【摘要】：目的:研究Ca2+通道蛋白Orai3在人臍靜脈內(nèi)皮細(xì)胞(HUVECs)外鈣敏感受體(CaR)介導(dǎo)的鈣內(nèi)流和一氧化氮(NO)生成中的作用和機(jī)制。方法:(1)利用轉(zhuǎn)染技術(shù)將構(gòu)建的Orai3干擾質(zhì)粒(Orai3 shRNA)轉(zhuǎn)染HUVECs,采用激光共聚焦顯微鏡觀察細(xì)胞轉(zhuǎn)染效果,實(shí)時定量RT-PCR和Western blotting檢測Orai3 shRNA轉(zhuǎn)染后Orai3 mRNA和蛋白的表達(dá)以及抑制效率。(2)取第2~5代細(xì)胞隨機(jī)分組:特異性質(zhì)粒轉(zhuǎn)染組即實(shí)驗(yàn)組(Orai3shRNA組)、未轉(zhuǎn)染組即空白對照組(control組)和空質(zhì)粒組(vehicle組),將上述3組細(xì)胞分別與CaR激動劑精胺[鈣池操縱性鈣通道(SOC)和受體操縱性鈣通道(ROC)均激活]、ROC模擬劑12-O-十四烷酰佛波醇-13-乙酸酯(TPA)+CaR負(fù)性變構(gòu)調(diào)節(jié)劑Calhex 231(激活ROC、阻斷SOC)、蛋白激酶C(PKC)抑制劑Ro31-8220及經(jīng)典型PKCs和PKCμ抑制劑Go6967(阻斷ROC、激活SOC)孵育,用熒光探針Fura-2/AM和DAF-FM負(fù)載方法同步檢測[Ca2+]i和NO生成的變化。結(jié)果:(1)實(shí)時定量RT-PCR及Western blotting檢測結(jié)果顯示:與control組相比較,shOrai3-77干擾后,Orai3 mRNA的表達(dá)明顯降低(抑制率為84.5%,P0.05),Orai3蛋白的表達(dá)亦明顯降低(抑制率為70.68%,P0.05)。(2)[Ca2+]iΔratio值和NO凈熒光強(qiáng)度值的測定結(jié)果顯示:與control組及vehicle組相比,在4種不同處理因素作用下,Orai3 shRNA轉(zhuǎn)染組中[Ca2+]iΔratio值和NO凈熒光強(qiáng)度值均明顯降低(P0.05),而vehicle組無顯著差異(P0.05)。結(jié)論:在HUVECs中Orai3參與了CaR經(jīng)SOC和ROC激活介導(dǎo)的鈣內(nèi)流和NO生成。
[Abstract]:Aim: to investigate the role and mechanism of Ca2 channel protein Orai3 in calcium influx mediated by (HUVECs) receptor (CaR) and nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs). Methods: (1) the constructed Orai3 interference plasmid (Orai3 shRNA) was transfected into HUVECs by transfection technique and the transfection effect was observed by confocal laser microscope. Real-time quantitative RT-PCR and Western blotting were used to detect the expression and inhibitory efficiency of Orai3 mRNA and protein after Orai3 shRNA transfection. (2) the cells of passage 2 were randomly divided into specific plasmid transfection group (Orai3shRNA group), non-transfection group (control group) and blank control group (control group). In the empty plasmid group (vehicle group), the above three groups of cells were treated with CaR agonist spermine [Ca cell manipulation calcium channel (SOC) and receptor operable calcium channel (ROC)] and the CaR analogue 12-O- tetradecanoyl phorbol alcohol 13 acetate (TPA) CaR negative structural regulation. Calhex 231 (Activation of Roc, C (PKC) inhibitor of SOC), protein Kinase, Ro31-8220) and PKCs and PKC 渭 inhibitor Go6967 (blocking roc, activating SOC) were incubated. Fluorescence probe Fura-2/AM and DAF-FM loading method were used to detect the changes of [Ca2] I and no production simultaneously. Results: (1) the results of real-time quantitative RT-PCR and Western blotting detection showed that: compared with control group, the expression of Orai3 mRNA decreased significantly (inhibition rate was 84.5%, P0.05). (inhibition rate was 70.68%, P0.05). (2) [Ca2] I 螖 ratio value and no net fluorescence intensity value. The results showed that compared with control group and vehicle group, In Orai3 shRNA transfection group, [Ca2] I 螖 ratio value and no net fluorescence intensity value were significantly decreased (P0.05), but there was no significant difference in vehicle group (P0.05). Conclusion: in HUVECs, Orai3 is involved in calcium influx and no production mediated by SOC and ROC activation by CaR.
【作者單位】： 新疆地方病與民族高發(fā)病教育部重點(diǎn)實(shí)驗(yàn)室 石河子大學(xué)醫(yī)學(xué)院;石河子大學(xué)醫(yī)學(xué)院病理生理教研室;石河子大學(xué)醫(yī)學(xué)院病理教研室;石河子大學(xué)醫(yī)學(xué)院醫(yī)學(xué)機(jī)能實(shí)驗(yàn)中心;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(No.31160239;No.81160018)
【分類號】：R363

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