RNA干扰HSP27基因表达对膀胱逼尿肌细胞收缩特性的影响  

Effect of RNA interference of heat shock protein 27 expression on contraction function of human detrusor cells in vitro

作　　者：熊智勇[1,2] 周鹏[1] 谢秋波[1] 卢根生[1] 王永权[1] 

机构地区：[1]第三军医大学西南医院全军泌尿外科研究所 [2]中国人民解放军95333部队

出　　处：《第三军医大学学报》2015年第6期500-504,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(81000288;81270845)~~

摘　　要：目的探讨热休克蛋白27(heat shock protein 27,HSP27)低表达对体外培养人膀胱逼尿肌细胞(bladder smooth muscle cells,BSMCs)肌动蛋白骨架结构及其收缩功能的影响。方法构建HSP27基因干扰慢病毒载体作用于BSMCs后,Western blot检测其抑制HSP27表达水平和干扰效率;通过鬼笔环肽荧光染色、Western blot检测BSMCs内F/G-actin比值检测等方法观察,干扰HSP27表达对BSMCs肌动蛋白骨架形态结构和聚合状态的影响;构建离体BSMCs三维培养模型,检测HSP27低表达对膀胱逼尿肌细胞收缩力的影响。结果成功构建HSP27干扰质粒p Green Puro-sh-HSP27,慢病毒载体高效感染BSMCs;与正常对照组比较,HSP27干扰组BSMCs细胞内肌动蛋白骨架结构紊乱和皱缩,检测F/G-actin比值(0.116±0.001)亦较正常BSMCs(8.717±0.500)明显下降(P<0.05);正常BSMCs三维培养模型在80 mmol/L KCl刺激下可检测到的最大收缩力为(8.4±0.6)mg显著大于HSP27干扰组[(3.8±0.2)mg,P<0.05]。结论 HSP27低表达状态可导致BSMCs肌动蛋白结构变化并导致其收缩功能受损。Objective To investigate the effect of low expression of heat shock protein 27 （HSP27） on actin skeleton structure and contraction function of human bladder smooth muscle cells （BSMCs） in vitro. Methods To inhibit the expression of HSP27, 3 pairs of siRNA were designed, inserted into lentiviral vector pGreenPuro and transferred into human BSMCs. Phalloidin staining and F-actin/G-actin ratio detection were used to observe the actin skeleton structure and polymerization status in the cultured BSMCs. The bladder smooth muscle fibers were reconstructed by culturing BSMCs with collagens to evaluate the contractile force. Results Lentiviral vector pGreenPuro-sh-HSP27 was successfully constructed and transferred into the cultured BSMCs effectively. Low-expression of HSP27 caused significant disruption of actin skeleton structure and decrease of F-actin/G-actin ratio （0. 116 ±0.001 ） in BSMCs compared with the normal control （8.717 ±0.500, P 〈 0. 05 ）. The contractile force （ 3. 8±0.2 mg ） of the reconstructed fibers was significantly attenuated by low-expression of HSP27 compared to the normal control （ 8.4 ± 0.6 mg, P 〈 0. 05 ） in the stimulation of 80 mmol/L KC1. Conclusion Low expression of HSP27 can impair contractile force of human BSMCs through altering the actin skeleton structure.

关 键 词：热休克蛋白27 肌动蛋白 逼尿肌细胞 膀胱收缩功能障碍 

分 类 号：R322.62[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]