after breaking up and clean using 1% hydrochloride alcohol solution
2min, is subsequently placed in each dehydration of 100% and 95% ethanol twice, each 5min；Finally place 5min in dimethylbenzene stand-by.
SABC adopts SP method, transplanted tumor piece of tissue is fixed, cuts into slices after paraffin embedding, in conventional dewaxing to water,
Microwave method antigen, 3% 37 DEG C of hydrogen peroxide is incubated 15 min, adds sCLU mono- to resist, and it is to resist through Dako that described sCLU mono- resists
SCLU mono- after 50 times of body diluted resists, and, adds phosphate buffered saline(PBS) to be placed in shaking table rinsing under 4 DEG C of environment overnight
3 times, each 5min；The sheep anti mouse two of Deca biotin labeling resists, and it is dilute through Dako antibody diluent that described sheep anti mouse two resists
Release the sheep anti mouse two after 50 times to resist, incubated at room 10 min, phosphate buffered saline(PBS) rinses 3 times, each 5min；Deca is fresh to join
Four diaminobenzidine hydrochloride solution 100 l of system, color development at room temperature, distilled water wash 3 times, haematoxylin redyes 5min, anhydrous second
Dehydration of alcohols is transparent, mounting.
Beneficial effects of the present invention are as follows：
（1）The method of the present invention is simply efficient, and clearly indicating silence secreting type clusterin can substantially suppress human liver cancer thin
Born of the same parents' nude mice by subcutaneous one-tenth knurl ability.
（2）The present invention effectively indicates silence secreting type clusterin transcription elongation human liver cancer cell model of nude mice bearing tumor and dives
Fu Qi.
（3）The present invention effectively indicates silence secreting type clusterin and delays the hepatocellular carcinoma in nude mice speed of growth.
（4）The present invention passes through albumen and gene analysiss, and effectively indicating silence secreting type clusterin makes Liver Cancer Bearing Nude Mice move
Plant secreting type clusterin and its mRNA expression in tumor and be less than blank control group and negative control group.
Brief description
Fig. 1 is to measure blank control group, negative control group and plasmid intervention group hepatoma carcinoma cell lotus in embodiment of the present invention
The preclinical comparison diagram of tumor nude mice model.
Fig. 2 is each group hepatoma carcinoma cell Xenografts in nude mice growth curve.
Fig. 3 is each group hepatoma carcinoma cell Xenografts in nude mice inhibition comparison diagram.
Fig. 4 measures secreting type clusterin mRNA relative amount in each group Xenografts in nude mice tissue for RT-PCR.
Fig. 5 is PBCA algoscopy and enzyme-linked immunosorbent assay each group Xenografts in nude mice group
Knit middle secreting type clusterin relative amount.
Fig. 6 be transplanted tumor histopathologic examination (× 100) and secreting type clusterin ImmunohistochemistryResults Results figure (×
200).
Specific embodiment
Hereinafter embodiments of the present invention are illustrated by particular specific embodiment, those skilled in the art can be by this explanation
Content disclosed by book understands other advantages and effect of the present invention easily.
A kind of method that silence secreting type clusterin suppresses hepatocellular carcinoma in nude mice ability, including intervention and detection two
Step；
Intervention concretely comprises the following steps：
（1）For people's secreting type clusterin sequence NCBI:NM_001831.3 designs interference sequence:SCLU-shRNA, 5 '-
GTAAGTACGTCAATAAGGA-3 ', negative control sequence NC-shRNA, 5 '-TTCTCCGAACGTGTCACGT-3 ', and build
Plasmid vector pRNAT-U6.1/Neo- shRNA1, sets up blank control group simultaneously；
（2）By above-mentioned Transfected Recombinant Plasmid to human hepatoma HepG2 cell, and processed using Geneticin, screening obtains steady
The HepG2 cell of fixed transfection；
（3）The HepG2 cell that aforementioned stable is transfected is inoculated nude mice by subcutaneous and is built human liver cancer cell Transplanted tumor model；
（4）The HepG2 cell measuring stable transfection becomes tumor situation and tumor endocrine type clusterin mRNA and egg in nude mice by subcutaneous
White expression；
Detection includes RT-PCR to the detection of sCLU-mRNA level, PBCA algoscopy in transplanted tumor to shifting
Plant the detection of total protein levels in tumor, enzyme-linked immunosorbent assay dyes to the detection of sCLU protein level in transplanted tumor and HE
And immunohistochemical analysis.
