The Study Of Lentivirus Transfecting High Expression GALC Gene HFL1Cells Senescence Model And Interaction With Colon Cancer Cells

ObjectiveUnderstanding and further defined of the interaction between colorectal cancercells and senescent stroma cells.Methods1.16patients with colorectal cancer adopting new adjuvant chemotherapy withXELOX were analyzed retrospectively． Tumor-stroma ratio (TSR) of thepathological sections.Activity of interstitial β-galactosidase and expression of P53、Villin、Ki67、E-cadherin were detected with immunohistochemistry. Survivalanalysis and observation of adverse reaction of chemotherapy were performed at thesame time.2.Constructing HFL1cells with high expression GALC gene by transfection oflentivirus and inducing senescence of HFL1cells by hydrogen peroxide.3.Detection of related indicators in senescence of HFL1cells with high expressionGALC gene: stained β-galactosidase by immunohistochemistry；detected the cellcycle by PI method；expressed on p16gene，p21gene and p53by qRT-PCR andWestern-blot analyses; expressed on TERTmRNA by qRT-PCR.4.Co-cultured colorectal cancer cell lines with HFL1cells with high expressionGALC gene; detected the activity of colorectal cancer cell lines by CCK8colorimeter; detected the cell cycle of colorectal cancer cell lines by PI method;examined the cell migration by transwell migration assays.Results1. ORR was25%. DCR was68.75%. Median OS was12months and PFS was13.43±6.10months in these16patients with locally advanced colorectal cancer adopting new adjuvant chemotherapy with XELOX. III-IV adverse reaction was6.25%of hand-foot syndrome，6.25%of diarrhoea，12.5%of neutropenia，6.25%of anaemia，37.50%(6patients) of TSR increasing after chemotherapy，37.5%(6patients)expression of intense positive reaction in interstitial β-Galactosidase.Positive reaction in interstitial β-galactosidase after chemotherapy was associatedwith survival.2. HFL1cells with high expression GALC gene that constructed by transfection oflentivirus can be stable transfect.The cell morphology showed as senescentinterstitial cells which were similar to that induced by hydrogen peroxide.3. The expression is intense positive reaction in HFL1cells with high expressionGALC gene and G0/G1stage cells were significantly increased. There weredifferences in statistics that mRNA of p21gene increased than normal HFL1cellsand TERTmRNA decreased significantly. Protein expression of p16gene and p21gene was significantly increased than normal HFL1cells.4. In the condition of co-cultured with HFL1cells with high expression GALC gene，the activity of LOVO cells was significantly increased. After24hours and48hoursco-cultured， the proportion of G0/G1stage decreased dramatically while theproportion of G2/M stage increased. Migration of PKO cells were increased. Therewere differences in statistics.Conclusions1. Adopting additional adjuvant chemotherapy with XELOX in locally advancedcolorectal cancer patients has reliable curative effect and adverse reactions can betolerated. Positive reaction in interstitial β-galactosidase after chemotherapy wasassociated with survival.2. HFL1cells with high expression GALC gene that constructed by transfection oflentivirus can be stable transfect and have senescent interstitial cells associated cellmorphous and molecular biological feature as well.3. Co-culturing colorectal cancer cell lines with HFL1cells with high expressionGALC gene can affect the cell cycle，cell vitality and cell migration of colorectal cancer cell lines.