稳定表达GFP-V12Rac1的NIH3T3细胞系的构建

发布时间：2018-02-28 00:04

本文关键词： GFP-V12Rac1 慢病毒载体 NIH3T3细胞稳定表达细胞迁移　出处：《南京医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： Rac1的活性在运动细胞中往往呈现前端富集,这一定位模式对于细胞运动的实现有着重要作用,但此定位的机制尚未完全阐明,要对此定位的机制展开深入研究,首先需要建立稳定表达GFP-V12Rac1的NIH3T3细胞系。本研究在构建GFP-V12Rac1和GFP的质粒和慢病毒表达载体的基础上,采用慢病毒感染和流式细胞术分选的方法构建稳定表达GFP和GFP-V12Rac1的NIH3T3小鼠成纤维细胞系。我们通过流式细胞术检测稳定表达细胞系的目的基因阳性率和表达水平;通过7-AAD和APC Annexin V双染和流式细胞术检测的方法确认表达GFP-V12Rac1的NIH3T3细胞在血清饥饿条件下的生存能力;通过观察细胞在I型胶原上的铺展和细胞质膜皱褶形成来判断外源表达GFP-V12Rac1融合蛋白是否具备正常功能,通过细胞迁移实验研究细胞的自发运动和趋化运动能力,同时初步检测了运动细胞中GFP-V12Rac1的定位。流式细胞术检测结果显示,稳定表达GFP和GFP-V12Rac1的NIH3T3细胞系在多次传代后仍然保持目的基因的高阳性率,且两个细胞系的平均GFP荧光强度均显著高于背景荧光强度;7-AAD和APC Annexin V双染结果显示,血清饥饿12h,外源GFP-V12Rac1不影响细胞的生存能力;铺展实验和活细胞工作站观测表明,外源GFP-V12Rac1具备促进细胞铺展和细胞质膜皱褶的能力;细胞迁移实验证实,细胞具备趋化运动能力。划痕实验结果提示,划痕引发的细胞运动中,少数细胞出现了GFP-V12Rac1的前端定位。本研究结果表明,慢病毒感染和流式细胞术分选是构建NIH3T3稳定表达细胞系的有效途径,外源表达的GFP-V12Rac1具备正常功能,构建的细胞系可用于对运动细胞中活化的Rac1进行定位观察。
[Abstract]:The activity of Rac1 tends to be preferentially enriched in the motor cells. This localization model plays an important role in the realization of cell movement. However, the mechanism of this localization has not been fully clarified, and the mechanism of this localization should be deeply studied. Firstly, we need to establish a stable NIH3T3 cell line expressing GFP-V12Rac1. Based on the construction of GFP-V12Rac1 and GFP plasmids and lentivirus expression vectors, The fibroblasts of NIH3T3 mice expressing GFP and GFP-V12Rac1 stably were constructed by lentivirus infection and flow cytometry. The positive rate and expression level of target genes in stable expression cell lines were detected by flow cytometry. The viability of NIH3T3 cells expressing GFP-V12Rac1 was confirmed by using 7-AAD and APC Annexin V double staining and flow cytometry. The expression of GFP-V12Rac1 fusion protein was evaluated by observing cell spreading on type I collagen and the formation of cytoplasmic membrane fold. The ability of spontaneous and chemotaxis of cells was studied by cell migration experiment. At the same time, the localization of GFP-V12Rac1 in motor cells was preliminarily detected. The results of flow cytometry showed that the NIH3T3 cell lines stably expressing GFP and GFP-V12Rac1 maintained the high positive rate of the target gene after repeated passages. The average GFP fluorescence intensity of the two cell lines was significantly higher than that of background fluorescence intensity 7-AAD and APC Annexin V. The results showed that exogenous GFP-V12Rac1 did not affect the viability of the cells after 12h of serum starvation. Exogenous GFP-V12Rac1 has the ability to promote cell spreading and cytoplasmic membrane fold. Cell migration experiments have proved that cells have chemotaxis ability. The results of scratch test suggest that in the cell movement induced by scratch, The results of this study indicate that lentivirus infection and flow cytometry are effective ways to construct stable expression cell lines of NIH3T3, and exogenous GFP-V12Rac1 has normal function. The constructed cell line can be used to localize activated Rac1 in motor cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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