目的:探讨Kindlin-2对小鼠子宫发育及雌鼠生育能力的影响及其作用机制。方法:利用Cdh16-Cre工具鼠和Kindlin-2^(flox/flox)小鼠构建在子宫内膜中特异性敲除Kindlin-2的小鼠模型,观察敲除Kindlin-2对雌鼠子宫内膜发育和生殖力的影响。在...目的:探讨Kindlin-2对小鼠子宫发育及雌鼠生育能力的影响及其作用机制。方法:利用Cdh16-Cre工具鼠和Kindlin-2^(flox/flox)小鼠构建在子宫内膜中特异性敲除Kindlin-2的小鼠模型,观察敲除Kindlin-2对雌鼠子宫内膜发育和生殖力的影响。在子宫内膜癌细胞系HEC-1和Ish中分别进行高表达和敲低Kindlin-2的实验,检测雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的激活变化,并且提取特异性敲除Kindlin-2的雌鼠(实验组,基因型为Cdh16-Cre;Kindlin-2^(flox/flox))和未特异性敲除Kindlin-2的雌鼠(对照组,基因型为Kindlin-2^(flox/flox))子宫蛋白,每组包含6~8只小鼠,重复3次独立实验,检测mTOR信号通路和Hippo信号通路关键分子的蛋白水平。结果:成功构建了子宫内膜特异性敲除Kindlin-2的小鼠模型,通过鼠尾聚合酶链式反应(polymerase chain reaction,PCR)、Western blot、免疫组织化学染色(immunohistochemistry,IHC)等方法鉴定和验证Kindlin-2在小鼠子宫中的敲除效率。子宫内膜特异性敲除Kindlin-2的雌鼠与对照组相比体质量减轻、生殖能力严重受损、出生仔鼠数量减少,但出生仔鼠中雌鼠和雄鼠的比例未发生改变,通过苏木精-伊红染色实验观察表明实验组子宫内膜发育不完整、子宫壁厚度变薄。机制方面,子宫内膜癌细胞系HEC-1和Ish中敲除Kindlin-2能够下调mTOR、磷酸化mTOR、腺嘌呤核糖核苷酸激活蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、磷酸化的AMPK和磷酸化的核糖体蛋白(ribosomal protein S6,S6)的蛋白水平,在雌鼠子宫中发现特异性敲除Kindlin-2能够上调Mps结合1(Mps one binding 1,MOB1)、磷酸化的Yes相关蛋白(Yes-associated protein,YAP)的蛋白水平。结论:Kindlin-2通过抑制mTOR信号通路、激活Hippo信号通路抑制子宫内膜的发育,进而抑制雌鼠的生育能力。展开更多
目的:观察Kindlin-2 RNA干扰(RNA interference,RNAi)对大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖的影响并探讨相关的作用机制。方法:构建并制备Kindlin-2 siRNA慢病毒载体并感染大鼠VSMC,CCK-8和BrdU技术检测重组Wnt3...目的:观察Kindlin-2 RNA干扰(RNA interference,RNAi)对大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖的影响并探讨相关的作用机制。方法:构建并制备Kindlin-2 siRNA慢病毒载体并感染大鼠VSMC,CCK-8和BrdU技术检测重组Wnt3a蛋白诱导的VSMC增殖情况,实时定量PCR测定VSMC中Kindlin-2、c-Myc和cyclin D1 mRNA的表达水平,免疫共沉淀了解Kindlin-2和β-catenin的关系,Western blot检测VSMC中Kindlin-2、β-catenin、磷酸化β-catenin(Ser675)、GSK-3β和磷酸化GSK-3β(Ser9)蛋白的表达。结果:Kindlin-2 siRNA慢病毒载体能有效感染大鼠VSMC。CCK-8和BrdU结果表明Kindlin-2 RNAi能够显著抑制Wnt3a诱导的VSMC增殖(1.12±0.14 vs. 2.25±0.15,P=0.000;0.162±0.017 vs. 0.288±0.019,P=0.000)。与阴性对照组相比,Kindlin-2 RNAi组和Kindlin-2 RNAi+Wnt3a组中Kindlin-2、c-Myc和cyclin D1 mRNA表达水平均明显下降(0.964±0.014 vs. 0.530±0.029,P=0.000;0.980±0.025 vs. 0.572±0.022,P=0.000;0.979±0.009 vs. 0.590±0.035,P=0.002和0.964±0.014 vs. 0.569±0.027,P=0.000;0.980±0.025 vs. 0.741±0.026,P=0.001;0.979±0.009 vs. 0.769±0.017,P=0.023)。免疫共沉淀证实VSMC中Kindlin-2可以与β-catenin结合,而且Kindlin-2 RNAi+Wnt3a组中Kindlin-2、磷酸化β-catenin(Ser675)和磷酸化GSK-3β(Ser9)蛋白的表达水平较阴性对照+Wnt3a组明显降低(0.468±0.029 vs. 0.725±0.033,P=0.001;1.058±0.109 vs. 1.478±0.045,P=0.001;0.624±0.048 vs. 0.809±0.067,P=0.020)。但各组中总β-catenin和总GSK-3β蛋白水平没有明显变化(F=0.638,P=0.647;F=0.781,P=0.563)。结论:Kindlin-2 RNAi可以通过Wnt信号通路发挥作用并抑制VSMC增殖。展开更多
