CN103743902A - Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse - Google Patents
Method for detecting inhibiting ability of silent GPC-3 gene transcription on hepatocellular carcinoma transplanted tumor in nude mouse Download PDFInfo
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Abstract
The invention discloses a method for detecting the inhibiting ability of silent GPC-3 (glypican-3) gene transcription on a hepatocellular carcinoma transplanted tumor in a nude mouse. The method includes: 1) designing and synthesizing 4 pairs of miRNA oligomeric single-strand DNA specific to a target gene consensus sequence, then synthesizing the corresponding dsDNA, inserting the dsDNA into a vector to construct 4 recombinant plasmids, and picking out the recombinant plasmid with the highest interference efficiency through fluorescence quantitative PCR; 2) transfecting the recombinant plasmid with the highest interference efficiency into HepG2 cells to construct transfected HepG2 cells, and performing screening, thus obtaining a stably transfected HepG2 cell; 3) inoculating the stably transfected HepG2 cell to the nude mouse subcutaneously to construct a human hepatocellular carcinoma tumor-bearing nude mouse model; and 4) then determining the growth state of the stably transfected HepG2 cell in the human hepatocellular carcinoma tumor-bearing nude mouse model. The method provided by the invention can be employed to detect the inhibiting ability of silent GPC-3 gene transcription on a hepatocellular carcinoma transplanted tumor in the nude mouse, and can be used for research of GPC-3 on transplanted tumors.
Description
Technical field
The present invention relates to GPC-3 related gene technology, particularly a kind of method that detects reticent GPC-3 genetic transcription inhibition hepatocellular carcinoma in nude mice ability.
Background technology
Phosphatidylinositols proteoglycan-3 (glypican-3, GPC-3) as a kind of cell surface protein, specificity overexpression in hepatocellular carcinoma (hepatocellular carcinoma, HCC), is considered to the potential target spot of hepatocarcinoma early diagnosis and specific treatment.Monophosphoinositideproteoglycans proteoglycans-3 (GPC-3) belongs to the fixed heparan sulfate proteoglycan family of glycosyl-phosphatidyl inositol anchor.The GPC-3 assignment of genes gene mapping is in human chromosome X26.10, because the sudden change of this molecule or unconventionality expression are normal and disease association, so be subject to extensive concern.In the middle of prior art, based on reticent GPC-3 genetic transcription, suppress the not simple means efficiently of hepatocellular carcinoma in nude mice research, the checking of the ability of reticent GPC-3 genetic transcription to hepatocellular carcinoma in nude mice is easy not.
Summary of the invention
The object of the invention is to, a kind of method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability that detects is provided, method comprises the steps:
1) for target gene human GPC3, the consensus sequence of 4 transcripts of Gene ID:2719 designs and synthesizes 4 pairs of miRNA oligomerization single stranded DNAs, synthetic corresponding dsDNA again, dsDNA insertion vector is built to 4 kinds of GPC-3-miRNA recombinant plasmids, and pick out by quantitative fluorescent PCR a kind of GPC-3-miRNA recombinant plasmid that jamming effectiveness is the highest;
2) Transfected Recombinant Plasmid the highest above-mentioned jamming effectiveness is built to transfection HepG 2 cell to HepG2 cell, and it is screened, obtain stable transfection HepG2 cell;
3) aforementioned stable transfection HepG 2 cell inoculation nude mice by subcutaneous is built to human liver cancer cell model of nude mice bearing tumor;
4) then measure aforementioned stable transfection HepG 2 cell growing state in human liver cancer cell model of nude mice bearing tumor.
