Effect of bone marrow mesenchymal stem cells on MHCC97-H cells after transforming growth factor beta1 and osteopontin gene interference / 中国组织工程研究

ABSTRACT

BACKGROUND:Bone marrow mesenchymal stem cel s (BMSCs) are the focus of research on the proliferation and metastasis of hepatocel ular carcinoma cel s. By genetic engineering techniques, the hepatocel ular carcinoma cel s can be induced to reduce the expression of bioactive factors, thereby seeking suitable intervention targets for improving the interventional effect of BMSCs. OBJECTIVE:To silence the expression of transforming growth factor beta1 (TGFβ1) and osteopontin (OPN) in high metastatic potential hepatocel ular carcinoma cel s (MHCC97-H) fol owed by co-culture with BMSCs and then to observe the change of MHCC97-H cel invasion ability as wel as the interventional effect of BMSCs on the animal model of hepatocel ular carcinoma tissue MHCC97-H by fluorescence imaging in vivo. METHODS:MHCC97-H cel s were divided into four groupsMHCC97-H group was set as a blank control group, and MHCC97-H NC siRNA as negative control group, and MHCC97-H siRNA TGFβ1 and siRNA OPN were experimental groups. Transwel s assay was carried out for co-culture experiments. After 48 hours of co-culture, crystal violet staining was performed for cel counting in three randomly selected fields of vision. Combined with the red fluorescence protein gene, MHCC97-H cel lines in each group were inoculated via the right subaxil ary subcutaneous transplantation to make a tumor model in nude mice. When the tumor volume was up to about 50 mm3, BMSCs were injected into the tumor in the nude mice, and 4 weeks later, fluorescence images were analyzed using software for fluorescence intensity. Frozen hepatocel ular carcinoma tissue sections were taken for 4’,6-diamidino-2-phenylindole staining and fluorescence microscope observation. RESULTS AND CONCLUSION:Cel counting results showed that BMSCs significantly decreased MHCC97-H cel s after gene silencing, and crystal violet staining showed that the migration ability of MHCC97-H cel s was significantly decreased. Tumor volume shown by the fluorescence imaging was significantly reduced after the OPN gene transfection, the fluorescence intensity was lower than that in the other groups, and quantitative results showed that the absorbance value of OPN shRNA cel s decreased significantly compared with other groups, indicating the BMSCs exhibit best interventional effectiveness in OPN-silenced MHCC97-H cel s. Pathological sections showed that BMSCs were mainly distributed in the tumor necrosis area, and the fluorescence expression in the OPN siRNA group was more than that in the TGFβ1 siRNA group and the blank control group, indicating that after OPN gene silencing of MHCC97-H cel s, the distribution of BMSCs in the tumor was increased. To conclude, it is able to reduce the invasive ability of hepatocel ular carcinoma cel s by inhibiting the expression of OPN and TGFβ1 factors, and OPN silencing may be more conductive to BMSCs biotherapy.