两种糖基转移酶（ppGalNAcT2及β3GnT8）与人脑胶质瘤细胞株U251相关性探索研究

发布时间：2018-08-18 10:29

【摘要】： 目的:探讨两种糖基转移酶(ppGalNAcT2及β3GnT8)对胶质瘤细胞U251侵袭转移的影响。方法:(1)将ppGalNAcT2及β3GnT8的真核表达正义载体及空载体、RNA干扰载体及其对照载体通过脂质体转染入胶质瘤细胞U251,并通过G418筛选出稳定细胞株。(2)荧光显微镜观察转入荧光载体的细胞株,RT-PCR检测ppGalNAcT2的表达;流式细胞术及MTT检测ppGalNAcT2对细胞周期及增殖的影响;划痕修复实验检测各组细胞的迁移能力差异;采用RT-PCR方法检测各组细胞MMP-2、TIMP-2 mRNA水平表达变化。(3)通过激光共聚焦显微镜观察检测GFP蛋白和β3GnT8的蛋白表达;RT-PCR及western blotting方法检测细胞β3GnT8 mRNA及蛋白水平表达变化;集落形成实验检测β3GnT8对单细胞形成集落能力的影响;划痕修复实验及Boyden小室检测细胞迁移及侵袭能力差异;采用RT-PCR及western blotting方法检测各组细胞MMP-2、TIMP-2 mRNA及蛋白水平表达变化。结果:(1)通过荧光显微镜观察、RT-PCR、western blotting及免疫荧光检测,载体成功转入细胞,得到稳定转染细胞株;(2)流式细胞术及MTT实验检测发现ppGalNAc-T2与细胞的周期时相及增殖能力间不存在显著相关性;划痕结果显示,ppGalNAc-T2上调组细胞迁移能力随ppGalNAc-T2的上调而减弱,干扰组划痕修复较快,其他对照组相近;RT-PCR检测发现随着ppGalNAc-T2的增高,MMP-2降低,而TIMP-2升高;(3)集落形成实验显示:与对照组相比,β3GnT8上调组单个细胞集落形成能力强,干扰组弱;划痕结果显示,β3GnT8上调组细胞迁移能力增强,而干扰组细胞划痕修复较慢。与划痕结果相对应的,通过Boyden小室实验发现,与对照组相比,β3GnT8上调组细胞的穿膜能力增强,细胞侵袭能力较强,而下调组穿膜率较对照组都低,侵袭能力弱;RT-PCR及western blotting检测发现随着β3GnT8表达的增高,MMP-2在mRNA水平及蛋白水平也相应升高,而TIMP-2表达水平未有显著改变。结论:(1)成功构建ppGalNAc-T2及β3GnT8上调及下调细胞株。(2)通过流式细胞术及MTT实验检测发现,ppGalNAc-T2与细胞周期及增殖能力间无显著相关性;划痕实验显示ppGalNAc-T2抑制细胞的划痕修复;进一步通过RT-PCR检测发现,ppGalNAc-T2与MMP-2呈负相关,与TIMP-2呈正相关。(3)细胞集落形成实验显示β3GnT8能促进集落形成;划痕修复及Boyden侵袭小室实验发现,β3GnT8促进细胞的划痕修复,同时促进细胞侵袭;进一步通过RT-PCR及western blotting检测显示,β3GnT8与MMP-2呈正相关,而与TIMP-2间无统计学意义。综上所述,我们推测这两种糖基转移酶ppGalNAc-T2及β3GnT8可能与胶质细胞的侵袭转移密切相关。
[Abstract]:Aim: to investigate the effects of two glycosyltransferases (ppGalNAcT2 and 尾 3GnT8) on the invasion and metastasis of glioma cell U251. Methods: (1) ppGalNAcT2 and 尾 3GnT8 were transfected into glioma cell line U251 by liposome, and stable cell lines were screened by G418. (2) fluorescence microscope was used to observe the transfer into fluorescent carrier. The expression of ppGalNAcT2 was detected by RT-PCR. Flow cytometry and MTT were used to detect the effect of ppGalNAcT2 on cell cycle and proliferation. The expression of MMP-2 and TIMP-2 mRNA was detected by RT-PCR method. (3) the expression of GFP protein and 尾 3GnT8 protein was detected by laser confocal microscopy, and the expression of 尾 3GnT8 mRNA and protein was detected by western blotting method. Colony formation assay was used to detect the effect of 尾 3GnT8 on the colony forming ability of single cell, the difference of cell migration and invasion ability was detected by scratch repair test and Boyden chamber, and the expression of MMP-2TIMP-2 mRNA and protein were detected by RT-PCR and western blotting methods. Results: (1) RT-PCR blotting and immunofluorescence were observed by fluorescence microscope, and the vector was successfully transferred into cells. (2) flow cytometry and MTT assay showed that there was no significant correlation between ppGalNAc-T2 and cell cycle phase and proliferative ability, and the result of scratch showed that the migration ability of PG-GalNAc-T2 up-regulated cells decreased with the up-regulation of ppGalNAc-T2. The results of RT-PCR showed that MMP-2 decreased with the increase of ppGalNAc-T2, while TIMP-2 increased. (3) compared with the control group, 尾 3GnT8 upregulated the ability of single cell colony formation was stronger than that of the control group, but the interference group was weak. The results of scratch showed that the cell migration ability of 尾 3GnT8 upregulated group was enhanced, while that of interference group was slower. In contrast to the scratch results, Boyden chamber experiments showed that the cells in 尾 3GnT8 upregulated group had stronger membrane penetration and more invasive ability than those in the control group, but the membrane penetration rate in the down-regulated group was lower than that in the control group. RT-PCR and western blotting analysis showed that the expression of MMP-2 increased with the increase of 尾 3GnT8 expression in mRNA and protein level, but the expression of TIMP-2 did not change significantly. Conclusion: (1) ppGalNAc-T2 and 尾 3GnT8 up-regulated and down-regulated cell lines were successfully constructed. (2) ppGalNAc-T2 GalNAc-T2 was not significantly correlated with cell cycle and proliferation ability by flow cytometry and MTT assay, and scratch test showed that ppGalNAc-T2 inhibited cell scratch repair. Furthermore, the results of RT-PCR detection showed that MMP-2 was negatively correlated with MMP-2, and positively correlated with TIMP-2. (3) Cell colony formation assay showed that 尾 3GnT8 could promote colony formation, and that 尾 3GnT8 promoted cell scratch repair and Boyden invasion chamber experiment, and 尾 3GnT8 promoted cell scratch repair. Furthermore, RT-PCR and western blotting showed that 尾 3GnT8 was positively correlated with MMP-2, but had no statistical significance with TIMP-2. In conclusion, we speculate that these two glycosyltransferases ppGalNAc-T2 and 尾 3GnT8 may be closely related to the invasion and metastasis of glial cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.41;R341

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