In some embodiments, the GPC-3-miRNA recombinant plasmid of 4 kinds is respectively:
p-CMV-GPC-3-miRNA-1；
p-CMV-GPC-3-miRNA-2；
p-CMV-GPC-3-miRNA-3；
p-CMV-GPC-3-miRNA-4;
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-1 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGTGAATTAGTTCCCTTCTTCGGGTTTTGGCCACTGACTGACCCGAAGAAGAACTAATTCA-3
R:5-CCTGTGAATTAGTTCTTCTTCGGGTCAGTCAGTGGCCAAAACCCGAAGAAGGGAACTAATTCAC-3
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-2 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGTAATTTGACTGACCACTGGCTGTTTTGGCCACTGACTGACAGCCAGTGCAGTCAAATTA-3
R:5-CCTGTAATTTGACTGCACTGGCTGTCAGTCAGTGGCCAAAACAGCCAGTGGTCAGTCAAATTAC-3
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-3 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGTTCATTAGCTGGGTATAGATGGTTTTGGCCACTGACTGACCATCTATACAGCTAATGAA-3
R:5-CCTGTTCATTAGCTGTATAGATGGTCAGTCAGTGGCCAAAACCATCTATACCCAGCTAATGAAC-3
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-4 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGAATACTTTCAGGTCACGTCTTGTTTTGGCCACTGACTGACAAGACGTGCTGAAAGTATT-3
R:5-CCTGAATACTTTCAGCACGTCTTGTCAGTCAGTGGCCAAAACAAGACGTGACCTGAAAGTATTC-3
In some embodiments, the recombinant plasmid that jamming effectiveness is the highest is:
p-CMV-GPC-3-miRNA-1。
In some embodiments, carrier is pcDNA
tM6.2-GW/EmGFP-miR.
In some embodiments, the highest Transfected Recombinant Plasmid of jamming effectiveness is used GenjetTM transfection reagent to building in transfection HepG 2 cell in HepG2 cell, and obtaining the screening reagent that stable transfection HepG2 cell uses is blasticidin S.
In some embodiments, in step (3), the method for transfection HepG 2 cell inoculation nude mice by subcutaneous structure human liver cancer cell model of nude mice bearing tumor is: the transfection HepG 2 cell of collecting exponential phase, with 2 × 107/ (0.2ml only), be inoculated in the right scapular region of nude mice subcutaneous, form the subcutaneous protuberance of diameter 4mm left and right.
In some embodiments, the method for measuring transfection HepG 2 cell growing state is for being survey tumor growth curve and immunohistochemical analysis.
In some embodiments, surveying tumor growth curve method is:
After inoculation, survival state, the injection site of observing nude mice every day has or not to have or not spontaneous regression and record every group of tumour after infection and ulceration, tumor growth and grows the time; After tumour occurs, use weekly the long and short footpath of vernier caliper measurement tumour, according to formula: gross tumor volume V (mm3)=0.5 × a × b2 (a=major diameter, b=minor axis), calculate gross tumor volume, and draw tumor growth curve according to the equal numerical value of volume.In inoculation, within latter 35 days, with cervical vertebra dislocation method, put to death nude mice, the upgrowth situation of gross examination of skeletal muscle transplantable tumor and the surrounding organ situation of getting involved; Peel off normal saline flushing for tumour, dry, take pictures; Sample is immersed in 4% paraformaldehyde and fixes 24h, standby;
Immunohistochemical analysis method is:
By sample dewaxing standby in above-mentioned survey tumor growth curve method, aquation, hydrogen peroxide blocking-up endogenous peroxydase, hyperbaric heating method is repaired antigen, Normal animal serum sealing non-specific binding; Drip respectively GPC-3, β-catenin, p-GSK3 β and cyclinD1 antibody, 4 ℃ are spent the night, PBS rinsing; Drip biotin labeled second antibody, incubated at room 10min, PBS rinsing; Drip streptomysin avidin-peroxidase, incubated at room 10min, PBS rinses; Drip freshly prepared four diaminobenzidine hydrochloride solution, color development at room temperature, distilled water washing, haematoxylin is redyed, transparent, the mounting of absolute ethyl alcohol dehydration; Observation by light microscope, shooting; With the alternative first antibody of 0.0l mol/L PBS (pH=7.5), second antibody, make negative control.