目的探讨Kindlin-2介导足细胞损伤在局灶节段肾小球硬化症模型小鼠发病中的作用。方法 8周龄雄性BALB/C小鼠分为正常对照组和模型组,各18只。模型组小鼠注射阿霉素(ADR,浓度2mg/m L,剂量10mg/kg鼠重)造模,正常对照组小鼠给予等量的生理...目的探讨Kindlin-2介导足细胞损伤在局灶节段肾小球硬化症模型小鼠发病中的作用。方法 8周龄雄性BALB/C小鼠分为正常对照组和模型组,各18只。模型组小鼠注射阿霉素(ADR,浓度2mg/m L,剂量10mg/kg鼠重)造模,正常对照组小鼠给予等量的生理盐水,于第4、8、12周每组各处死6只小鼠,检测24h尿蛋白定量、血清白蛋白(ALB)、总胆固醇(TC),光镜观察肾脏组织病理学改变,免疫组织化学技术检测足细胞数量,RT-q PCR技术检测肾脏组织Kindlin-2、肾病蛋白(Nephrin)及Podocin m RNA表达水平。结果与正常对照组比较,模型组4、8、12周24h尿蛋白定量、血总胆固醇(TC)均增加[24h尿蛋白定量:(5.66±1.06)mg/24h比(1.72±0.62)mg/24h、(4.63±1.16)mg/24h比(1.77±0.63)mg/24h、(4.78±0.87)mg/24h比(1.69±0.49)mg/24h;血TC:(8.84±1.30)mmol/L比(2.01±0.49)mmol/L、(7.45±1.27)mmol/L比(2.06±0.49)mmol/L、(6.79±1.38)mmol/L比(2.10±0.54)mmol/L,P均<0.05];血ALB水平降低[(11.98±1.33)g/L比(22.27±1.41)g/L、(12.78±1.47)g/L比(22.65±1.49)g/L、(12.99±1.38)g/L比(23.31±1.49)g/L,P均<0.05];模型组第4周可见部分肾小球出现局灶、节段硬化,伴有足细胞肿胀;第8周部分肾小球出现球性硬化,伴有明显的足细胞增生和肥大;第12周弥漫性肾小球出现球形硬化。模型组足细胞数量随着时间的进展逐渐减少[(77.40±7.56)个/肾小球比(104.00±10.05)个/肾小球,(63.27±8.51)个/肾小球比(103.80±12.23)个/肾小球,(53.15±8.23)个/肾小球比(98.86±8.89)个/肾小球,P均<0.05]。模型组第4周开始Kindlin-2 m RNA水平明显上调[(1.14±0.06)、(1.13±0.08)、(1.45±0.10)比(0.82±0.07),P均<0.05],Podocin m RNA水平明显下调[(1.79±0.08)、(1.64±0.06)、(1.45±0.07)比(2.05±0.05),P均<0.05],第8周开始Nephrin m RNA水平明显下调[(1.42±0.13)、(1.17±0.11)比(2.06±0.20),P均<0.05]。Kindlin-2 m RNA水平与足细胞数量(r=-0.02,P<0.01)、Nephrin m RNA水平(r=-0.8,P<0.01)、Podocin m RNA水平(r=-0.9,P<0.01)呈显著负相关。结论 Kindlin-2可能通过介导足细胞损伤参与小鼠局灶节段肾小球硬化症的发病过程。展开更多
Objective: Recent studies have shown that tumor-associated macrophages(TAMs) play an important role in cancer invasion and metastasis. Our previous studies have reported that TAMs promote the invasion and metastasis o...Objective: Recent studies have shown that tumor-associated macrophages(TAMs) play an important role in cancer invasion and metastasis. Our previous studies have reported that TAMs promote the invasion and metastasis of gastric cancer(GC) cells through the Kindlin-2 pathway. However, the mechanism needs to be clarified.Methods: THP-1 monocytes were induced by PMA/interleukin(IL)-4/IL-13 to establish an efficient TAM model in vitro and M2 macrophages were isolated via flow cytometry. A dual luciferase reporter system and chromatin immunoprecipitation(Ch IP) assay were used to investigate the mechanism