曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

目的 研究曲尼司特(Tran)对环孢素A(CsA)诱导的人肾小管上皮细胞(HK-2)向间充质转变的影响,并探讨该药抗纤维化的机制.方法 所有用于实验的HK-2细胞株均为8～12代细胞,分为4组:(1)空白对照组,收获细胞,不做任何处理;(2)CsA组,加入4.2μmol/LCsA;(3)CsA+Tran组,预先加入100μmol/L Tran,作用2 h后再加入4.2 μmol/L CsA;(4)Tran组,仅加入100μmol/L Tran.72 h后于共聚焦显微镜下观察各组细胞形态学变化;用免疫荧光法以及免疫印迹法检测各组钙黏蛋白(E-cadherin)、平滑肌肌动蛋白α(α-SMA)和骨桥蛋白(OPN)的表达.结果 HK-2细胞在正常情况下表现为典型的"鹅卵石"样形态,细胞圆钝,且与邻近的细胞连接较为紧密;空白对照组和Tran组细胞表现为典型的HK-2细胞形态;CsA组细胞变狭长,甚至向周边伸出"伪足"样改变,细胞间连接较为稀疏;CsA+Tran组的细胞形态学改变有明显改善.CsA组细胞E-cadherin荧光表达强度明显弱于对照组,α-SMA、OPN荧光表达强于对照组;CsA+Tran组细胞E-cadherin荧光表达强于CsA组,α-SMA、OPN荧光表达弱于CsA组.免疫印迹检查中,CsA组细胞E-cadherin 的表达明显低于对照组,而α-SMA、OPN的表达明显高于对照组,CsA+Tran组细胞E-cadherin的表达高于CsA组,而α-SMA、OPN的表达低于CsA组.结论 曲尼司特能抑制CsA诱导的HK-2细胞由肾小管上皮向间充质细胞转化的过程,其机制可能与抑制OPN的表达有关.

Abstract：

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

微信平台在宫颈癌高危患者自我管理行动力和健康知识水平的影响

目的 分析微信平台在宫颈癌高危患者自我管理行动力和健康知识水平的影响.方法 本研究所选对象为宫颈癌高危患者100例,经数字随机方式将全部患者分成两组,50例对照组患者仅在入院时实施健康教育,50例观察组患者则在入院健康教育的同时,采用微信平台加强随访,观察比较患者的自我管理行动力和健康知识水平.结果 ①自我管理能力评分:观察组疾病认知能力(7.26±1.24)、按时复诊(6.04±1.31)、治疗依从性(8.39±1.32)、健康行为(8.52±1.34)以及情绪管理(8.38±1.33),各项评分均显著高于对照组(P<0.05);②健康知识水平掌握情况:观察组基础知识43例、感染途径44例、易感因素41例、宫颈癌相关知识44例、预防与治疗41例、定期TCT检查40例,观察组各项健康知识水平掌握例数均高于对照组(P<0.05).结论 应用微信平台能让宫颈癌高危患者的自我管理行动力和健康知识水平显著提高,具有临床应用和推广价值.     

骨桥蛋白靶向干扰对环孢素A诱导的肾小管上皮细胞向间充质转变的影响

目的 研究骨桥蛋白(0PN)靶向干扰对环孢素A(CsA)诱导的大鼠肾小管上皮细胞(NRK52E)向间充质转变的影响.方法 NRK52E细胞按以下方法随机分组:(1)空白对照(A)组;(2)4.2 μmol/L CsA(B)组;(3)0PN shRNA表达载体转染+4.2 μmol/L CsA(C)组;(4)阴性对照shRNA表达载体转染+4.2 μmo1/L CsA(D)组.共聚焦显微镜下观察不同处理组细胞形态学变化,并用免疫荧光法及Westem blot法检测各组细胞上皮型钙黏蛋白(E-cad)、α平滑肌肌动蛋白(α-SMA)、OPN蛋白的表达.结果 与B组相比,C组细胞形态学得到明显改善.B组细胞E-cad蛋白表达明显低于A组,而α-SMA、OPN蛋白表达明显高于A组(P＜0.05);C组细胞E-cad蛋白的表达高于B组,而α-SMA、OPN蛋白表达则低于B组(P＜0.05).结论 OPN的靶向干扰可以减轻CsA诱导的NRK52E细胞由上皮向间充质转变的过程.     

RNA干扰沉默骨桥蛋白对大鼠移植肾小管上皮细胞向间充质转分化的作用

目的:探讨骨桥蛋白(osteopontin,OPN)的shRNA干扰质粒在大鼠慢性移植肾肾病(chronic allograft nephropathy,CAN)模型中对大鼠移植肾上皮细胞向间充质转分化(epithelial to mesenchymal transition,EMT)的作用,初步探讨OPN在慢性移植物...     