In some embodiments, for 4 kinds of GPC-3-miRNA recombinant plasmid design p-CMV-Neg-miRNA plasmids in contrast;
The positive and negative adopted chain of p-CMV-Neg-miRNA insetion sequence (5 '-3 ') is respectively:
F;5-tgctgAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT-3
R:5-cctgAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTc-3
The method of patented claim of the present invention can the research to transplantable tumor for reticent GPC-3.
The method of patented claim of the present invention can detect reticent GPC-3 genetic transcription and suppress hepatocellular carcinoma in nude mice ability.
Can draw following result:
Reticent GPC-3 genetic transcription prolonged human hepatoma carcinoma cell model of nude mice bearing tumor latent period.
Reticent GPC-3 genetic transcription delays the hepatocellular carcinoma in nude mice speed of growth.
The obvious hepatocellular carcinoma in nude mice of reticent GPC-3 genetic transcription.
Reticent GPC-3 genetic transcription makes GPC-3 in hepatocellular carcinoma in nude mice, β-catenin, p-GSK3 β, cyclinD1 expression lower than blank group.
And the method for patented claim of the present invention is simply efficient, the clear and definite reticent GPC-3 genetic transcription that shown has obvious ability to suppressing hepatocellular carcinoma in nude mice.
Accompanying drawing explanation
Fig. 1 is the preclinical comparison diagram of mensuration human liver cancer cell model of nude mice bearing tumor of an embodiment of the present invention;
Fig. 2 is the mensuration subcutaneous transplantation knurl growth curve of an embodiment of the present invention;
Fig. 3 is that the detection liver cancer of an embodiment of the present invention is examined transplantable tumor inhibition comparison diagram;
Fig. 4 is that the SABC of an embodiment of the present invention detects tumor tissue Wnt signal path correlative protein expression figure.
Embodiment
Embodiment
The preparation of 1.1 materials:
Human HepG2 cell's strain is purchased from Shanghai cell institute of the Chinese Academy of Sciences; Hyclone, trypsin solution and phosphate buffer (PBS) are purchased from American I nvitrogen company; Dimethyl sulfoxide (DMSO) and blasticidin S are purchased from U.S. Sigma company; GenJetTM DNA In Vitro Transfection Reagent is purchased from U.S. SignaGen company; Mouse-anti people GPC-3 antibody is purchased from U.S. Abcam company; β-catenin, p-GSK3 β, cyclinD1 and the anti-human antibody of β-actin rabbit are purchased from U.S. CST company; The goat-anti rabbit of horseradish peroxidase-labeled and sheep anti-mouse igg antibody are purchased from Britain Everest Biotech company.
The preparation of 1.2 animals used as test:
18 BALB/c nude mices are by (Nantong University's Experimental Animal Center provides), 4～6 weeks ages of mouse, body weight 18～20g, male and female half and half, random point control group (untreated group), miRNA negative group (miRNA-neg group) and miRNA interference group (GPC-3-miRNA group), every group 6, under SPF condition, raise.
The cultivation of 1.3HepG2 cell
The DMEM nutrient culture media that contains 10% hyclone for HepG2 cell lines, adhere-wall culture in 37 ℃, 5%CO2 incubator goes down to posterity when Fusion of Cells degree > 80%.
Under the condition completing in early-stage preparations, specifically adopt following steps to detect reticent GPC-3 genetic transcription and suppress hepatocellular carcinoma in nude mice ability:
2.1 4 kinds of GPC-3-miRNA recombinant plasmid designs are with synthetic
For target gene human GPC3, the consensus sequence of 4 transcripts of Gene ID:2719, utilizes Invitrogen miRNA design system Software for Design 4 to miRNA oligomerization single stranded DNA and negative control sequence oligomerization single stranded DNA, and sequence is in Table 1.
