骨髓间充质干细胞对经转化生长因子β1、骨桥蛋白基因沉默肝癌细胞MHCC97-H的影响

发布时间：2018-06-20 05:44

本文选题：干细胞 + 间质干细胞　；参考：《中国组织工程研究》2017年05期

【摘要】：背景:骨髓间充质干细胞对肝癌增殖能力和转移能力的影响是研究的热点,可通过基因工程技术对肝癌细胞进行改造,使其降低生物活性因子的表达,从而寻找合适的干预靶点,提高骨髓间充质干细胞的干预效能。目的:经转化生长因子β1、骨桥蛋白基因沉默的高转移潜能肝癌细胞(MHCC97-H)与骨髓间充质干细胞共培养,观察MHCC97-H细胞侵袭能力的变化,并通过动物模型荧光成像观察骨髓间充质干细胞对MHCC97-H肝癌组织动物模型的干预作用。方法:(1)将MHCC97-H细胞细胞分为4组,MHCC97-H设为空白对照组,MHCC97-H基因沉默转染阴性对照为阴性对照组,MHCC97-H-转化生长因子β1为转化生长因子β1基因沉默组,MHCC97-H-骨桥蛋白为骨桥蛋白基因沉默组;(2)Transwells法进行各组细胞与骨髓间充质干细胞共培养实验,48 h后结晶紫染色,随机取3个视野,计数;(3)结合有红色荧光蛋白基因的各组MHCC97-H细胞株,分别自裸鼠右侧腋下接种复制皮下移植瘤模型。肿瘤体积到50 mm3开始瘤内注射骨髓间充质干细胞,4周后进行荧光强度分析;肝癌组织冰冻切片二脒基苯基吲哚细胞核染色后行荧光显微镜观察。结果与结论:(1)细胞计数结果表明,骨髓间充质干细胞使经基因沉默后的MHCC97-H细胞迁移数量明显减少;结晶紫染色病理图片显示,经基因修饰的MHCC97-H细胞迁移能力明显降低;(2)体内荧光成像结果显示,骨桥蛋白基因沉默组肿瘤体积明显缩小,荧光亮度低于其他组别,定量结果显示骨桥蛋白基因沉默组吸光度值明显降低,表明骨髓间充质干细胞对经骨桥蛋白干扰的MHCC97-H高转移潜能肝癌动物模型干预效能最佳;(3)病理切片荧光观察结果显示,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;(4)结果提示,通过抑制骨桥蛋白和转化生长因子β1两种因子的表达可以降低肝癌细胞侵袭能力,经骨桥蛋白沉默可能更有利于基于骨髓间充质干细胞的生物治疗。
[Abstract]:Background: the effect of bone marrow mesenchymal stem cells (BMSCs) on the proliferation and metastasis of hepatocellular carcinoma (HCC) is a hot topic. In order to find appropriate intervention targets, improve the intervention effectiveness of bone marrow mesenchymal stem cells. Objective: to observe the change of invasion ability of MHCC97-H cells by co-culture of MHCC97-Hand bone marrow mesenchymal stem cells (BMSCs) by transforming growth factor 尾 1, osteopontin gene silencing (TGF- 尾 1) and osteopontin gene silencing (TGF- 尾 1). The effects of bone marrow mesenchymal stem cells (BMSCs) on MHCC97-H liver cancer tissue were observed by fluorescence imaging. Methods MHCC97-H cells were divided into four groups: MHCC97-H as blank control group, MHCC97-H gene silencing as negative control group, MHCC97-H- H- transforming growth factor 尾 _ 1 as transforming growth factor 尾 _ 1 gene silencing group, MHCC97-H- osteopontin as osteopontin group. In the silencing group, the cells were co-cultured with bone marrow mesenchymal stem cells (BMSCs) for 48 h and then stained with crystal violet. MHCC97-H cell lines with red fluorescent protein gene were randomly selected from 3 fields, and subaxillary inoculation of MHCC97-H cell lines with red fluorescent protein gene was carried out to establish subcutaneous transplanted tumor model from right axillary of nude mice. The fluorescence intensity of bone marrow mesenchymal stem cells was analyzed 4 weeks after intratumoral injection of bone marrow mesenchymal stem cells (BMSCs) from 50 mm3 to 50 mm3, and the staining of diamidine phenyl indole nuclei in frozen sections of liver cancer was observed by fluorescence microscope. Results and conclusion the cell count showed that bone marrow mesenchymal stem cells significantly decreased the migration of MHCC97-H cells after gene silencing. The results of fluorescence imaging showed that the tumor volume of osteopontin gene silencing group was significantly smaller and the fluorescence brightness was lower than that of other groups. The quantitative results showed that the absorbance value of osteopontin gene silencing group was significantly decreased, which indicated that bone marrow mesenchymal stem cells had the best intervention effect on MHCC97-H high metastatic potential liver cancer model with osteopontin interference. Bone marrow mesenchymal stem cells (BMSCs) mainly distributed near the necrotic area of tumor. The fluorescence expression of osteopontin gene silencing group was more than that of transforming growth factor 尾 1 gene silencing group and blank control group. The results indicated that the distribution of bone marrow mesenchymal stem cells (BMSCs) increased after osteopontin gene silencing in MHCC97-H cells. The results suggested that the expression of osteopontin and transforming growth factor 尾 1 could decrease the invasion ability of hepatocellular carcinoma cells by inhibiting the expression of osteopontin and transforming growth factor 尾 1. Osteopontin silencing may be more beneficial to the biotherapy based on bone marrow mesenchymal stem cells.
【作者单位】：解放军总医院第一附属医院放射科;解放军第九五医院放射科;解放军南京总医院医学影像科;
【基金】：国家自然科学基金资助项目(81271607) 南京军区医药卫生重点资助项目(11Z035) 中国博士后基金(2015M572810)~~
【分类号】：R735.7

【相似文献】