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目的：利用慢病毒载体筛选人脑小胶质细胞(HM1900)清道夫受体SRB1基因敲减稳定细胞系，检测该细胞系对AD 致病蛋白Aβ的吞噬水平变化。方法：采用反转录PCR确认人脑小胶质细胞(HM1900)SRB1(GeneID：949)基因表达情况，利用软件设计三组不同序列的针对人SRB1 基因的慢病毒shRNA干扰载体，包装为慢病毒感染HM1900 细胞，实时荧光定量PCR 检测各慢病毒干扰载体的靶基因干扰效率。选用干扰效果最佳的RNA 干扰慢病毒筛选并获得SRB1 基因敲减细胞系，稳定传代后用 realtime-PCR 检测SRB1 受体表达下调情况。通过蛋白内吞实验测定基因敲减后该细胞系对病理蛋白Aβ的吞噬能力，与正常小胶质细胞进行比较。结果：利用筛选出的干扰载体完成对HM1900细胞系的SRB1 基因敲减，荧光定量PCR检测显示SRB1 基因在靶细胞HM1900 中表达抑制率达83.7%。蛋白内吞实验显示该基因敲减细胞系对病理蛋白Aβ的吞噬能力下降到对照组的 57%(P＜0.05)。结论：通过慢病毒载体成功建立SRB1基因稳定敲减的人脑小胶质细胞系，SRB1受体参与了小胶质细胞对病理蛋白A茁的吞噬及清除过程。

英文摘要:

Objective:To establish SRB1 gene-knockdown microglia cell line and investigate the function of microglia SRB1 receptor in the phagocytosis of Aβduring the process of Alzheimer's disease (AD).Methods:The expression of SRB1 gene in human microglia 1900 was determined by reverse transcription-PCR, three lentiviral shRNA targeting SRB1 were designed and the best interferring shRNA sequence was selected. The resulting lentiviral construct was used to knockdown SRB1 gene in human microglia. Intracellular Aβ42 Quantification was performed to test the phagocytosis function of the SRB1 knockdown microglia cell line.Results:The lentiviral particles were successfully prepared and showed effective infection capacity and knockdown effects. SRB1 gene silencing was performed in Human microglia1900 with a knock-down efficiency of 83.7%. The phagocytosis function was significantly decreased to 57%in SRB1 gene-silencing microglia cell line.Conclusion:The SRB1 knock-down cell line was established. The function of SRB1 gene was confirmed by oligomer Aβ(oAβ) phagocytosis test. These findings have identified a significant role of SRB1 in Aβclearance.

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