论文部分内容阅读
目的:建立稳定表达Rab5a及其失活突变体Rab5a N133I巨噬细胞系,并分析Rab5a对LPS刺激的RAW264.7巨噬细胞中i NOS、TNF-α和IL-6表达的影响。方法:将Rab5a及其失活突变载体Rab5a N133I转染RAW264.7细胞,通过G418筛选稳定表达细胞系。通过Real-time PCR鉴定稳定表达细胞系。MTT法分析其生长特性,LPS刺激稳定表达细胞系不同时间,检测i NOS、TNF-α和IL-6的表达水平。结果:转染Rab5a/Rab5a N133I细胞Rab5a mRNA水平显著高于对照(P<0.05)。Rab5a过表达促进RAW264.7细胞增殖,而过表达Rab5a N133I细胞增殖显著慢于对照(P<0.05)。Rab5a过表达后,LPS诱导的RAW264.7细胞中i NOS、TNF-α和IL-6的表达显著增高(P<0.01)。然而,过表达Rab5a N133I对LPS诱导的i NOS、TNF-α和IL-6的表达没有显著影响。结论:Rab5a过表达显著促进了LPS活化的RAW264.7细胞中i NOS、TNF-α和IL-6的表达,这种促进作用依赖于其结合GTP的能力。
OBJECTIVE: To establish Rab5a and its inactivated Rab5a N133I macrophage cell line stably expressing Rab5a and analyze the effect of Rab5a on the expression of iNOS, TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages. METHODS: Rab5a and its inactivated mutant Rab5a N133I were transfected into RAW264.7 cells, and the stable cell lines were screened by G418. Stable expression cell lines were identified by Real-time PCR. MTT assay was used to analyze the growth characteristics. LPS stimulation of stable expression of cell lines at different times, detection of iNOS, TNF-α and IL-6 expression levels. Results: Rab5a mRNA expression in Rab5a / Rab5a N133I cells was significantly higher than that in control (P <0.05). Rab5a overexpression promoted the proliferation of RAW264.7 cells, while the over-expression of Rab5a N133I cells was significantly slower than the control (P <0.05). After overexpression of Rab5a, the expression of iNOS, TNF-α and IL-6 in LPS-induced RAW264.7 cells were significantly increased (P <0.01). However, overexpression of Rab5a N133I had no significant effect on LPS-induced iNOS, TNF-α and IL-6 expression. Conclusion: Overexpression of Rab5a can significantly promote the expression of iNOS, TNF-α and IL-6 in LPS-activated RAW264.7 cells. The promoting effect depends on its ability to bind to GTP.