Embodiment 1
The present embodiment is the intervention of the present invention and the tool of detection silence secreting type clusterin suppression hepatocellular carcinoma in nude mice ability
Body step.
1st, cell culture：HepG2 cell lines are cultivated in 10%FCS DMEM culture medium, and cell strain is placed in 5%
CO2, in 37 DEG C of incubators, cellar culture.
2nd, transfect shRNA：According to people secreting type clusterin sequence (NCBI:NM_001831.3) design interference sequence:
ShRNA-1,5 '-GTAAGTACGTCAATAAGGA-3 ' and negative control group sequence NC-shRNA, 5 '-
TTCTCCGAACGTGTCACGT-3 ', and build plasmid vector pRNAT-U6.1/Neo-shRNA1；HepG2/ADM cell is connect
Plant in six orifice plates, treat length to complete opening 80%, according to transfection reagent GenJetTM description, respectively will be right to interference group shRNA and feminine gender
Proceed to HepG2 cell according to NC-shRNA, and set blank control wells；Change former training liquid after 12h and continue culture, fluorescence microscope after 24h
Observe transfection efficiency.
3rd, liver cancer xenograft models build：The BALB/c nude mice of predetermined 36 4-6 week old, male and female half and half, it is randomly divided into sky
White matched group, negative control group and intervention group, intervention group is sCLU-shRNA group, every group 6, raises under the conditions of being placed in SPF；
0.2ml is concentrated the HepG2 pallium cell injection transfecting to intervention group nude mice shoulder back, the cell in HepG2 cell suspension
Number is 2 107Individual/ml, the HepG2 cell that equivalent is untreated and negative plasmid transfection is crossed is injected in matched group and the moon respectively
Property matched group, formed 3mm about subcutaneous protuberance.Daily tumor growth situation and tumor size, measure gross tumor volume V=0.5
ⅹaⅹb2, a represents, b represents minor axis, according to gross tumor volume equal numeric renderings tumor growth curve；Injection cell suspension 35 days
Afterwards, draw neck to put to death nude mice, take out tumor tissues, with normal saline flushing, dry, take pictures, specimen is immersed in 4% paraformaldehyde
Fixing, standby；Observation index：Observe a tumorous size every 3 days after injection cancerous cell, measure tumor body with slide gauge long
Minor axis, draws growth curve afterwards；After taking out tumor tissues, observe 3 groups of tumor sizes, outward appearance, form.
4th, RT-PCR surveys sCLU-mRNA level in tumor：By blank, negative control and intervention group transplanted tumor tissue homogenate
Extract total serum IgE using Trizol, ultraviolet spectrophotometer measures rna content and absorbance ratio A260/A280；Closed according to cDNA
Become test kit explanation synthesis the first chain；Carry out RT-PCR using SYBPremixEx TaqTM II test kit, with glyceraldehyde -3- phosphorus
Acidohydrogenase as internal reference, RT-PCR reaction condition is：95 DEG C of 30s, 60 DEG C of 45s, 40 circulations；Result uses 2-ΔΔCtMethod is entered
Row analysis；MRNA relative to change calculations formula is：Ratio=2-ΔΔCt, Δ Ct=CtsCLU-CtGAPDH, Δ Δ Ct=Δ CtSample-Δ
CtBlank, relative expression quantity=2-ΔΔCt；Primer sequence:SCLU, F:ATCACTGTGACGGTCCC TGTA, R:
TCACTCCTCCCGGTGCTT；GAPDH, F:CAAGGTCATC CATGACAACTTTG, R:
GTCCACCACCCTGTTGCTGTAG.
