目的 研究PPAR-γ、PDGF-βR及FAK在肺动脉平滑肌细胞迁移中的分子调控机制。方法 分离大鼠肺动脉平滑肌细胞,培养至3-7代细胞用于实验。分为4组,CON组:常规培养细胞;PDGF-BB组:细胞同步化后加入PDGF-BB(20 ng/ml),刺激1 h后裂解细胞;Y1...目的 研究PPAR-γ、PDGF-βR及FAK在肺动脉平滑肌细胞迁移中的分子调控机制。方法 分离大鼠肺动脉平滑肌细胞,培养至3-7代细胞用于实验。分为4组,CON组:常规培养细胞;PDGF-BB组:细胞同步化后加入PDGF-BB(20 ng/ml),刺激1 h后裂解细胞;Y15组:细胞同步化后加入PDGF-BB(20 ng/ml)预刺激1 h,再加入FAK抑制剂Y15(10 μmol/L)刺激2 h后裂解细胞;ROSI组:细胞同步化后加入罗格列酮(10 μmol/L)刺激1 h,再加入PDGF-BB刺激1 h后裂解细胞。裂解各组处理细胞,Western blot检测各组p-PDGF-βR、p-FAK(Tyr397)水平;划痕实验及Transwell方法比较细胞迁移能力。结果 PDGF-BB组细胞迁移能力较CON组明显增强(77.3%± 4.2% vs 61.3%± 3.5%, P <0.05),p-PDGF-βR、p-FAK(Tyr397)水平提高(2.2 ± 0.12 vs 0.93 ± 0.11;1.07 ± 0.08 vs 0.39 ± 0.02, P <0.05)。Y15组细胞迁移能力较PDGF-BB组明显降低(72.7%± 4.7% vs 77.3%± 4.2%, P <0.05),p-PDGF-βR无明显变化(2.0 ± 0.10 vs 2.2 ± 0.12, P >0.05),p-FAK(Tyr397)水平降低(0.48 ± 0.09 vs 1.07 ± 0.08, P <0.05)。ROSI组细胞迁移能力较Y15组差异无统计学意义(75.3%± 4.04% vs 72.7%± 4.70%, P >0.05),p-PDGF-βR水平降低(1.25 ± 0.10 vs 2.0 ± 0.10, P <0.05);p-FAK(Tyr397)水平提高(0.52 ± 0.07 vs 0.48 ± 0.09, P <0.05);而与PDGF-BB组比较,ROSI组细胞迁移能力(77.3%± 4.2% vs 75.3%± 4.04%)及p-PDGF-βR(2.2 ± 0.12 vs 1.25 ± 0.10)、p-FAK(Tyr397)水平(1.07 ± 0.08 vs 0.52 ± 0.07)均显著降低( P <0.05)。Transwell迁移小室实验结果同划痕实验结果。结论 PPAR-γ/PDGF-βR/FAK(Tyr397)通路,可能是抑制肺动脉平滑肌细胞迁移机制之一。展开更多
目的探讨G蛋白耦联受体30(GPR30)介导的雌激素非基因效应促乳腺癌细胞侵袭的作用及其机制。方法体外培养乳腺癌细胞株SK-BR-3(GPR30+,ERα-/ERβ-),分别给予不同浓度的17β-雌二醇(E2)和GPR30特异性激动剂(G1)处理不同时间,应用免疫印...目的探讨G蛋白耦联受体30(GPR30)介导的雌激素非基因效应促乳腺癌细胞侵袭的作用及其机制。方法体外培养乳腺癌细胞株SK-BR-3(GPR30+,ERα-/ERβ-),分别给予不同浓度的17β-雌二醇(E2)和GPR30特异性激动剂(G1)处理不同时间,应用免疫印迹法观察黏着斑激酶(FAK)的磷酸化;转染GPR30 si RNA抑制GPR30表达后,采用Transwell侵袭小室法观察E2和G1对细胞迁移和侵袭的影响。结果与对照组比较,不同浓度的E2(10-10、10-9、10-8、10-7、10-6mol/L)或G1(10-8、10-7、10-6 mol/L)处理SK-BR-3细胞20 min,均能促进FAK的磷酸化。转染GPR30 si RNA 48h抑制GPR30表达后,E2、G1促进FAK磷酸化的效果均明显减弱。E2、G1均可明显促进SK-BR-3细胞的侵袭。而转染si RNA GPR30抑制GPR30表达后,E2、和G1促进细胞侵袭的能力明显减弱。结论雌激素可通过作用于GPR30激活非基因效应诱导FAK的磷酸化,增加乳腺癌细胞的侵袭能力。展开更多
CD133+ prostate cancer stem cells (PCSCs) have recently been identified in human prostate cancer tissues. The present study reports the integrin profile of prostate cancer progenitor cells and the role of α1 and ...CD133+ prostate cancer stem cells (PCSCs) have recently been identified in human prostate cancer tissues. The present study reports the integrin profile of prostate cancer progenitor cells and the role of α1 and β1 integrins in the homing and differentiation of PCSCs in vitro. PCSCs were isolated from the tissue specimens of patients with prostate cancer and the expression of surface integrins and adhesion patterns were determined. Our analysis of the expression of surface integrins and their adhesion patterns of prostate cancer stem cells derived from prostate cancer tissues revealed that the levels of β1 and α2β1 integrins were significantly higher (P 〈 0.05) than those of the other integrins. By contrast, peripheral blood-derived CD 133+ cells from prostate cancer patients showed a high level of expression (P 〈 0.01) of α2β1,αvβ3, αvβ5, β1 and α1 integrins and a minimal expression of α4β1 integrins. Moreover, CD 133+ cells derived from both prostate cancer tissues and peripheral blood exhibited an increased degree of attachment to extraceUular matrix proteins (P 〈 0.001) and a high expression level of α2β1 integrin. In vitro experiments using blocking antibodies indicated that α1 and β1 integrins have a role in the homing and differentiation of PCSCs. This is the first report to suggest the importance of integrins in mediating the homing and differentiation of PCSCs.展开更多
