目的观察雌二醇对去卵巢骨质疏松大鼠骨组织Wnt/β-catenin信号通路的影响,探讨其防治绝经后骨质疏松症的作用机制。方法将80只雌性SD大鼠随机分为对照组、假手术组、去势组、药物组,构建绝经后骨质疏松大鼠模型,检测骨组织骨密度(bone ...目的观察雌二醇对去卵巢骨质疏松大鼠骨组织Wnt/β-catenin信号通路的影响,探讨其防治绝经后骨质疏松症的作用机制。方法将80只雌性SD大鼠随机分为对照组、假手术组、去势组、药物组,构建绝经后骨质疏松大鼠模型,检测骨组织骨密度(bone mineral density,BMD)和生物力学指标,运用qRT-PCR和免疫组化分别检测骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达,通过HE染色观察骨组织形态学。结果去势组股骨BMD、生物力学指标较对照组和假手术组均显著降低(P<0.05),药物组股骨BMD、生物力学指标较去势组均显著升高(P<0.05)。去势组骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达较对照组和假手术组均显著降低(P<0.05),药物组骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达较去势组均显著升高(P<0.05)。去势组骨小梁明显减少,排列稀疏不规则,连接不完整,骨髓腔变大,有大量纤维组织。药物组骨小梁数量减少不明显,粗细均匀稍致密,排列尚规则,连续性完整性较好。结论雌二醇通过Wnt/β-catenin信号通路作用于BMP-2,增加了去卵巢骨质疏松大鼠骨密度,提高了骨生物力学性能,能够改善骨组织结构,发挥了抗骨质疏松作用。展开更多
目的:研究分泌型卷曲相关蛋白5(secreted frizzled related protein 5,SFRP5)对P-糖蛋白(P-glycoprotein,P-gp)介导的白血病多药耐药性的作用。方法:采用转基因方法构建过表达SFRP5的KG1a/SFRP5细胞,real-time PCR检测MDR1 mRNA表达,Wes...目的:研究分泌型卷曲相关蛋白5(secreted frizzled related protein 5,SFRP5)对P-糖蛋白(P-glycoprotein,P-gp)介导的白血病多药耐药性的作用。方法:采用转基因方法构建过表达SFRP5的KG1a/SFRP5细胞,real-time PCR检测MDR1 mRNA表达,Western blot检测细胞P-gp表达。免疫荧光显微镜观察细胞膜表面P-gp表达。流式细胞仪检测细胞内药物浓度。MTT方法检测细胞耐药性。结果:与KG1a细胞及表达绿色荧光蛋白的KG1a/eGFP细胞相比,KG1a/SFRP5细胞中MDR1 mRNA水平显著下降(P<0.01),总P-gp表达水平亦被下调,细胞膜表面P-gp荧光强度减弱,细胞内的罗丹明浓度显著升高(P<0.01),对ADR的IC50显著降低(P<0.01),细胞耐药性下降。结论:SFRP5蛋白表达可以下调MDR1转录及P-gp表达,增加细胞内药物浓度,逆转白血病多药耐药。展开更多
Background A 65-kD mdrl （multi-drug resistance protein 1, P-glycoprotein）-Iike protein has been suggested to be the regulatory protein to the chloride channel protein 3 （CIC-3） mediating insulin granules acidifica...Background A 65-kD mdrl （multi-drug resistance protein 1, P-glycoprotein）-Iike protein has been suggested to be the regulatory protein to the chloride channel protein 3 （CIC-3） mediating insulin granules acidification and release in mouse pancreatic beta cells. But the protein has not been deeply investigated. In this study, we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions. Methods Total RNAs of rat kidneys served as positive controls, and pancreas, islets and INS-1 cells were extracted for reverse-transcript PCR （RT-PCR）, respectively. The cDNAs were run with specific primers selected from the mRNA of abcblb encoding P-glycoprotein. All PCR products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE. P-glycoprotein was detected by a specific monoclonal antibody, C219. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay. Results Compared with positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein. A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. Insulin secretion of rat islets were stimulated by high glucose （16.7mmol/L）, and showed biphasic curve during 60-minute incubation. After co-incubation with cyclosporine A （CsA）, specific inhibitor to P-glycoprotein, the second phase of insulin secretion was reduced significantly while the first phase was not influenced. Conclusions The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein. It may play a key role regulating biphasic insulin release.展开更多
