摘要
目的:探讨携带S1P3 siRNA慢病毒载体对自发性高血压大鼠(SHR)勃起功能的改善作用。方法:5只12周龄健康雄性WKY大鼠为A组,随机将12周龄健康雄性SHR大鼠分组(每组5只):B1组:携带S1P3 siRNA慢病毒转染的SHR;B2组:空慢病毒载体(GFP)转染的SHR;C组:SHR大鼠对照组。B1组阴茎海绵体中部注射20μl携带S1P3 siRNA慢病毒(2×10~8TU/ml),B2组注射GFP 20μl (2×10~8TU/ml),每侧10μl。1周后测各组大鼠ICPmax/MAP值,免疫组化、Western印迹、RT-qPCR检测S1P3、ROCK1、ROCK2、eNOS mRNA及蛋白在各组大鼠阴茎海绵体中的表达。结果:各组大鼠体重、血清T水平无明显差异。A、B1、B2、C组大鼠在0、3、5 V电压刺激下ICPmax/MAP分别为:0V:0.16±0.01、0.15±0.01、0.10±0.00、0.11±0.01,3 V:0.55±0.03、0.55±0.01、0.22±0.01、0.22±0.01,5 V:0.82±0.02、0.79±0.03、0.43±0.01、0.42±0.02,C、B2组ICPmax/MAP较A、B1组显著降低(P<0.05)。免疫组化结果显示,S1P3、ROCK1、ROCK2在A、B1组大鼠阴茎海绵体组织中表达水平与B2、C组相比明显减少(P<0.05),eNOS则明显增加(P<0.05);Western印迹结果显示,与B2、C组相比,A、B1组大鼠阴茎海绵体组织中S1P3、ROCK1、ROCK2蛋白表达显著减低(P<0.05),eNOS显著升高(P<0.05);A、B1、B2、C组S1P3基因相对表达量分别为:0.72±0.04、0.71±0.07、1.00±0.06、1.00±0.10,ROCK1:0.99±0.05、1.08±0.16、1.85±0.44、2.02±0.38,ROCK2:1.00±0.03、1.08±0.16、2.16±0.78、2.46±0.69,eNOS:1.04±0.15、0.81±0.23、0.32±0.08、0.32±0.04,S1P3、ROCK1、ROCK2在A组和B1组中的表达水平与C组、B2组相比明显减少(P<0.05),eNOS明显增加(P<0.05)。结论:携带靶向S1P3 siRNA慢病毒载体抑制阴茎海绵体组织内S1P3基因的表达,并下调RhoA/Rho激酶信号通路,从而改善SHR大鼠勃起功能。
Objective: To investigate the improving effect of S1 P3 silencing on the erectile function of spontaneous hypertension rats(SHR). Methods: Five 12-week-old healthy male WKY rats were included in group A and another 15 12-week-old healthy male SHRs equally randomized into groups B1(intracavernously injected with 20 μl S1 P3 siRNA lentiviral vectors [2 ×10~8TU/ml]), B2(intracavernously injected with 20 μl GFP lentiviral vectors [2 ×10~8TU/ml]), and C(control). At 1 week after transfection, the ratio of the maximum intracavernous pressure/mean arterial pressure(ICPmax/MAP) of the rats was measured, and the expressions of S1 P3, ROCK1, ROCK2 and eNOS in the corpus cavernosal tissue were determined by immunohistochemistry, Western blot and RT-qPCR. Results: There were no statistically significant differences in the body weight and serum T level among the three groups of rats. Under 0 V, 3 V and 5 V electrical stimulation, the ratios of ICPmax/MAP were 0.16 ± 0.01, 0.55 ± 0.03 and 0.82 ± 0.02 in group A, 0.15 ± 0.01, 0.55 ± 0.01 and 0.79 ± 0.03 in group B1, 0.10 ± 0.00, 0.22 ± 0.01 and 0.43 ± 0.01 in group B2, and 0.11 ± 0.01, 0.22 ± 0.01 and 0.42 ± 0.02 in group C, significantly lower in the latter two than in the former two groups(P < 0.05). The protein expressions of S1 P3, ROCK1 and ROCK2 in the corpus cavernosum were remarkably down-regulated while that of eNOS up-regulated in groups A and B1 compared with those in groups B2 and C(P < 0.05). Groups A and B1, in comparison with B2 and C, also showed markedly decreased mRNA expressions of S1 P3(0.72 ± 0.04 and 0.71 ± 0.07 vs 1.00 ± 0.06 and 1.00 ± 0.10, P < 0.05), ROCK1(0.99 ± 0.05 and 1.08 ± 0.16 vs 1.85 ± 0.44 and 2.02 ± 0.38, P < 0.05) and ROCK2(1.00 ± 0.03 and 1.08 ± 0.16 vs 2.16 ± 0.78 and 2.46 ± 0.69, P < 0.05), but increased eNOS(1.04 ± 0.15 and 0.81 ± 0.23 vs 0.32 ± 0.08 and 0.32 ± 0.04, P < 0.05). Conclusion: S1 P3 siRNA can improve the erectile function of SHRs by inhibiting the expression of the S1P3 gene and down-regulating the RhoA/Rho kinase signaling pathway in the corpus cavernosum.
作者
陈建国
姜睿
CHEN Jian-guo;JIANG Rui(Department of Urology,The Affiliated Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China)
出处
《中华男科学杂志》
CAS
CSCD
北大核心
2019年第10期874-882,共9页
National Journal of Andrology
基金
四川省科技厅基金(14JC0803)
四川省教育厅基金(15ZA0164)
四川省人力资源和社会保障厅回国人员科研基金(川人社办发[2016]64号)~~
