TGF-β1基因转染BM-MSC对肝癌转移潜能影响的实验研究  

Experimental study of the impact of TGF-β1 transfected BM-MSC on the metastatic potential of MHCC97-H

作　　者：李天然[1] 周军[2] 杜湘珂[3] 魏正茂[3] 霍天龙[3] 

机构地区：[1]南京军区福州总医院九五临床部放射科,莆田351100 [2]南京军区福州总医院内科,莆田351100 [3]北京大学人民医院放射科

出　　处：《功能与分子医学影像学（电子版）》2014年第2期16-20,共5页Functional and Molecular Medical Imaging(Electronic Edition)

基　　金：国家自然基金项目资助(81271607)

摘　　要：目的观察经TGF-β1基因修饰的人骨髓间充质干细胞(hMSC)对高转移潜能肝癌细胞(MHCC97-H)增殖能力及侵袭能力的影响。方法 1利用Gateway Technology法构建TGF-β1慢病毒载体,eGFP为报告基因,转染hMSC,病毒滴度测定转染率;2 Transwell法检测hMSC与肝癌细胞共培养实验,下室加入500μl含20﹪FBS的培养基和1×104个基因修饰前后hMSC,上室加入100μl浓度为1×106个/ml的人高转移肝癌细胞(MHCC97-H)悬液,小室置于37℃,5﹪CO2培养箱内培养48 h,结晶紫染色,细胞计数,检测MHCC97-H细胞侵袭能力的变化;3 CCK-8法检测与hMSC共培养前后MHCC97-H增殖能力的变化;4 PCR法检测转基因hMSC与MHCC97-H细胞共培养前后转移相关因子基因α-V,肿瘤生长因子TGF-β1和肿瘤凋亡因子PDCD4等基因表达。结果 (1)hMSC TGF-β1过表达组细胞的转导率达90﹪以上。(2)MHCC97-H细胞增殖能力检测结果 :1 MHCC97-H与hMSC细胞共培养后,MHCC97-H增殖能力增强;2 hMSC TGF-β1基因过表达组对MHCC97-H细胞增殖具有抑制作用。(3)MHCC97-H细胞侵袭能力检测结果 :1 MHCC97-H与hMSC细胞共培养后,侵袭能力增强;2 hMSC TGF-β1基因过表达组对MHCC97-H细胞侵袭能力具有明显的抑制作用。(4)Q-PCR检测结果:1hMSC基因转染组TGF-β1表达明显增高;2 hMSC基因转染共培养组MHCC97-H细胞TGF-β1基因表达明显低于对照组;3 hMSC基因转染共培养组MHCC97-H细胞α5基因表达降低;4 hMSC基因转染共培养组MHCC97-H细胞PDCD4基因表达降低。结论 TGF-β1基因hMSC过表达组细胞MHCC97-H细胞的转导率达90﹪以上;转基因hMSC具有抑制MHCC97-H细胞增殖和细胞侵袭的作用。Objective The paper is to study the impact of TGF-β1 gene-modified bone marrow mesenchymal stem cells（BM-MSCs） on the proliferation and invasion potential of highly metastatic HCC cells（MHCC97-H）. Methods Using the Gateway Technology TGF-β1 lentiviral vector was built with eGFP as the reporter gene, and was used to transfect BM-MSC. Transwell assay was used to evalute BM-MSC and HCC cells co-culture experiments; in the lower chamber 500 μl medium containing 20 ﹪ FBS and 1 × 10^4 the genetically modified BM-MSC 100 μl was added, and in the upper chamber concentration of 1 × 10^6 cells / ml metastatic liver cancer cells（MHCC97-H） was added. The suspension chamber was set into a 37 °C, 5 ﹪ CO2 incubator for 48 h, then crystal violet staining and cell counting was performed to evaluate the invasion potential of MHCC97-H cell. CCK-8 assay was used to detect the change of the proliferative capacity before and after the MHCC97-H coculture test. PCR assay was performed to detect the difference in the relevant factor gene alpha-V, tumor growth factor TGF-β1 and tumor apoptotic factors PDCD4 gene expression before and after the transgenic BM-MSC and MHCC97-H cells were co-cultured. Results The transduced rate of TGF-β1 expressing BM-MSC cells was more than 90 ﹪. MHCC97-H cell proliferation test results： The proliferation potential of MHCC97-H co-cultured with BM-MSC cell increased. TGF-β1 gene overexpressing BM-MSC group inhibited cell proliferation of MHCC97-H. MHCC97-H cell invasion ability test results： MHCC97-H co-cultured with BM-MSC cells invasion ability increased; BM-MSC of TGF-β1 overexpressing group was significantly inhibited in the cell invasion ability of MHCC97-H. Q-PCR test results： 1 BM-MSC gene transfected group was significantly higher in the TGF-β1 expression than control group. 2 TGF-β1 gene expression of the MHCC97-H cells in the co-cultured group with the gene transfected BM-MSC was significantly lower than the control group. 3 α-V gene expression of MHCC97-H in t

关 键 词：髓间充质干细胞 高转移潜能肝癌细胞 肿瘤生长因子 细胞程序化凋亡因子4 整合素 

分 类 号：R735.7[医药卫生—肿瘤] R341[医药卫生—临床医学]