文摘目的 研究PPAR-γ、PDGF-βR及FAK在肺动脉平滑肌细胞迁移中的分子调控机制。方法 分离大鼠肺动脉平滑肌细胞,培养至3-7代细胞用于实验。分为4组,CON组:常规培养细胞;PDGF-BB组:细胞同步化后加入PDGF-BB(20 ng/ml),刺激1 h后裂解细胞;Y15组:细胞同步化后加入PDGF-BB(20 ng/ml)预刺激1 h,再加入FAK抑制剂Y15(10 μmol/L)刺激2 h后裂解细胞;ROSI组:细胞同步化后加入罗格列酮(10 μmol/L)刺激1 h,再加入PDGF-BB刺激1 h后裂解细胞。裂解各组处理细胞,Western blot检测各组p-PDGF-βR、p-FAK(Tyr397)水平;划痕实验及Transwell方法比较细胞迁移能力。结果 PDGF-BB组细胞迁移能力较CON组明显增强(77.3%± 4.2% vs 61.3%± 3.5%, P <0.05),p-PDGF-βR、p-FAK(Tyr397)水平提高(2.2 ± 0.12 vs 0.93 ± 0.11;1.07 ± 0.08 vs 0.39 ± 0.02, P <0.05)。Y15组细胞迁移能力较PDGF-BB组明显降低(72.7%± 4.7% vs 77.3%± 4.2%, P <0.05),p-PDGF-βR无明显变化(2.0 ± 0.10 vs 2.2 ± 0.12, P >0.05),p-FAK(Tyr397)水平降低(0.48 ± 0.09 vs 1.07 ± 0.08, P <0.05)。ROSI组细胞迁移能力较Y15组差异无统计学意义(75.3%± 4.04% vs 72.7%± 4.70%, P >0.05),p-PDGF-βR水平降低(1.25 ± 0.10 vs 2.0 ± 0.10, P <0.05);p-FAK(Tyr397)水平提高(0.52 ± 0.07 vs 0.48 ± 0.09, P <0.05);而与PDGF-BB组比较,ROSI组细胞迁移能力(77.3%± 4.2% vs 75.3%± 4.04%)及p-PDGF-βR(2.2 ± 0.12 vs 1.25 ± 0.10)、p-FAK(Tyr397)水平(1.07 ± 0.08 vs 0.52 ± 0.07)均显著降低( P <0.05)。Transwell迁移小室实验结果同划痕实验结果。结论 PPAR-γ/PDGF-βR/FAK(Tyr397)通路,可能是抑制肺动脉平滑肌细胞迁移机制之一。
文摘目的探讨G蛋白耦联受体30(GPR30)介导的雌激素非基因效应促乳腺癌细胞侵袭的作用及其机制。方法体外培养乳腺癌细胞株SK-BR-3(GPR30+,ERα-/ERβ-),分别给予不同浓度的17β-雌二醇(E2)和GPR30特异性激动剂(G1)处理不同时间,应用免疫印迹法观察黏着斑激酶(FAK)的磷酸化;转染GPR30 si RNA抑制GPR30表达后,采用Transwell侵袭小室法观察E2和G1对细胞迁移和侵袭的影响。结果与对照组比较,不同浓度的E2(10-10、10-9、10-8、10-7、10-6mol/L)或G1(10-8、10-7、10-6 mol/L)处理SK-BR-3细胞20 min,均能促进FAK的磷酸化。转染GPR30 si RNA 48h抑制GPR30表达后,E2、G1促进FAK磷酸化的效果均明显减弱。E2、G1均可明显促进SK-BR-3细胞的侵袭。而转染si RNA GPR30抑制GPR30表达后,E2、和G1促进细胞侵袭的能力明显减弱。结论雌激素可通过作用于GPR30激活非基因效应诱导FAK的磷酸化,增加乳腺癌细胞的侵袭能力。
文摘CD133+ prostate cancer stem cells (PCSCs) have recently been identified in human prostate cancer tissues. The present study reports the integrin profile of prostate cancer progenitor cells and the role of α1 and β1 integrins in the homing and differentiation of PCSCs in vitro. PCSCs were isolated from the tissue specimens of patients with prostate cancer and the expression of surface integrins and adhesion patterns were determined. Our analysis of the expression of surface integrins and their adhesion patterns of prostate cancer stem cells derived from prostate cancer tissues revealed that the levels of β1 and α2β1 integrins were significantly higher (P 〈 0.05) than those of the other integrins. By contrast, peripheral blood-derived CD 133+ cells from prostate cancer patients showed a high level of expression (P 〈 0.01) of α2β1,αvβ3, αvβ5, β1 and α1 integrins and a minimal expression of α4β1 integrins. Moreover, CD 133+ cells derived from both prostate cancer tissues and peripheral blood exhibited an increased degree of attachment to extraceUular matrix proteins (P 〈 0.001) and a high expression level of α2β1 integrin. In vitro experiments using blocking antibodies indicated that α1 and β1 integrins have a role in the homing and differentiation of PCSCs. This is the first report to suggest the importance of integrins in mediating the homing and differentiation of PCSCs.