摘要
该文主要研究慢病毒介导的T细胞免疫球蛋白黏液素3(T-cell immunoglobulin mucin-3,Tim-3)对宫颈癌细胞的促肿瘤作用及其相关作用机制。首先,采用PCR方法获取Tim-3片段,将其克隆入pCDH载体中,并通过酶切和测序进行鉴定;将三个表达载体,即重组阳性表达载体(pCDH-Tim-3)、空载体(pCDH-NC)和绿色荧光蛋白载体(pCDH-GFP),分别与psPAX2和pMD2.G共转染至293T细胞中,最后形成相应的慢病毒组。然后,将三组慢病毒分别感染HeLa细胞,获得稳定细胞株(HeLa-Tim-3、HeLa-NC、HeLa-GFP),其中设置HeLa-NC为空载病毒阴性对照组;最后,通过观察HeLa-GFP组,初步获取慢病毒感染效率;运用流式细胞术、Western blot和细胞免疫荧光检测Tim-3的相对表达水平;细胞划痕和Transwell检测细胞的迁移及侵袭能力;Western blot检测上皮间质转化(epithelial-to-mesenchymal,EMT)相关蛋白(E-cadherin、N-cadherin、Snail)的表达变化;流式细胞术检测细胞凋亡率变化。该文成功构建了pCDH-Tim-3慢病毒表达载体并将其包装成慢病毒,获得对应的稳定细胞株;在HeLa-GFP中可以观察到大量的绿色荧光;与HeLa-NC组相比,Western blot、流式细胞术和细胞免疫荧光检测结果均表明,HeLa-Tim-3组的Tim-3蛋白表达水平显著升高(P<0.05);细胞划痕实验和Transwell检测结果均表明,HeLa-Tim-3组的迁移侵袭能力明显提高(P<0.01);另外,Western blot检测结果表明,与HeLa-NC组相比,HeLa-Tim-3组N-cadherin和Snail显著增多但E-cadherin明显减少(P<0.05),而流式细胞术检测结果表明,HeLa-Tim-3组凋亡率显著减少(P<0.001)。因此,高表达Tim-3宫颈癌细胞可以通过EMT转化和抑制细胞凋亡等相关作用机制来加强肿瘤的恶性进展。
The aim of this study was to investigate effects of Tim-3(T-cell immunoglobulin mucin-3)mediated by lentivirus on cervical cancer cells and its related mechanism.Tim-3 fragments were amplified by PCR and cloned into a pCDH vector for enzyme digestion and sequencing.Firstly,the recombinant positive expression vectors pCDH-Tim-3/pCDH-NC/pCDH-GFP,psPAX2,and pMD2.G were co-transfected into 293T cells,forming three types of lentivirus groups.Next,three groups of lentiviruses were respectively infected with HeLa cells to obtain stable cell lines(HeLa-Tim-3,HeLa-NC,HeLa-GFP).HeLa-NC was set as the control group.Finally,the efficiency of infecting lentivirus was observed in HeLa-GFP group preliminarily.The relative expression levels of Tim-3 were detected by flow cytometry,Western blot,and cellular immunofluorescence.Abilities including migration and invasion were detected by cell scratch and Transwell.Expression of EMT(epithelial-to-mesenchymal)related to proteins(E-cadherin,N-cadherin,Snail)was detected by Western blot.The apoptosis rate was detected by flow cytometry assay.We successfully constructed a lentivirus plasmid containing Tim-3 and then packaged it as lentivirus particles.The stable cell lines of HeLa-Tim-3,HeLa-NC,and HeLa-GFP were obtained,separately.Comparing with HeLa-NC group,the expression of Tim-3 in HeLa cells was increased after infection in the HeLa-Tim-3 group(P<0.05).The ability of invasion and metastasis were significantly enhanced in HeLa cells with high expression of Tim-3(P<0.01).The observed effect was associated with downregulation of E-cadherin and upregulation of N-cadherin and snail(P<0.05).Meanwhile,the apoptosis rate was also markedly inhibited(P<0.001).Our findings suggest that the expression of Tim-3 in cervical cancer may facilitate the tumor invasion and metastasis by promoting EMT transformation and inhibiting apoptosis.
作者
余师师
张秉强
张炳兰
YU Shishi;ZHANG Bingqiang;ZHANG Binglan(Department of Gastroenterology,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2020年第4期557-565,共9页
Chinese Journal of Cell Biology
基金
国家青年科学基金(批准号:81703057/H1611)资助的课题。
