| Part ?: Construction and identification of a PDX model for liver cancerObjective: A mouse xenograft model derived from fresh tumor tissue of liver cancer patients was established as a platform for evaluation of drug efficacy.Methods: Inoculation of fresh liver cancer tissue from liver cancer patients into the skin of mice for constructing a subcutaneous xenograft model of liver cancer PDX.The tumor block of the liver cancer PDX model was successfully constructed in the first generation,and the liver in situ PDX model of liver cancer was constructed.HE staining,Western blot and immunohistochemical staining were used to identify the genetic characteristics of PDX model and clinical tumor tissue,as well as the stability of biological characteristics during passage.Results: We successfully established 17 cases of PDX subcutaneous xenograft model of liver cancer patients(30.9% success rate)and 2 liver transplantation models of liver cancer mice(100% success rate).The morphological changes of hepatocellular carcinoma cells and the expression of Ki67 in immunohistochemistry were observed by HE staining,and the consistency was observed in both P1 and P3 generations.It indicated that the liver cancer PDX xenograft model showed high consistency with liver cancer tissues in patients with histomorphology and protein expression.Conclusion: Successfully established a clinically similar PDX model of liver cancer,which can be a suitable anti-tumor drug evaluation platform.Part ?: Preliminary evaluation of the therapeutic effect of ATRA on liver cancer based on PDX modelObjective: Based on the PDX model of liver cancer,the role of Pin1 inhibitor ATRA sustainedrelease tablets in the treatment of liver cancer was evaluated.Methods: Five cases of 3~5 generation liver cancer PDX models were selected.After passage,they were divided into two groups,8 in each group,sham-operated control group and ATRA sustained-release tablets subcutaneously implanted plus drug group.Tumor volume was recorded weekly and tumor growth curves were plotted.The tumor block was taken out until the control group grew to 700-1000 mm3.The immunohistochemical staining,immunofluorescence staining and Western blot were used to detect the difference of the expression level of Pin1 and its downstream Cyclin D1 protein and tumor proliferation-related protein Ki67 in the control group and the drug-added group.effect.Results: In 5 cases of liver cancer PDX model,1 case(sample number: 17016422),the liver cancer PDX model showed good efficacy under the dosage of 2.5mg ATRA sustained-release tablets.Pin1 and its downstream Cyclin D1 protein in the tumor group.Both proliferation and proliferation-related proteins showed a significant decrease.Three cases(sample numbers: 17013572,18026063,17061051)showed efficacy under the action of 5 mg of ATRA sustainedrelease tablets,and the protein level was the same as above.However,there was one case(sample number: 17013276).The model was ineffective after treatment with 2.5 mg and 5 mg of ATRA,and there was no significant difference in the expression levels of Pin1,Cyclin D1 and Ki67 protein in the control group and the drug-added group.Further,by immunohistochemical analysis,the level of Glypican-3 expression is closely related to the sensitivity of ATRA.Conclusion: The Pin1 inhibitor ATRA has a significant inhibitory effect on the growth of some liver cancers.However,there are still some cases where liver cancer is not sensitive to ATRA,and the specific mechanism remains to be further studied.Part ?: Preliminary study on assessing the drug resistance of liver cancer to ATRAObjective: In this section,we observed whether the growth of these two cells was inhibited by knocking down the Pin1 gene on the liver cancer cell lines Huh7 and PLC.Then,it was propagated for many generations to observe whether the cell growth rate was restored.Then,the changes in gene expression before and after cell growth recovery were examined to find possible mechanisms of drug resistance.A PDX model of ATRA-resistant liver cancer was initially constructed.Methods: We purposely knocked out its Pin1 gene by lentivirus infection of the liver cancer cell line Huh-7 and PLC,and studied its changes at the molecular and cellular levels.First,we used a 24-well plate,30,000/well plate,to detect the proliferation rate of Huh7-scramble-4 and Huh7-shpin1-4 and PLC-scramble-4 and PLC-shpin1-4 by cytometry,and then pass Western.The expression levels of Pin1 and related proteins were detected by blot.Secondly,we passed the two cells to the 20 th generation by continuous culture,and again tested Huh7-scramble-4 and Huh7-shpin1-20 and PLC-scramble-4 and PLC-shpin1-20 cell lines with the above methods.Changes in Pin1 and its related proteins and detection of IC50 values of different concentrations(0,5,10,20,40,80 and 160 ?M)of ATRA using CCK8.Finally,we selected Huh7-scramble-4 and Huh7-shpin1-20;PLC-scramble-4 and PLC-shpin1-20 to extract cellular RNA,and sent the company to perform transcriptome sequencing analysis of these six cells.According to the data of the differential genes obtained by the analysis,q-PCR and Western blot were used for verification.Further continuous dosing experiments in the ATRA-sensitive PDX model,it is expected that the ATRA-resistant liver cancer PDX model can be screened by multiple generations of drug administration.Results: After knocking down Pin1,the growth rate of Huh7 and PLC in liver cancer cells was significantly slower,and the protein samples were detected by Western blot to reduce the expression of Pin1 protein.Subculture was continued,and when the cells were passed for about 20 generations,the growth rate of the two cells returned to normal.The expression of Pin1 protein was detected by Western blot.It was found that Pin1 protein was still knocked down and Cyclin D1 expression was restored.The CCK8 assay found that the IC50 values of the two cells after the Pin1 knockdown growth recovery became larger,indicating that it was insensitive to Pin1 inhibitors such as ATRA.The final analysis of transcriptome sequencing showed that the expression levels of EPHA3 in these two cells were significantly increased after growth recovery.The results of q-PCR and Western blot were consistent with those of transcriptome sequencing.It is speculated that EPHA3 may knock down cells with Pin1.Growth rate recovery is related.In the PTX model of ATRA liver cancer,which was initially constructed,it showed that Cyclin D1 expression gradually recovered with the tolerance of ATRA.Conclusion: Preliminary analysis of liver cancer has certain resistance to ATRA,and its tolerance may be related to up-regulation of EPHA3 and pathway leading to recovery of Cyclin D1 protein. |
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