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目的:探讨Rab GTP酶是否参与了对细菌脂蛋白(BLP)耐受的骨髓诱导分化巨噬细胞(BMDMs)杀菌能力增强的过程。方法:首先利用real-time PCR比较BLP耐受巨噬细胞和非耐受细胞中Rab5a、Rab5b、Rab7、Rab9、Rab9b、Rab11a、Rab11b、Rab12、Rab32和Rab34的表达变化,筛选出水平升高的Rab7分子;进一步用real-time PCR和Western blot实验证实Rab7的mRNA和蛋白表达是否在BLP耐受细胞感染大肠杆菌后继续升高;接下来用RNAi技术下调BLP耐受巨噬细胞Rab7表达,观察细胞对细菌的吞噬能力及杀灭能力的影响。结果:在检测的10个Rab GTP酶中,Rab7在BLP耐受BMDMs的mRNA水平升高,是非耐受细胞的1.4倍;进一步用real-time PCR和Western blot实验证实Rab7的mRNA和蛋白表达在BLP耐受细胞感染大肠杆菌后,随着时间延长均明显升高;下调Rab7表达不影响BLP耐受巨噬细胞对细菌的吞噬能力,但是显著降低其对细菌的杀灭能力。结论:BLP耐受通过上调巨噬细胞Rab7的表达,在增强巨噬细胞对细菌的杀灭过程中发挥重要作用。
AIM: To investigate whether Rab GTPase is involved in the enhancement of bactericidal activity of bacterial lipoprotein (BLP) -mediated bone marrow-derived macrophages (BMDMs). Methods: The expression of Rab5a, Rab5b, Rab7, Rab9, Rab9b, Rab11a, Rab11b, Rab12, Rab32 and Rab34 in BLP-tolerant macrophages and non-tolerant cells was compared by real- Rab7 molecule was further confirmed by real-time PCR and Western blot to confirm whether Rab7 mRNA and protein expression continued to increase after BLP-resistant cells were infected with E.coli. Next, RNAi technology was used to down-regulate Rab7 expression in BLP-tolerant macrophages Effects of cells on phagocytosis and killing of bacteria. Results: Among the 10 Rab GTPases tested, the mRNA level of Rab7 in BLP-resistant BMDMs was 1.4 times higher than that in non-tolerant cells. The mRNA and protein expression of Rab7 were confirmed by real-time PCR and Western blot BLP-resistant cells infected with Escherichia coli, with the prolonged significantly increased; down-regulation of Rab7 expression does not affect BLP tolerance macrophage phagocytosis of bacteria, but significantly reduced its ability to kill bacteria. Conclusion: BLP tolerance plays an important role in enhancing the killing of macrophages by up-regulating the expression of Rab7 in macrophages.