4 pairs of miRNA oligomerization single stranded DNAs and negative control sequence oligomerization single stranded DNA are dissolved into 100 μ M with distilled water respectively, and complementary strand is respectively got 5 μ L and is mixed, 10 × annealing buffer, 2 μ L, distilled water polishing to 20 μ L, 95 ℃ of heating 5min, the cooling 20min of room temperature, forms corresponding dsDNA.
The dsDNA of corresponding annealing is continued to be diluted to 10nM, get 4 μ L, carrier pcDNA
tM6.2-GW/EmGFP-miR2 μ L, 5 × connect damping fluid 4 μ L, T4DNA ligase 1 μ L, distilled water polishing to 20 μ L, room temperature connects 30min, obtains 5 kinds and connects product.
Get respectively 10 μ l and connect products and transform 100 μ l competent cell DH5 α, after be inoculated on LB/ spectinomycin plating medium, overnight incubation in 37 ℃ of incubators, the bacterium colony growing is positive bacterium colony, chooses bacterium colony amplification, extracts recombinant plasmid, order-checking is identified.Four kinds of GPC-3-miRNA recombinant plasmids are respectively: p-CMV-GPC-3-miRNA-1, p-CMV-GPC-3-miRNA-2, p-CMV-GPC-3-miRNA-3 and p-CMV-GPC-3-miRNA-4, the negative control of GPC-3-miRNA recombinant plasmid is p-CMV-Neg-miRNA, and sequence is in Table 1.
By 4 kinds of recombinant plasmids that build, by quantitative fluorescent PCR, screen best a pair of of jamming effectiveness, i.e. p-CMV-GPC-3-miRNA-1.
Table 1GPC-3-miRNA vector plasmid title and sequence
2.2 cell transfecting and screening
Two groups of HepG2 cells (miRNA-neg and GPC-3-miRNA group) are planted to plate 24h, when naked eyes sight cell density reaches 70% left and right, utilize Genjet
tMtransfection reagent is distinguished transfection to miRNA-neg and GPC-3-miRNA group HepG2 cell by p-CMV-Neg-miRNA, p-CMV-GPC-3-miRNA-1, HepG2 cell screening after utilizing blasticidin S to transfection, obtain aforementioned stable transfection HepG 2 cell clone, amplification cultivation.
2.3 transplantable tumor nude mice models are set up
Collect respectively exponential phase miRNA-neg group stable transfection HepG2 cell, GPC-3-miRNA group stable transfection HepG2 cell and untreated group (original HepG2) cell, with 2 × 10
7it is subcutaneous that/(0.2ml only) is inoculated in the right scapular region of nude mice, forms the subcutaneous protuberance of diameter 4mm left and right.
2.4 measure transfection HepG2 growing state:
2.4.1 tumor growth curve
After inoculation, survival state, the injection site of observing nude mice every day has or not to have or not spontaneous regression and record every group of tumour after infection and ulceration, tumor growth and grows the time.After tumour occurs, use weekly the long and short footpath of vernier caliper measurement tumour, according to formula: gross tumor volume V (mm3)=0.5 × a × b2 (a=major diameter, b=minor axis), calculate gross tumor volume, and draw tumor growth curve according to the equal numerical value of volume.In inoculation, within latter 35 days, with cervical vertebra dislocation method, put to death nude mice, the upgrowth situation of gross examination of skeletal muscle transplantable tumor and the surrounding organ situation of getting involved.Peel off normal saline flushing for tumour, dry, take pictures.Sample is immersed in 4% paraformaldehyde and fixes 24h, standby.
