Effect of bone marrow mesenchymal stem cells on MHCC97-H cells after transforming growth factor beta1 and osteopontin gene interference

doi:10.3969/j.issn.2095-4344.2017.05.006

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are the focus of research on the proliferation and metastasis of hepatocellular carcinoma cells. By genetic engineering techniques, the hepatocellular carcinoma cells 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 hepatocellular carcinoma cells (MHCC97-H) followed by co-culture with BMSCs and then to observe the change of MHCC97-H cell invasion ability as well as the interventional effect of BMSCs on the animal model of hepatocellular carcinoma tissue MHCC97-H by fluorescence imaging in vivo.
METHODS: MHCC97-H cells were divided into four groups: MHCC97-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. Transwells assay was carried out for co-culture experiments. After 48 hours of co-culture, crystal violet staining was performed for cell counting in three randomly selected fields of vision. Combined with the red fluorescence protein gene, MHCC97-H cell lines in each group were inoculated via the right subaxillary 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 hepatocellular carcinoma tissue sections were taken for 4’,6-diamidino-2-phenylindole staining and fluorescence microscope observation.
RESULTS AND CONCLUSION: Cell counting results showed that BMSCs significantly decreased MHCC97-H cells after gene silencing, and crystal violet staining showed that the migration ability of MHCC97-H cells 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 cells decreased significantly compared with other groups, indicating the BMSCs exhibit best interventional effectiveness in OPN-silenced MHCC97-H cells. 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 cells, the distribution of BMSCs in the tumor was increased. To conclude, it is able to reduce the invasive ability of hepatocellular carcinoma cells by inhibiting the expression of OPN and TGF β1 factors, and OPN silencing may be more conductive to BMSCs biotherapy.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Mesenchymal Stem Cells, Bone Marrow, Liver Neoplasms, Models, Animal

CLC Number:

R394.2

Li Tian-ran, Huang Xiao-bin, Huang Chu-heng, Lu Guang-ming, Li Yan-jun . Effect of bone marrow mesenchymal stem cells on MHCC97-H cells after transforming growth factor beta1 and osteopontin gene interference[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 687-692.

Figures/Tables 1

2.1 实验动物数量分析 24只裸鼠制备皮下移植瘤模型，所有动物肿瘤均接种成功，分为4组进行实验干预。 2.2 Transwells法迁移MHCC97-H细胞计数结果实验中阴性对照组迁移细胞数为(139.67±14.01)个，转化生长因子β1基因沉默组迁移细胞数为(45.67±3.51)个，骨桥蛋白基因沉默组迁移细胞数为(69.67±7.02)个，空白对照组迁移细胞数为(196.00±14.42)个。组间比较见图1。结果显示，各组间比较差异有显著性意义(F=7.461，P < 0.001)，表明经基因沉默后细胞迁移数量明显减少，尤其是骨桥蛋白基因沉默组和转化生长因子β1基因沉默组细胞迁移数量显著减少。 2.3 Transwells法迁移MHCC97-H细胞的病理学染色结果 MHCC97-H细胞结晶紫染色，光镜下观察。结果显示，经基因修饰的MHCC97-H细胞迁移能力明显降低(见图2)。 2.4 骨髓间充质干细胞对MHCC97-H细胞的干预情况荧光成像与阴性对照组比较，骨桥蛋白基因沉默组肿瘤体积明显缩小，荧光亮度降低；而转化生长因子β1基因沉默组肿瘤大小和荧光强度与阴性对照组大致相仿(见图3)。 2.5 Image J软件分析荧光成像数据吸光度(A)定量分析分析骨髓间充质干细胞对经基因干扰肝癌的影响情况，由图4可见，骨桥蛋白基因沉默组A值明显降低，与阴性对照组比较差异有显著性意义(P < 0.05)，表明骨髓间充质干细胞对经骨桥蛋白干扰的高转移潜能肝癌(MHCC97-H)动物模型干预效能最佳。 2.6 骨髓间充质干细胞对高转移潜能肝癌(MHCC97-H)干预情况病理切片荧光成像由镜下观察可见，骨髓间充质干细胞主要分布在肿瘤坏死区附近，骨桥蛋白基因沉默组荧光表达较转化生长因子β1基因沉默组和空白对照组多，表明MHCC97-H细胞经骨桥蛋白基因沉默后，骨髓间充质干细胞在肿瘤中的分布增多(图5)。"

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