Establishment Of Models For Mice Hepatocellular Carcinoma And The Effct Of Hepatopoietin Cn On Heptoma Growth

PartⅠEstablishment of mice primary inducement hepatocellular carcinoma modelObjective: To establish HPPCn liver-specific overexpression transgenic mice and primary hepatoma mice models. Methods:①Establishment of HPPCn liver-specific overexpression transgenic mice model: the plasmid pGEM-his-hppcn were digested by ApaⅠand MluⅠ, and the digested DNA fragment (including ALB promoter and enhancer suquences, his-hppcn, SV40 tail) was injected into the pronucleus of fertilized ovum, and injected eggs were transplanted into the oviduct of pseudopregnant FVB mice. Founder mice were identified by PCR analysis. For this analysis, genomic DNA was extracted from tails at 10 days of age, and amplified using his-hppcn primers P3: 5'-CAAATGGGAGACAAAGAG-3', P3: 5'- AGATGCGTGAGGTTCG-3'which produced a 600bp fragment from mice carrying the transgene.②Establishment of a primary hepatoma mice model: firstly, C57BL/6♀, C57BL/6♂, and C3H/Hen♂were given different doses of DEN by intraperitoneal injection (C57BL/6♀, 300mg/kg weight; C57BL/6♂, 300mg/kg weight; C3H/Hen♂, 200mg/kg weight). A week later, mice were fed 2-AAF for three days at a dose of 20mg/kg weight, and then 34% of livers of mice were removed by ligating and excising the left lobe at their root. 2-AAF was consecutively used to feed these mice for three days. Three weeks later, the mice were executed, and the partial remnat liver tissues after PH were fixed with 4% (w/v) freshly prepared paraformaldehyde in PBS for immunohistochemisty and the other was homogenated and the total RNA and protein of liver tissue were prepared. Finally, effects were evaluated by RIA, IHC, Western blot, Q-PCR and so on. Results:①We obtained three batches of transgenic mice by repetatus microinjections.②Initialized by DEN, the mortality was as following: C57BL/6♀, 64.7%; C57BL/6♂, 0; C3H♂, 41.2%. Each parameter or index related to HCC of C57BL/6♀showed significant changes, indicating that HCC was induced successfully. Conclusion:①We have successfully established the HPPCn liver-specific overexpression transgenic mice models.②A short-term primary hepatoma model of mice was successfully established.PartⅡEstablishment of hepatocellular carcinoma xenograft models in nude mice and assessment of effct of Hepatopoietin Cn on heptoma growthObjectives: To establish subcutaneous and orthotopic hepatocellular carcinoma transplanted models in nude mice and to confirm the function of HPPCn on the growth and migration of hepatoma cells in vivo. Methods:①Establishmet of subcutaneous hepatocellular carcinoma transplanted models in nude mice: firstly, HepG2-siNC, HepG2-siHPPCn were screened by G418, MTT and Transwell assay were conducted to determine cell proliferation and migration, then 2-4×10~6 cells of HepG2, HepG2-siNC, HepG2-siHPPCn were subcutaneously implanted in nude mice armpit. 30 days later, the mude mice were executed, the hepatomas, lung, livers were obtained and fixed with 4% (w/v) freshly prepared paraformaldehyde in PBS for pathological analysis.②Establishment of orthotopic hepatocellular carcinoma transplanted models in nude mice: HepG2 cells were injected subcutaneously at the armpit at a concentration of 2-4×10~6/100μl to establish a model of tumor-bearing mice. About 20 days later, the bearing tumors were extracted. After necrotic tissue and noncancerous tissues of specimens were removed, the cancerous tissues were cut into small pieces of about 4-6mm~3 in size. The orthotopic models were established by implanting tumor bits directly under the envelope of the mice livers. The left lobe of the liver was exposed and the liver capsule was mechanically injured with a needle. Then a piece of tumor tissue was filled into the liver tissue with forceps and abdominal wall was finally closed, 30 days later, the mice was executed and the hepatomas, lung, livers were obtained and fixed with 4% (w/v) freshly prepared paraformaldehyde in PBS for pathological analysis. Results:①MTT and Transwell analysis suggested that the interference of HPPCn significantly suppressed HepG2 cell proliferation and migration, and subcutaneous hepatocellular carcinoma transplanted models showed that the interference of HPPCn significantly suppressed HepG2 cell proliferation apparently in vivo.②Tumor formation rate was 100%, and there was dubious metastasis in some livers. Conclusions:①Subcutaneous transplanted hepatocellular carcinoma model was successfully established in nude mice, and interfered HPPCn expression in HepG2 cell would inhibite the migration and proliferation.②We established a orthotopic transplantation model in nude mice successfully.