2.4.2 immunohistochemical analysis
By conventional the sample of upper step dewaxing, aquation, hydrogen peroxide blocking-up endogenous peroxydase, hyperbaric heating method is repaired antigen, Normal animal serum sealing non-specific binding.Drip respectively GPC-3, β-catenin, p-GSK3 β and cyclinD1 antibody, 4 ℃ are spent the night, PBS rinsing; Drip biotin labeled second antibody, incubated at room 10min, PBS rinsing; Drip streptomysin avidin-peroxidase, incubated at room 10min, PBS rinses; Drip freshly prepared four diaminobenzidine hydrochloride solution, color development at room temperature, distilled water washing, haematoxylin is redyed, transparent, the mounting of absolute ethyl alcohol dehydration.The Japan Olympus BX50 of company observation by light microscope, shooting; With the alternative first antibody of 0.0l mol/L PBS (pH=7.5), second antibody, make negative control.
2.4.3 statistical procedures
Utilize SPSS18.0 software to carry out One-way ANOVA (One-way ANOVA).Measured value represents with mean ± standard deviation, the relatively employing chi-square criterion of positive cell percentage, and tumor average volume adopts t check.P<0.05 thinks that difference has statistical significance.
Implementation result
3.1HepG2 human liver cancer cell model of nude mice bearing tumor latent period
Experimental group (GPC-3-miRNA group) nude mice by subcutaneous tumour forms and compares prolongation of latency with control group (untreated and miRNA-neg group), average out to 11.17 ± 0.98 days, miRNA-neg and untreated group are respectively 5.5 ± 0.55 days, 5.33 ± 0.52 days swollen neoplastic latent period, experimental group tumour forms obvious delay, P<0.01.MiRNA-neg group is compared and is had no statistics notable difference, P>0.05 with untreated group.See Fig. 1.Demonstration is stablized strain by the HepG2 of reticent GPC-3 and is given mouse bare subcutaneous injection, and after GPC-3-miRNA blocking-up GPC-3 expresses, HepG2 cell transplantation knurl has been subject to remarkable inhibition in nude mice by subcutaneous growth.
3.2 human hepatoma HepG2 cell lotus knurl growth curves are got 0.2ml HepG2 cell suspension in the right scapular region hypodermic injection of nude mice, form the subcutaneous protuberance of diameter 4mm left and right, and second day afterwale is absorbed gradually; About control group the 5th day, there is flat tubercle in inoculation position, and grow up gradually.Experimental group nude mice by subcutaneous tumor growth rate is considerably slower than control group, and when experiment finishes, difference is the most obvious.When experiment finishes, GPC-3-miRNA group knurl volume is 65.48 ± 13.66mm
3be significantly less than control group, control group is respectively 404.83 ± 52.63mm
3, 365.67 ± 14.47mm
3, experimental group is compared P<0.01 with control group, shows that reticent GPC-3 has the effect of certain inhibition tumor growth.Control group is compared and is had no statistics notable difference, P>0.05.In experimentation, all animals is all without natural death.See Fig. 2.The one-tenth knurl ability of HepG2 cell in nude mice obviously reduces, and the expression of prompting inhibition GPC-3 can suppress the multiplication capacity of HepG2 cell to a certain extent.
3.3 liver cancer are examined transplantable tumor inhibition transplantable tumor outward appearance pinkiness, surface irregularity, and without ulceration, tumor tissues surface fiber coating is complete easily separated, in tumor tissue matter, is nodositas or lobulated.Its hetero-organization and organ have no the cancerous swelling focus of transfer and see Fig. 3.
3.4 SABC detection Wnt/ β-catenin signal path key molecules expression immunohistochemical methods detect respectively the expression of GPC-3, β-catenin, p-GSK3 β, cyclinD1 albumen in tumor tissue.In GPC-3-miRNA group, GPC-3, β-catenin, p-GSK3 β, cyclinD1 expression are lower than control group.See Fig. 4.Which kind of mechanism in order to study GPC-3, to affect the propagation of tumour cell with, utilize SABC to detect the expression of Wnt signal path key molecule p-GSK3 β and downstream signaling molecule β-catenin and cyclinD1, result shows that, after blocking-up GPC-3, β-catenin, p-GSK3 β and cyclinD1 express same minimizing.This method shows that blocking-up GPC-3 reduces the expression of β-catenin and cyclinD1 in cell, causes the cell cycle G1 phase to be blocked, and cell proliferation is suppressed.