5th, PBCA algoscopy surveys total protein levels in tumor：Tumor tissue is shredded, and even using tissue
Tissue homogenate made by slurry device；Follow the explanation of PBCA test kit and prepare PBCA work
Liquid, fully mixes；Then 10 μ l standard substance phosphate buffered saline(PBS) are taken to be diluted to 100 μ l so that ultimate density is 0.5mg/
ml；Standard substance are added to the protein standard substance of 96 orifice plates by 0 μ l, 1 μ l, 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l and 20 μ l
Kong Zhong, plus phosphate buffered saline(PBS) supplies 20 μ l；Dilute sample is added in 96 orifice plate sample wells, each hole adds 200 μ l
BCA working solution, places 30 min under 37 DEG C of environment；Then it is cooled to room temperature, measures A562 with microplate reader, according to standard curve
Calculate total protein concentration in transplanted tumor.
6th, enzyme-linked immunosorbent assay surveys sCLU protein level in transplanted tumor：Follow test kit operation instruction by 1000pg/ml,
500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.3 pg/ml and 15.6 pg/ml standard substance each
0.1ml sequentially adds in ELISA Plate one row 7 hole, and 1 hole only adds conduct zero hole of sample diluting liquid；Diluted with sample diluting liquid
Good test serum homogenised sample is pressed every hole 0.1 ml and is added in ELISA Plate remaining hole；Using shrouding membrane closure ELISA Plate, put into
37 DEG C of calorstats are incubated 90 min, firmly have the final say, discard liquid；Anti-human secreting type clusterin antibody working solution is pressed every hole
0.1 ml sequentially adds, and reacts 60 min under 37 DEG C of environment；Question response washes plate 3 times with Tris buffer salt solution, often after terminating
Secondary immersion 1min；Biotin-avidin working solution is sequentially added by every hole 0.1ml, reacts 30 min under 37 DEG C of environment,
Wash plate 5 times with Tris buffer salt solution, sequentially add TMB nitrite ion by every hole 90 μ l after washing hardened bundle, keep away under 37 DEG C of environment
Photoreaction 10 min, then sequentially adds TMB terminate liquid by every hole 0.1ml, and now blue the standing of in the hole turns yellow；Finally use enzyme mark
Instrument measures OD value in absorbance 450nm, calculates sCLU protein concentration in surveyed tissue homogenate according to standard curve.
7th, HE dyeing and immunohistochemical analysis：Using HE staining：The roasting piece of 60 DEG C of paraffin section 4 hours, using dimethylbenzene
Dewaxing 3 times, each 10min, it is subsequently placed in 100% and 95% ethanol respectively dehydration 2 times, each 5min；Using Harris haematoxylin
Dyeing 5min, using 0.5% eosin stains 2min after being broken up and cleaned using 1% hydrochloride alcohol solution, is subsequently placed in 100% and 95%
Ethanol is each to be dehydrated twice, each 5min；Finally place 5min in dimethylbenzene stand-by.
SABC adopts SP method, transplanted tumor piece of tissue is fixed, cuts into slices after paraffin embedding, in conventional dewaxing to water,
Microwave method antigen, 3% 37 DEG C of hydrogen peroxide is incubated 15 min, adds sCLU mono- to resist, it is dilute through Dako antibody that sCLU mono- resists
Release the sCLU mono- after liquid dilutes 50 times to resist, add phosphate buffered saline(PBS) to be placed in shaking table and rinse 3 times under 4 DEG C of environment overnight,
5min every time；The sheep anti mouse two of Deca biotin labeling resists, and it is after Dako antibody diluent dilutes 50 times that sheep anti mouse two resists
Sheep anti mouse two resist, incubated at room 10 min, phosphate buffered saline(PBS) rinse 3 times, each 5min；Freshly prepared four salt of Deca
Sour diaminobenzidine solution 100 l, color development at room temperature, distilled water wash 3 times, haematoxylin redyes 5min, and dehydrated alcohol dehydration is thoroughly
Bright, mounting.
8th, data analysiss：Measured value is represented with mean ± standard deviation, carries out data analysiss using SPSS17.0 software；Use
Ipwin32 software analysis microscope photograph；Statistical method analysis has t to check, or variance analyses etc.；Compare with matched group,P<
0.05 expression difference has statistical significance.
