【摘要】：目的分析巨噬細(xì)胞RAW264.7的Rac1表達(dá)水平,通過構(gòu)建Rac1載體和特異性siRNA探討其對RAW264.7細(xì)胞生物學(xué)活性的影響。方法經(jīng)反轉(zhuǎn)錄法克隆Rac1的cDNA并構(gòu)建Rac1基因表達(dá)載體,Western blot技術(shù)檢測Rac1的蛋白表達(dá)水平,定量熒光PCR(qRT-PCR)測定Rac1 mRNA,流式細(xì)胞術(shù)分析細(xì)胞表面膜分子表達(dá)的改變,Transwell法觀察細(xì)胞的遷移能力,ELISA法檢測細(xì)胞培養(yǎng)上清中相關(guān)細(xì)胞因子的表達(dá)。結(jié)果經(jīng)測序等手段鑒定表明,Rac1載體構(gòu)建成功,且將Rac1載體轉(zhuǎn)入RAW264.7細(xì)胞可見其mRNA和蛋白表達(dá)均明顯上調(diào)、細(xì)胞遷移能力增強(qiáng),但對RAW264.7細(xì)胞膜表面CD80、CD86和MHC-Ⅱ類分子的表達(dá)沒有明顯影響。然而,Rac1抑制劑及其特異性siRNA卻可上調(diào)上述3種膜分子的表達(dá)。此外,Rac1轉(zhuǎn)染的RAW264.7細(xì)胞分泌的IL-1β水平顯著低于siRNA處理組及Rac1抑制劑組,而IL-6、IL-33、TNF-α的表達(dá)各組之間沒有顯著差異。結(jié)論 Rac1的表達(dá)有助于RAW264.7細(xì)胞的遷移和抑制IL-1β的分泌,而對細(xì)胞的抗原提呈作用無顯著影響。
[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，

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