This detects the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, can the research to transplantable tumor for GPC-3, show that reticent GPC-3 genetic transcription suppresses the effect of hepatocellular carcinoma in nude mice, has good supporting function to the research of liver cancer.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, without departing from the concept of the premise of the invention, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (9)
1. detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, the method comprises the steps:
1) for target gene human GPC3, the consensus sequence of 4 transcripts of Gene ID:2719 designs and synthesizes 4 pairs of miRNA oligomerization single stranded DNAs, synthetic corresponding dsDNA again, dsDNA insertion vector is built to 4 kinds of GPC-3-miRNA recombinant plasmids, and pick out by quantitative fluorescent PCR a kind of GPC-3-miRNA recombinant plasmid that jamming effectiveness is the highest;
2) Transfected Recombinant Plasmid the highest above-mentioned jamming effectiveness is built to transfection HepG 2 cell to HepG2 cell, and it is screened, obtain stable transfection HepG2 cell;
3) aforementioned stable transfection HepG 2 cell inoculation nude mice by subcutaneous is built to human liver cancer cell model of nude mice bearing tumor;
4) then measure aforementioned stable transfection HepG 2 cell growing state in human liver cancer cell model of nude mice bearing tumor.
2. a kind ofly according to claim 1 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, 4 kinds of described GPC-3-miRNA recombinant plasmids are respectively:
P-CMV-GPC-3-miRNA-1, p-CMV-GPC-3-miRNA-2, p-CMV-GPC-3-miRNA-3 and p-CMV-GPC-3-miRNA-4;
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-1 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGTGAATTAGTTCCCTTCTTCGGGTTTTGGCCACTGACTGACCCGAAGAAGAACTAATTCA-3
R:5-CCTGTGAATTAGTTCTTCTTCGGGTCAGTCAGTGGCCAAAACCCGAAGAAGGGAACTAATTCAC-3
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-2 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGTAATTTGACTGACCACTGGCTGTTTTGGCCACTGACTGACAGCCAGTGCAGTCAAATTA-3
R:5-CCTGTAATTTGACTGCACTGGCTGTCAGTCAGTGGCCAAAACAGCCAGTGGTCAGTCAAATTAC-3
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-3 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGTTCATTAGCTGGGTATAGATGGTTTTGGCCACTGACTGACCATCTATACAGCTAATGAA-3
R:5-CCTGTTCATTAGCTGTATAGATGGTCAGTCAGTGGCCAAAACCATCTATACCCAGCTAATGAAC-3
The positive and negative adopted chain of p-CMV-GPC-3-miRNA-4 insetion sequence (5 '-3 ') is respectively
F:5-TGCTGAATACTTTCAGGTCACGTCTTGTTTTGGCCACTGACTGACAAGACGTGCTGAAAGTATT-3
R:5-CCTGAATACTTTCAGCACGTCTTGTCAGTCAGTGGCCAAAACAAGACGTGACCTGAAAGTATTC-3?。
3. a kind ofly according to claim 2 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, the GPC-3-miRNA recombinant plasmid that described jamming effectiveness is the highest is p-CMV-GPC-3-miRNA-1.
4. a kind ofly according to claim 1 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, described carrier is pcDNA
tM6.2-GW/EmGFP-miR.
5. a kind ofly according to claim 1 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability,
The highest Transfected Recombinant Plasmid of described jamming effectiveness is used Genjet to building in HepG2 cell in transfection HepG 2 cell
tMtransfection reagent, described in obtain stable transfection HepG2 cell use screening reagent be blasticidin S.
6. a kind ofly according to claim 1 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, in described step (3), the method for transfection HepG 2 cell inoculation nude mice by subcutaneous structure human liver cancer cell model of nude mice bearing tumor is: collect the transfection HepG 2 cell of exponential phase, with 2 × 10
7it is subcutaneous that/(0.2ml only) is inoculated in the right scapular region of nude mice, forms the subcutaneous protuberance of diameter 4mm left and right.