Implementation result：
1st, hepatoma carcinoma cell model of nude mice bearing tumor incubation period：It is illustrated in figure 1 in embodiment of the present invention and measure blank control group, the moon
Property matched group and the preclinical comparison diagram of plasmid intervention group hepatoma carcinoma cell model of nude mice bearing tumor, blank control group, negative control group
And intervention group Xenografts in nude mice forms the time respectively 6.5 ± 0.55 days, 6.67 ± 0.52 days and 11.17 ± 0.98 days,
Intervention group tumor forms obvious postpone compared with blank control group（T=11.068,P<0.01）, negative control group and blank
Group no significant difference（t=0.542,P=0.611）.The HepG2 of display silence sCLU surely turns strain and gives mouse bare subcutaneous injection, shRNA
After blocking sCLU expression, HepG2 cell is suppressed in nude mice by subcutaneous one-tenth knurl ability.
2nd, Xenografts in nude mice growth curve：It is illustrated in figure 2 the growth of each group hepatoma carcinoma cell Xenografts in nude mice bent
Line, each group Xenografts in nude mice tumor growth rate obvious difference, blank control group at the end of experiment, negative control group and dry
Pre- group nude mice by subcutaneous gross tumor volume is respectively 715.58 ± 21.99mm3, 679.29 ± 27.35mm3, 187.05 ±
29.78mm3, intervention group is significantly less than matched group（t=27.237,P<0.01）, negative control group is no substantially poor with blank control group
Different（t=2.24,P= 0.075）.All animals all no natural deaths in experimentation.Intervention group HepG2 cell is in nude mice skin
Lower one-tenth knurl ability weakens, and points out suppression sCLU protein expression can significantly inhibit the multiplication capacity of HepG2 cell.
3rd, observe hepatoma carcinoma cell transplanted tumor and become tumor effect：It is illustrated in figure 3 the suppression of each group hepatoma carcinoma cell Xenografts in nude mice
Effectiveness comparison figure processed, observes subcutaneous transplantation tumor and shows that transplanted tumor outward appearance pinkiness shows uneven, no ulceration, tumor group
Knit and show that peplos are completely easily separated, be in nodositas or lobulated in tumor tissue.Neoplasm metastasis are had no in its hetero-organization.Compare feminine gender
Transplanted tumor volume in matched group and blank control group, in intervention group, subcutaneous transplantation tumor is significantly smaller.
4th, transplant tumor tissue sCLU Transcription inhibition：It is illustrated in figure 4 RT-PCR and measure each group Xenografts in nude mice tissue
Middle secreting type clusterin mRNA relative amount, RT-PCR result shows, sCLU-mRNA table in intervention group Xenografts in nude mice
The level that reaches is significantly lower than blank control group and expresses；And its expression in negative control group blank control group is compared no substantially poor
Different.
5th, nude mice model tumor tissue sCLU expressing quantity：Be illustrated in figure 5 PBCA algoscopy and
Secreting type clusterin relative amount in enzyme-linked immunosorbent assay each group Xenografts in nude mice tissue.Result shows, does
Disturb sCLU albumen relative amount in group transplanted tumor and be significantly lower than blank control group, and sCLU albumen relative amount in negative control group
No significant difference compared with blank control group.
6th, SABC surveys sCLU expression in transplanted tumor in nude mice：It is illustrated in figure 6 transplanted tumor histopathologic examination
(× 100) and secreting type clusterin ImmunohistochemistryResults Results figure (× 200), is existed using SABC and HE dyeing detection sCLU
Expression in Xenografts in nude mice, HE dyeing shows intervention group, negative control group and blank by histopathologic examination
Obvious difference between matched group.Showed by immune group result sCLU albumen dyes relatively deeply in negative control group and blank control group,
And dye shallower in intervention group or hardly dye.
Above-described embodiment is presently preferred embodiments of the present invention, is not the restriction to technical solution of the present invention, as long as
The technical scheme that can realize on the basis of above-described embodiment without creative work, is regarded as falling into patent of the present invention
Rights protection scope in.