文摘目的观察雌二醇对去卵巢骨质疏松大鼠骨组织Wnt/β-catenin信号通路的影响,探讨其防治绝经后骨质疏松症的作用机制。方法将80只雌性SD大鼠随机分为对照组、假手术组、去势组、药物组,构建绝经后骨质疏松大鼠模型,检测骨组织骨密度(bone mineral density,BMD)和生物力学指标,运用qRT-PCR和免疫组化分别检测骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达,通过HE染色观察骨组织形态学。结果去势组股骨BMD、生物力学指标较对照组和假手术组均显著降低(P<0.05),药物组股骨BMD、生物力学指标较去势组均显著升高(P<0.05)。去势组骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达较对照组和假手术组均显著降低(P<0.05),药物组骨组织Wnt、β-catenin、BMP-2的mRNA和蛋白表达较去势组均显著升高(P<0.05)。去势组骨小梁明显减少,排列稀疏不规则,连接不完整,骨髓腔变大,有大量纤维组织。药物组骨小梁数量减少不明显,粗细均匀稍致密,排列尚规则,连续性完整性较好。结论雌二醇通过Wnt/β-catenin信号通路作用于BMP-2,增加了去卵巢骨质疏松大鼠骨密度,提高了骨生物力学性能,能够改善骨组织结构,发挥了抗骨质疏松作用。
文摘目的:研究分泌型卷曲相关蛋白5(secreted frizzled related protein 5,SFRP5)对P-糖蛋白(P-glycoprotein,P-gp)介导的白血病多药耐药性的作用。方法:采用转基因方法构建过表达SFRP5的KG1a/SFRP5细胞,real-time PCR检测MDR1 mRNA表达,Western blot检测细胞P-gp表达。免疫荧光显微镜观察细胞膜表面P-gp表达。流式细胞仪检测细胞内药物浓度。MTT方法检测细胞耐药性。结果:与KG1a细胞及表达绿色荧光蛋白的KG1a/eGFP细胞相比,KG1a/SFRP5细胞中MDR1 mRNA水平显著下降(P<0.01),总P-gp表达水平亦被下调,细胞膜表面P-gp荧光强度减弱,细胞内的罗丹明浓度显著升高(P<0.01),对ADR的IC50显著降低(P<0.01),细胞耐药性下降。结论:SFRP5蛋白表达可以下调MDR1转录及P-gp表达,增加细胞内药物浓度,逆转白血病多药耐药。
基金Acknowledgements： This study was supported by EFSD Grant Award for Collaborative Diabetes Research between China and Europe supported by Bristol-Myers-Squibb to LI Dai-qing and Tianjin Municipal Natural Science Foundation to LI Dai-qing （No. 08JCZDJC25100 ）.
文摘Background A 65-kD mdrl （multi-drug resistance protein 1, P-glycoprotein）-Iike protein has been suggested to be the regulatory protein to the chloride channel protein 3 （CIC-3） mediating insulin granules acidification and release in mouse pancreatic beta cells. But the protein has not been deeply investigated. In this study, we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions. Methods Total RNAs of rat kidneys served as positive controls, and pancreas, islets and INS-1 cells were extracted for reverse-transcript PCR （RT-PCR）, respectively. The cDNAs were run with specific primers selected from the mRNA of abcblb encoding P-glycoprotein. All PCR products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE. P-glycoprotein was detected by a specific monoclonal antibody, C219. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay. Results Compared with positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein. A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. Insulin secretion of rat islets were stimulated by high glucose （16.7mmol/L）, and showed biphasic curve during 60-minute incubation. After co-incubation with cyclosporine A （CsA）, specific inhibitor to P-glycoprotein, the second phase of insulin secretion was reduced significantly while the first phase was not influenced. Conclusions The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein. It may play a key role regulating biphasic insulin release.
基金EFSD Grant Award for Collaborative Diabetes Research between China and Europesupported by Bristol-Myers-Squibbto Dai-QingLi天津市应用与基础及前沿技术研究计划重点项目（08JCZDJC25100）+1 种基金教育部留学回国人员科研启动基金天津市重大科技攻关项目（中瑞合作,09ZCZDSF04500,胰岛素抵抗的检测、改善和机制）
基金EFSD Grant Award for Collaborative Diabetes Research between China and Europe supported by Bristol-Myers-Squibb to Dai-Qing Li天津市应用与基础及前沿技术研究计划重点项目(08JCZDJC25100)
