新候选肿瘤抑制基因SLC8A2对胶质瘤细胞株U87的功能影响及相关机制研究

发布时间：2018-05-11 15:18

本文选题：SLC8A2 + 胶质瘤　；参考：《苏州大学》2014年硕士论文

【摘要】：第一部分SLC8A2对胶质瘤细胞株U87作用的体内外实验目的：构建过表达基因SLC8A2的U87稳定细胞株，通过体内外实验观察SLC8A2对胶质瘤细胞株U87的功能影响。方法：构建携带有SLC8A2全基因的慢病毒载体Lenti-SLC8A2-IRES-EGFP和阴性对照空病毒载体Lenti-EGFP，转染胶质瘤细胞株U87，筛选获得稳定表达靶基因及报告基因EGFP的胶质瘤细胞U87-SLC8A2（实验组）和仅稳定表达EGFP的胶质瘤细胞U87-NC（阴性对照组），并分别应用荧光定量PCR和Western blot检测SLC8A2在各组细胞中的表达；应用Transwell小室及预铺Matrigel基质胶的Transwell小室检测SLC8A2对U87细胞侵袭迁移能力的影响；应用流式细胞仪技术检测SLC8A2对U87细胞周期的影响；运用CCK-8方法检测SLC8A2对U87细胞增殖的影响；通过裸鼠皮下移植瘤实验检测SLC8A2对U87细胞致瘤性的影响。结果：成功构建过表达SLC8A2的细胞株U87-SLC8A2；SLC8A2显著抑制了U87细胞的侵袭和迁移能力，但对U87细胞的增殖及周期无显著影响；裸鼠皮下移植瘤实验显示，接种U87-SLC8A2细胞的裸鼠全都不致瘤，，而接种空白组与阴性对照组细胞的裸鼠全部致瘤成功。结论：SLC8A2能显著抑制胶质瘤细胞株U87的侵袭迁移能力和裸鼠致瘤性，表明SLC8A2有可能为胶质瘤相关抑癌基因。第二部分SLC8A2抑制胶质瘤细胞株U87侵袭迁移能力的机制研究目的：进一步探索SLC8A2抑制胶质瘤细胞株U87侵袭迁移能力的相关分子机制。方法：应用RT-PCR检测各组细胞中MMP-2、MMP-3、MMP-7、MMP-9、MT1-MMP、uPA及uPAR转录水平的差异，并应用Western blot检测各组细胞中MMP-2蛋白表达水平的差异；运用基质金属蛋白酶明胶酶谱法检测各组细胞分泌至培养基中MMP-2酶活性的变化；应用Western blot检测各组细胞中NF-κB、MAPK及Akt通路中相关蛋白表达水平的差异。结果：与阴性对照组细胞相比，U87-SLC8A2组细胞中MMP-2、MMP-3、MMP-7、MMP-9、MT1-MMP、uPA及uPAR的转录水平及MMP-2蛋白水平被显著下调（P＜0.01），且U87-SLC8A2细胞分泌至培养基中的MMP-2酶活性被显著抑制（P＜0.01）；与阴性对照组细胞相比，U87-SLC8A2组细胞中MMPs和uPA/uPAR系统的上游调控通路NF-κB及ERK1/2活性被显著抑制（P＜0.01）。结论：SLC8A2可能是通过ERK1/2-NF-κB-MMPs/uPA/uPAR信号轴抑制了胶质瘤细胞株U87的侵袭迁移能力。
[Abstract]:Part one: in vitro and in vivo effects of SLC8A2 on glioma cell line U87 Aim: to construct a stable U87 cell line with overexpression gene SLC8A2 and observe the effect of SLC8A2 on the function of U87 glioma cell line in vitro and in vivo. Methods: the lentivirus vector Lenti-SLC8A2-IRES-EGFP carrying the whole SLC8A2 gene and the negative control vector Lenti-EGFPwere constructed and transfected into glioma cell line U87. The glioma cells U87-SLC8A2 (experimental group) and U87-SLC8A2 (experimental group) with stable expression of target gene and reporter gene EGFP were obtained. The expression of SLC8A2 in U87-NCcells (negative control group) was detected by fluorescence quantitative PCR and Western blot, respectively. The effect of SLC8A2 on the invasion and migration of U87 cells was detected by Transwell chamber and Transwell chamber precoated with Matrigel matrix glue, the effect of SLC8A2 on U87 cell cycle was detected by flow cytometry, and the effect of SLC8A2 on the proliferation of U87 cells was detected by CCK-8 method. The effect of SLC8A2 on the tumorigenicity of U87 cells was detected by subcutaneous tumor transplantation in nude mice. Results: the cell line U87-SLC8A2SLC8A2 successfully constructed successfully inhibited the invasion and migration of U87 cells, but had no significant effect on the proliferation and cycle of U87 cells. All the nude mice inoculated with blank group and negative control group were successful in tumorigenesis. Conclusion WSLC8A2 can significantly inhibit the invasion and migration of glioma cell line U87 and the tumorigenicity of nude mice, suggesting that SLC8A2 may be a glioma-associated tumor suppressor gene. The Mechanism of SLC8A2 inhibiting invasion and Migration of Glioma Cell Line U87 Objective: to explore the molecular mechanism of SLC8A2 inhibiting invasion and migration of glioma cell line U87. Methods: RT-PCR was used to detect the transcription level of MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMPUPA and uPAR, and Western blot was used to detect the expression of MMP-2 protein. The activity of MMP-2 was detected by matrix metalloproteinase gelatinase assay and the expression level of NF- 魏 B MMP-2 and related proteins in Akt pathway were detected by Western blot. Results: in U87-SLC8A2 group, the transcription levels of MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMPUPA and MMP-2 protein were significantly down-regulated (P < 0.01), and the activity of MMP-2 enzyme secreted by U87-SLC8A2 cells into culture medium was significantly inhibited (P < 0.01), and compared with the negative control group, the activity of MMP-2 enzyme was significantly inhibited (P < 0.01), while that of MMPU UPA and MMPU UPA protein in U87-SLC8A2 group was significantly decreased than that in negative control group (P < 0.01). In U87-SLC8A2 group, the upstream regulatory pathway NF- 魏 B and ERK1/2 of MMPs and uPA/uPAR system were significantly inhibited (P < 0.01). Conclusion the invasion and migration ability of glioma cell line U87 may be inhibited by the ERK1 / 2-NF- 魏 B-MMPs/uPA/uPAR signal axis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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