Claims (6)
1. a kind of silence secreting type clusterin suppress hepatocellular carcinoma in nude mice ability method it is characterised in that：Including intervention
With two steps of detection；
Described intervention concretely comprises the following steps：
（1）For people's secreting type clusterin sequence NCBI:NM_001831.3 designs interference sequence:SCLU-shRNA, 5 '-
GTAAGTACGTCAATAAGGA-3 ', negative control sequence NC-shRNA, 5 '-TTCTCCGAACGTGTCACGT-3 ', and build
Plasmid vector pRNAT-U6.1/Neo- shRNA1, sets up blank control group simultaneously；
（2）By above-mentioned Transfected Recombinant Plasmid to human hepatoma HepG2 cell, and processed using Geneticin, screening obtains steady
The HepG2 cell of fixed transfection；
（3）The HepG2 cell that aforementioned stable is transfected is inoculated nude mice by subcutaneous and is built human liver cancer cell Transplanted tumor model；
（4）The HepG2 cell measuring stable transfection becomes tumor situation and tumor endocrine type clusterin mRNA and egg in nude mice by subcutaneous
White expression；
Described detection includes RT-PCR to the detection of sCLU-mRNA level, PBCA algoscopy in transplanted tumor
To the detection of total protein levels, enzyme-linked immunosorbent assay in transplanted tumor to the detection of sCLU protein level and HE in transplanted tumor
Dyeing and immunohistochemical analysis.
2. the method that detection silence secreting type clusterin according to claim 1 suppresses hepatocellular carcinoma in nude mice ability,
It is characterized in that：Described RT-PCR is to the concretely comprising the following steps of the detection of sCLU-mRNA level in transplanted tumor：By blank, cloudy
Property comparison and intervention group transplanted tumor tissue homogenate use Trizol to extract total serum IgE, ultraviolet spectrophotometer measures rna content and suction
Luminosity ratio A260/A280；Synthesis the first chain is illustrated according to cDNA synthetic agent box；Tried using SYBPremixEx TaqTM II
Agent box carries out RT-PCR, and using glyceraldehyde-3-phosphate dehydrogenase as internal reference, described RT-PCR reaction condition is：95 DEG C of 30s, 60
DEG C 45s, 40 circulations；Result uses 2-ΔΔCtMethod is analyzed；MRNA relative to change calculations formula is：Ratio=2-ΔΔCt, Δ Ct=
CtsCLU-CtGAPDH, Δ Δ Ct=Δ CtSample-ΔCtBlank, relative expression quantity=2-ΔΔCt；Primer sequence:SCLU, F:
ATCACTGTGACGGTCCCTGTA, R: TCACTCCTCCC GGTGCTT；GAPDH, F:
CAAGGTCATCCATGACAACTTTG, R: GT CCACCACCCTGTTGC TGTAG.
3. the method that detection silence secreting type clusterin according to claim 1 suppresses hepatocellular carcinoma in nude mice ability,
It is characterized in that：The concrete steps to the detection of total protein levels in transplanted tumor for the described PBCA algoscopy
For：Tumor tissue is shredded, and makes tissue homogenate using tissue homogenate device；Follow PBCA and measure test kit
Illustrate to prepare PBCA working solution, fully mix；Then take 10 μ l standard substance phosphate buffered saline(PBS) dilute
Release to 100 μ l so that ultimate density is 0.5mg/ml；Standard substance are pressed 0 μ l, 1 μ l, 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ
L and 20 μ l is added in the protein standard sample wells of 96 orifice plates, plus phosphate buffered saline(PBS) supplies 20 μ l；Dilute sample is added 96
In orifice plate sample well, each hole adds 200 μ l BCA working solutions, places 30 min under 37 DEG C of environment；Then it is cooled to room temperature, use
Microplate reader measures A562, total protein concentration in transplanted tumor is calculated according to standard curve.
