Rac1对巨噬细胞RAW264.7细胞生物学作用的影响
发布时间：2018-12-26 19:01
【摘要】：目的分析巨噬细胞RAW264.7的Rac1表达水平,通过构建Rac1载体和特异性siRNA探讨其对RAW264.7细胞生物学活性的影响。方法经反转录法克隆Rac1的cDNA并构建Rac1基因表达载体,Western blot技术检测Rac1的蛋白表达水平,定量荧光PCR(qRT-PCR)测定Rac1 mRNA,流式细胞术分析细胞表面膜分子表达的改变,Transwell法观察细胞的迁移能力,ELISA法检测细胞培养上清中相关细胞因子的表达。结果经测序等手段鉴定表明,Rac1载体构建成功,且将Rac1载体转入RAW264.7细胞可见其mRNA和蛋白表达均明显上调、细胞迁移能力增强,但对RAW264.7细胞膜表面CD80、CD86和MHC-Ⅱ类分子的表达没有明显影响。然而,Rac1抑制剂及其特异性siRNA却可上调上述3种膜分子的表达。此外,Rac1转染的RAW264.7细胞分泌的IL-1β水平显著低于siRNA处理组及Rac1抑制剂组,而IL-6、IL-33、TNF-α的表达各组之间没有显著差异。结论 Rac1的表达有助于RAW264.7细胞的迁移和抑制IL-1β的分泌,而对细胞的抗原提呈作用无显著影响。
[Abstract]:Objective to analyze the expression of RAW264.7 Rac1 in macrophages and to investigate the effect of Rac1 vector and specific siRNA on the biological activity of RAW264.7 cells. Methods the cDNA of Rac1 was cloned by reverse transcription and the expression vector of Rac1 was constructed to detect the expression of Rac1 protein by, Western blot. The changes of membrane molecule expression on the cell surface were analyzed by Rac1 mRNA, flow cytometry with quantitative fluorescent PCR (qRT-PCR). The migration ability of cells was observed by Transwell and the expression of cytokines in supernatant of cell culture was detected by ELISA method. Results the results of sequencing showed that the Rac1 vector was successfully constructed, and the expression of mRNA and protein was up-regulated and the cell migration ability was enhanced by transferring Rac1 vector into RAW264.7 cells. However, the expression of CD80, on the surface of RAW264.7 cell membrane was enhanced. The expression of CD86 and MHC- class 鈪，
本文编号：2392553
[Abstract]:Objective to analyze the expression of RAW264.7 Rac1 in macrophages and to investigate the effect of Rac1 vector and specific siRNA on the biological activity of RAW264.7 cells. Methods the cDNA of Rac1 was cloned by reverse transcription and the expression vector of Rac1 was constructed to detect the expression of Rac1 protein by, Western blot. The changes of membrane molecule expression on the cell surface were analyzed by Rac1 mRNA, flow cytometry with quantitative fluorescent PCR (qRT-PCR). The migration ability of cells was observed by Transwell and the expression of cytokines in supernatant of cell culture was detected by ELISA method. Results the results of sequencing showed that the Rac1 vector was successfully constructed, and the expression of mRNA and protein was up-regulated and the cell migration ability was enhanced by transferring Rac1 vector into RAW264.7 cells. However, the expression of CD80, on the surface of RAW264.7 cell membrane was enhanced. The expression of CD86 and MHC- class 鈪，
本文编号：2392553
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