NIX在机械牵张诱导的急性肺损伤中的表达及其严重程度的相关性

目的急性肺损伤的本质是各种内外因素引起的全身失控性炎症反应，胸部撞击伤是急性肺损伤的常见原因之一。Nix作为Bcl-2的家族成员，研究发现，Nix在多种人组织细胞的凋亡途径中发挥着重要的作用。本研究旨在探讨NIX在机械牵张诱导的急性肺损伤中的表达及其严重程度的相关性； 方法小生物撞击仪器精准控制建立不同严重程度大鼠胸部撞击伤模型，分组轻伤、重伤、严重伤组，对照组(n＝10)不做处理；各模型组(n＝10)分别于精准撞击后0.5、2、12、24、48、72 h后无伤取其肺组织，利用HE染色观察其病理学变化。取不同损伤程度的AT-Ⅱ型细胞培养株，RT-PCR技术检测各模型组各个时间点肺泡细胞Nix的表达情况，Western blot检测蛋白表达情况。 结果撞击伤大鼠模型组肺组织出现不同程度的出血、水肿并大量炎性细胞浸润。与对照组相比，在撞击0.5、2、12、24、48、72 h后，各模型组大鼠肺组织中Nix mRNA都存在较为明显增高，Nix mRNA在伤后0.5 h已经开始增高，并在撞击48 h后Nix mRNA的表达达到峰值，撞击伤后72 h已有显著下降(P<0.05)，接近伤后0.5 h水平，并随着撞击伤的严重程度Nix mRNA表达水平也呈相应的增高(P<0.05)。与对照组相比，各模型组大鼠肺组织Nix蛋白表达水平也与Nix mRNA表达水平呈现相同的变化曲线(P<0.05)，且同样随撞击伤严重程度的增高而增高(P<0.05)。 结论在机械牵张诱导的急性肺损伤中，肺组织中Nix基因的表达情况与肺损伤严重程度呈正相关(P<0.05)。这为我们通过Nix作为靶点进行肺组织相关疾病的研究提供理论依据。     

慢性移植肾肾病模型中骨桥蛋白、α平滑肌肌动蛋白和E钙黏蛋白的表达

目的 观察骨桥蛋白(OPN)、α平滑肌肌动蛋白(α-SMA)、E钙黏蛋白在大鼠慢性移植肾肾病(CAN)模型移植肾中的表达及其表达的相关性.方法 以Fisher大鼠为供者,Lewis大鼠为受者,进行原位异体肾移植(CAN组);以供、受者均为Lewis大鼠的肾移植模型作为对照.术后12周处死受者,取血、尿样本检测移植肾功能,取移植肾进行HE染色和天狼星红染色,观察其组织形态学变化.采用免疫组织化学法和免疫印迹法观察移植肾组织OPN、α-SMA和E钙黏蛋白的表达.结果 CAN组大鼠移植肾组织形态学符合CAN的病理改变.免疫组化结果显示,与对照组相比较,CAN组肾小管上皮及肾间质中OPN和α-SMA的表达增强,而CAN组小管上皮E钙黏蛋白的表达明显下降.免疫印迹结果与免疫组化结果一致,CAN组移植肾组织中OPN和α-SMA的表达相对灰度值分别为85.74±2.29和88.79±4.44,对照组分别为14.25±0.71、11.21±0.56,两组间的差异有统计学意义(P＜0.05).CAN组E钙黏蛋白的相对灰度值为24.96±0.02,对照组为75.04±3.21,两组间的差异有统计学意义(P＜0.05).两组大鼠移植肾OPN的表达量与α-SMA的表达量呈正相关(r=0.746,P＜0.05),与E钙黏蛋白的表达量呈负相关(r=-0.526,P＜0.05).结论 CAN大鼠移植肾中OPN的表达增加,OPN可能参与了移植肾肾小管上皮细胞向间充质细胞转化的过程.

Abstract：

Objective To investigate the expression of OPN, α-SMA, E-cadherin and their correlation in the chronic allograft nephropathy (CAN) rat model, and to explore the possible role of OPN in CAN.Methods Orthotopic renal-transplantation using Fisher rats as donors and Lewis rats as recipients was done to establish CAN group, and Lewis to Lewis rats as control group. Rats in each group were sacrificed 12 weeks after the surgery. Blood and urine were collected for further test. Allograft samples were collected and sectioned for HE, Sirus-red staining, immunohistochemistry and Western blot.Results There were CAN morphological changes of the allograft in CAN group. As compared with control group, immunohistochemistry and Western blot revealed that the expression of OPN and α-SMA in CAN group was significantly increased, and that of E-Cadherin reduced. Its trend was correlated with the inflammatory response and the EMT of tubule epithelial cells.Conclusions OPN expression in rat CAN model is significantly up-regulated. OPN may play a role in CAN. OPN might affect the CAN by promoting EMT of tubule epithelial cells.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.