STIM2、TRPC3在人脐静脉内皮细胞外钙敏感受体介导的钙内流和NO生成中的作用

Roles of STIM2 and TRPC3 in the CaR mediated Ca^(2+) entry and NO generation in human umbilical vein endothelial cells

目的:研究Ca2+感受蛋白基质相互作用分子2(STIM2)及瞬时型感受器电位3(TRPC3)在人脐静脉内皮细胞(HUVEC)细胞外钙敏感受体(CaR)介导钙内流和NO生成中的作用。方法:1免疫荧光技术检测HUVEC中STIM2和TRPC3的蛋白表达。2利用转染技术将构建的STIM2干扰质粒(STIM2shRNA)和TRPC3干扰质粒(TRPC3shRNA)分别转染入HUVEC,实时定量RT-PCR和Western blot检测STIM2shRNA、TRPC3shRNA转染后的抑制效率。3取2~5代细胞随机分组:实验组(即精胺+Ca2++shSTIM2-002组和精胺+Ca2++shTRPC3-004组),其余两组分别为对照组(Control组,即精胺+Ca2+组),空质粒组(Vehicle组,即精胺+Ca2++Vehicle组),分别将上述4组细胞与CaR激动剂孵育,各组用荧光探针Fura-2/AM、DAF-FM负载方法同步测定[Ca2+]i和NO生成的变化。结果:1免疫荧光技术检测显示STIM2和TRPC3两者均表达于HUVEC胞浆。2实时定量RT-PCR及Western blot检测结果显示:与Control组相比,shSTIM2-002和shTRPC3-004干扰后,STIM2和TRPC3mRNA的表达均明显降低(抑制率分别为88.2%和74.0%,P<0.05);STIM2和TRPC3蛋白的表达亦明显降低(抑制率分别为79.9%和71.8%)(P<0.05)。3[Ca2+]i△ratio值和NO净荧光强度值的测定结果显示:与精胺+Ca2+组的[Ca2+]i、NO净荧光强度值相比,精胺+Ca2++ShSTIM2-002组、精胺+Ca2++ShTRPC3-004组及精胺+Ca2++Vehicle组均无显著差异(均P>0.05)。结论:STIM2和TRPC3均未单独参与CaR介导的Ca2+内流和NO的生成过程。

Objective： To study the roles of stromal interaction molecule 2 （Sq＇IM2） and transient receptor potential canonical 3 （TRPC3） in extracellular Ca^2＋ -sensing receptor （CaR）-induced extracellular Ca^2＋ influx and the production of nitric oxide （NO） in human umbilical vein endothelial cells （HUVEC）. Methods： ①The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. ②The expressions of SLIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmice. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. ③）The second to fifth passage of HUVEC were divided into： STIM2-002 short hairpin RNA （STIM2-002 shRNA ） ＋ spermine ＋ Ca^2＋ group and TRPC3-004 short hairpin RNA （TRPC3-004 shRNA ） ＋ spermine ＋ Ca^2＋ group; control group （spennine ＋ Ca^2＋ group） and vehicle＋ spermine ＋ Ca^2＋ group. The four groups of cells were incubated with CaR agonist spormine, the intraceUular Ca^2＋ concentration （ [ Ca^2＋ ]i） was detected using the fluorescence Ca^2 ＋ indicator Fura-2/AM, and the production of NO was determined by DAF-FM（NO fluorescent probe） of each group in HUVEC. Results： ①Immunofluorescence technique results showed that STIM2 and TRPC3 proteins were present in the cytoplasm of HUVEC. ②The results of transfection comtructed STIM2 and TRPC3 RNA interference plasmice demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRIV3 mRNA levels by 88.2% and 74.0%, respectively （ P 〈 0.05）, simul- taneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively （ P 〈 0.05）. ③Compared with spermine ＋ Ca^2＋ group, the [ Ca^2＋ ]i and the net NO fluorescence intensity of spermine ＋ Ca^2＋ ＋ ShSTIM2-002 group, spennine ＋ Ca^2＋ ＋ ShTRPC3- 004 group and spermine ＋ Ca^2＋ ＋ Vehicle group were not changed （ P 〉 0.05）. Conclusion： STIM2 and TRPC3 do not participate in CaR - mediated Ca^2＋ influx and NO production individually.