4. the method that detection silence secreting type clusterin according to claim 1 suppresses hepatocellular carcinoma in nude mice ability,
It is characterized in that：Described enzyme-linked immunosorbent assay is to the concretely comprising the following steps of the detection of sCLU protein level in transplanted tumor：Abide by
Follow test kit operation instruction by 1000pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.3 pg/
The each 0.1ml of standard substance of ml and 15.6 pg/ml sequentially adds in ELISA Plate one row 7 hole, and 1 hole only adds the conduct of sample diluting liquid
Zero hole；The test serum homogenised sample having been diluted with sample diluting liquid is added in ELISA Plate remaining hole by every hole 0.1 ml；
Using shrouding membrane closure ELISA Plate, put into 37 DEG C of calorstats and be incubated 90 min, firmly have the final say, discard liquid；By anti-human secreting type
Clusterin antibody working solution is sequentially added by every hole 0.1 ml, reacts 60 min under 37 DEG C of environment；Question response is used after terminating
Tris buffer salt solution washes plate 3 times, soaks 1min every time；Biotin-avidin working solution is sequentially added by every hole 0.1ml,
React 30 min under 37 DEG C of environment, wash plate 5 times with Tris buffer salt solution, sequentially add by every hole 90 μ l after washing hardened bundle
TMB nitrite ion, under 37 DEG C of environment, lucifuge reacts 10 min, then sequentially adds TMB terminate liquid by every hole 0.1ml, now hole
Interior blue standing turns yellow；Finally use microplate reader to measure OD value in absorbance 450nm, tissue is surveyed according to standard curve calculating even
SCLU protein concentration in slurry.
5. the method that detection silence secreting type clusterin according to claim 1 suppresses hepatocellular carcinoma in nude mice ability,
It is characterized in that：The concretely comprising the following steps of described HE dyeing and immunohistochemical analysis：HE staining：The roasting piece 4 of 60 DEG C of paraffin section is little
When, dewaxed 3 times using dimethylbenzene, each 10min, it is subsequently placed in 100% and 95% ethanol respectively dehydration 2 times, each 5min；Use
Harris haematoxylin dyeing 5min, using 0.5% eosin stains 2min after breaking up and clean using 1% hydrochloride alcohol solution, then
It is placed in each dehydration of 100% and 95% ethanol twice, each 5min；Finally place 5min in dimethylbenzene stand-by.
6. SABC adopts SP method, transplanted tumor piece of tissue is fixed, cuts into slices after paraffin embedding, in conventional dewaxing to water, micro-
Ripple repairs antigen, and 3% 37 DEG C of hydrogen peroxide is incubated 15 min, adds sCLU mono- to resist, it is through Dako antibody that described sCLU mono- resists
SCLU mono- after 50 times of diluted resists, and, adds phosphate buffered saline(PBS) to be placed in shaking table rinsing 3 under 4 DEG C of environment overnight
Secondary, each 5min；The sheep anti mouse two of Deca biotin labeling resists, and it is through the dilution of Dako antibody diluent that described sheep anti mouse two resists
Sheep anti mouse two after 50 times resists, incubated at room 10 min, and phosphate buffered saline(PBS) rinses 3 times, each 5min；Deca Fresh
Four diaminobenzidine hydrochloride solution 100 l, color development at room temperature, distilled water wash 3 times, haematoxylin redyes 5min, dehydrated alcohol
It is dehydrated transparent, mounting.
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| CN107715103A (en) * | 2017-11-20 | 2018-02-23 | 深圳大学 | Applications of the Clusterin in the cancer treatment drugs for preparing killing tumor stem cell |
| CN107885974A (en) * | 2017-11-22 | 2018-04-06 | 南宁科城汇信息科技有限公司 | Transcript profile and proteomic assays method in a kind of liver cancer biological process |
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| CN103743902A (en) * | 2013-11-25 | 2014-04-23 | 南通大学附属医院 | Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse |
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| CN107715103A (en) * | 2017-11-20 | 2018-02-23 | 深圳大学 | Applications of the Clusterin in the cancer treatment drugs for preparing killing tumor stem cell |
| CN107715103B (en) * | 2017-11-20 | 2020-08-11 | 深圳大学 | Application of Clusterin in preparation of cancer treatment drug for killing tumor stem cells |
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