7. a kind of method that suppresses hepatocellular carcinoma in nude mice based on reticent GPC-3 genetic transcription according to claim 2, the method for described mensuration transfection HepG 2 cell growing state is for surveying tumor growth curve and immunohistochemical analysis.
8. a kind ofly according to claim 7 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability,
Described survey tumor growth curve method is:
After inoculation, survival state, the injection site of observing nude mice every day has or not to have or not spontaneous regression and record every group of tumour after infection and ulceration, tumor growth and grows the time; After tumour occurs, use weekly the long and short footpath of vernier caliper measurement tumour, according to formula: gross tumor volume V (mm
3)=0.5 × a × b2 (a=major diameter, b=minor axis), calculates gross tumor volume, and draws tumor growth curve according to the equal numerical value of volume; In inoculation, within latter 35 days, with cervical vertebra dislocation method, put to death nude mice, the upgrowth situation of gross examination of skeletal muscle transplantable tumor and the surrounding organ situation of getting involved; Peel off normal saline flushing for tumour, dry, take pictures; Sample is immersed in 4% paraformaldehyde and fixes 24h, standby;
Described immunohistochemical analysis method is:
By the above-mentioned sample dewaxing of fixing, aquation, hydrogen peroxide blocking-up endogenous peroxydase, hyperbaric heating method is repaired antigen, Normal animal serum sealing non-specific binding; Drip respectively GPC-3, β-catenin, p-GSK3 β and cyclinD1 antibody, 4 ℃ are spent the night, PBS rinsing; Drip biotin labeled second antibody, incubated at room 10min, PBS rinsing; Drip streptomysin avidin-peroxidase, incubated at room 10min, PBS rinses; Drip freshly prepared four diaminobenzidine hydrochloride solution, color development at room temperature, distilled water washing, haematoxylin is redyed, transparent, the mounting of absolute ethyl alcohol dehydration; Observation by light microscope, shooting; With the alternative first antibody of 0.0l mol/L PBS (pH=7.5), second antibody, make negative control.
9. a kind ofly according to claim 1 detect the method that reticent GPC-3 genetic transcription suppresses hepatocellular carcinoma in nude mice ability, for the GPC-3-miRNA recombinant plasmid design p-CMV-Neg-miRNA plasmid described in 4 kinds in contrast;
The positive and negative adopted chain of p-CMV-Neg-miRNA insetion sequence (5 '-3 ') is respectively:
F;5-tgctgAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT-3
R:5-cctgAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTc-3?。
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| CN105838735A (en) * | 2016-02-19 | 2016-08-10 | 浙江中医药大学 | Human pancreatic cancer nude mouse model construction method and use thereof |
| CN106434747A (en) * | 2016-06-14 | 2017-02-22 | 南通大学附属医院 | Method for inhibiting hepatoma carcinoma nude mouse transplantation tumor capacity through silent secretory clusterin |
| CN113891897A (en) * | 2019-03-21 | 2022-01-04 | 新加坡科技研究局 | Composition comprising a metal oxide and a metal oxide |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838735A (en) * | 2016-02-19 | 2016-08-10 | 浙江中医药大学 | Human pancreatic cancer nude mouse model construction method and use thereof |
| CN105838735B (en) * | 2016-02-19 | 2019-12-31 | 浙江中医药大学 | Construction method and application of nude mouse model for human pancreatic cancer |
| CN106434747A (en) * | 2016-06-14 | 2017-02-22 | 南通大学附属医院 | Method for inhibiting hepatoma carcinoma nude mouse transplantation tumor capacity through silent secretory clusterin |
| CN113891897A (en) * | 2019-03-21 | 2022-01-04 | 新加坡科技研究局 | Composition comprising a metal oxide and a metal oxide |
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