| Objective:On the basis of the conclusion that the combination of rAAV-shRNA-CDK2 and GAPS GF could inhibit the subcutaneous implantation tumor of HepG2,the distribution of rAAV-shRNA-CDK2 in the gonadal gland and testis were observed.The effects of rAAV-shRNA-CDK2 on testis and ovaries were preliminarily judged,and the auxiliary effects of GAPS GF on testes and ovaries were observed.The results provided data support for preclinical safety evaluation of rAAV-shRNA-CDK2,and could be used to guide clinical drug use.In order to provide medication basis and diagnosis and treatment guidance for patients with liver cancer in pregnancy period,the growth and reproduction of offspring can be evaluated preliminarily,and the basis of choice for patients can be provided.Methods:1.A total of 24 nude mice(half male and female)were constructed,and the 1mm3 human HepG2 liver tissue was subcutaneously inoculated into the armpit of the forelimb of BALB/c-nu nude mice;2.The tumorigenic status of each group of nude mice was observed and measured every 5 days.The tumor growth curve was plotted according to the tumor length and diameter;3.Ten days after inoculation,divided the nude mice into 4 groups randomly:tumor control group,NC control group,rAAV-shRNA-CDK2 group,GAPS GF with rAAV-shRNA-CDK2 combination group,6 rats in each group(half male and female);4.rAAV-shRNA-CDK2 and rAAV-shRNA-NC drug were one-time administration by tail vein injection,GAPS GF was continuous administration for 15 days by intramuscular injection.24 hours after the administration,the nude mice were killed by the broken neck;5.The tumor tissue was stripped completely and weighed,and the tumor inhibition rate was calculated;6.The testes and ovaries were rapidly dissected,and fluorescence observation was performed with small animal imager;7.Judging tissue injury by HE staining;8.Western blot and immunohistochemistry were used to detect the expression of EGFP and CDK2 protein and cell location in testicular and ovarian tissues,to observe the distribution and influence of rAAV-shRNA-CDK2 on testes and ovaries,and the effect of GAPSGF on them.Result:1.All the nude mice were tumorigenic,and the tumorigenesis rate was 100%;2.The tumor inhibitory rates of rAAV-shRNA-CDK2 on HepG2 cells of male and female rats were 66.73%and 68.33%,respectively;the tumor inhibitory rates of GAPS GF and rAAV-shRNA-CDK2 on HepG2 cells in male and female mice were 68.64%and 70.38%respectively,and increased by 1.91%and 2.05%respectively;3.Western blot and small animals showed no protein EGFP expression in testis and ovaries;4.HE staining section of testis and ovaries showed normal morphology and no pathological changes;5.rAAV-shRNA-CDK2 has no effect on the expression of CDK2 in testes and ovaries,and CDK2 was located in the nucleus of ovarian and testicular cells.Conclusion:1.The rAAV-shRNA-CDK2 can effectively inhibit the growth of hematoma cells.The combination of GAPS GF and rAAV-shRNA-CDK2 can increase the inhibition rate of tumor,suggesting that GAPS GF can help to inhibit the proliferation of HepG2 cells;2.rAAV-shRNA-CDK2 was not found in testes and ovaries;3.The affect of rAAV-shRNA-CDK2 on the morphology of testes and ovaries was not observed,nor does it cause pathological changes. |
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