膀胱平滑肌细胞乙酰胆碱M_3受体干扰RNA慢病毒载体的构建

Construction of small interference RNA lentiviral Vector for M_3 muscarinic acetylcholine receptor gene of bladder smooth muscle cells

目的构建膀胱平滑肌细胞(bladder smooth muscle cells,BSMCs)M_3型受体干扰RNA慢病毒载体。方法合成4条M_3受体干扰序列,构建GV118-sh-M_3慢病毒载体,测序验证序列。将GV118-sh-M_3慢病毒载体与质粒p Helper1.0、p Helper 2.0三个载体共转染293T细胞,收集病毒颗粒并用荧光法检测4组病毒滴度。体外原代培养BSMCs,4组GV118-sh-M_3慢病毒(KD1、KD2、KD3和KD4)转染BSMCs,观察绿色荧光蛋白表达并通过RT-PCR、Western blotting法检测各组干扰效率。结果成功构建4个GV118-sh-M_3慢病毒载体,测序结果符合设计序列。4组GV118-sh-M_3慢病毒滴度分别为2×10~8、6×10~8、5×10~8和5×10~8TU/m L。分别转染BSMCs后,KD1组绿色荧光蛋白表达量最高且干扰效率最高。结论成功包装并筛选了GV118-sh-M_3慢病毒载体,为后续进一步研究干扰M_3受体在神经源性膀胱中的作用奠定了基础。

Objective To construct M3 acetylcholine receptor gene interference RNA lentiviral vectors for bladder smooth muscle cells. Methods Four pairs of small interference RNA（ siRNA） sequence for M3 muscarinic acetylcholine receptor gene were synthesized to construct GV118-sh-M3 lentivirus vectors. DNA sequencing was used to identify the sequence. GV118-sh-M3 lentivirus vectors were cotransfected with pHelper 1. 0 and pHelper 2. 0 vectors into 293 T cells. We harvested the viral particles and assayed the titer of lentivirus by fluorescence. BSMCs were primary cultured in vitro and transfected by lentivirus of four groups（ KD1,KD2,KD 3 and KD4）.Fluorescence expression was observed and the expression levels of M3 gene in BSMCs of groups were assessed by RT-PCR and Western blotting was used to calculate interference efficiency. Results Four GV118-sh-M3 lentivirus vectors were successfully constructed and the sequencing results were consistent with sequence of the design. Viral particles of groups were harvested with titer of 2 ×10^8,6 ×10^8,5 ×10^8 and 5 ×10^8 TU/mL,respectively. KD1 group was with the highest fluorescent expression and interference efficiency. Conclusion The GV118-sh-M3 interference lentivirus vector was successfully constructed and selected,which may lay a foundation for further study on the study of M3 muscarinic acetylcholine receptor gene interference in treating neurogenic bladder.