of transforming growth factor β2(TGFβ2) regulating Kindlin-2 expression. Immunohistochemistry was used to study the relationships among TAM infiltration in human GC tissues, Kindlin-2 protein expression, clinicopathological parameters and prognosis in human GC tissues. A nude mouse oncogenesis model was used to verify the invasion and metastasis mechanisms in vivo.Results: We found that Kindlin-2 expression was upregulated at both m RNA and protein levels in GC cells cocultured with TAMs, associated with higher invasion rate. Kindlin-2 knockdown reduced the invasion rate of GC cells under coculture condition. TGFβ2 secreted by TAMs regulated the expression of Kindlin-2 through the transcription factor NF-кB. TAMs thus participated in the progression of GC through the TGFβ2/NF-κB/Kindlin-2 axis. Kindlin-2 expression and TAM infiltration were significantly positively correlated with TNM stage, and patients with high Kindlin-2 expression had significantly poorer overall survival than patients with low Kindlin-2 expression. Furthermore, Kindlin-2 promoted the invasion of GC cells in vivo.Conclusions: This study elucidates the mechanism of TAMs participating in GC cell invasion and metastasis through the TGFβ2/NF-κB/Kindlin-2 axis, providing a possibility for new treatment options and approaches.展开更多
文摘目的:探讨Kindlin-2对小鼠子宫发育及雌鼠生育能力的影响及其作用机制。方法:利用Cdh16-Cre工具鼠和Kindlin-2^(flox/flox)小鼠构建在子宫内膜中特异性敲除Kindlin-2的小鼠模型,观察敲除Kindlin-2对雌鼠子宫内膜发育和生殖力的影响。在子宫内膜癌细胞系HEC-1和Ish中分别进行高表达和敲低Kindlin-2的实验,检测雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的激活变化,并且提取特异性敲除Kindlin-2的雌鼠(实验组,基因型为Cdh16-Cre;Kindlin-2^(flox/flox))和未特异性敲除Kindlin-2的雌鼠(对照组,基因型为Kindlin-2^(flox/flox))子宫蛋白,每组包含6~8只小鼠,重复3次独立实验,检测mTOR信号通路和Hippo信号通路关键分子的蛋白水平。结果:成功构建了子宫内膜特异性敲除Kindlin-2的小鼠模型,通过鼠尾聚合酶链式反应(polymerase chain reaction,PCR)、Western blot、免疫组织化学染色(immunohistochemistry,IHC)等方法鉴定和验证Kindlin-2在小鼠子宫中的敲除效率。子宫内膜特异性敲除Kindlin-2的雌鼠与对照组相比体质量减轻、生殖能力严重受损、出生仔鼠数量减少,但出生仔鼠中雌鼠和雄鼠的比例未发生改变,通过苏木精-伊红染色实验观察表明实验组子宫内膜发育不完整、子宫壁厚度变薄。机制方面,子宫内膜癌细胞系HEC-1和Ish中敲除Kindlin-2能够下调mTOR、磷酸化mTOR、腺嘌呤核糖核苷酸激活蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、磷酸化的AMPK和磷酸化的核糖体蛋白(ribosomal protein S6,S6)的蛋白水平,在雌鼠子宫中发现特异性敲除Kindlin-2能够上调Mps结合1(Mps one binding 1,MOB1)、磷酸化的Yes相关蛋白(Yes-associated protein,YAP)的蛋白水平。结论:Kindlin-2通过抑制mTOR信号通路、激活Hippo信号通路抑制子宫内膜的发育,进而抑制雌鼠的生育能力。
文摘目的:观察Kindlin-2 RNA干扰(RNA interference,RNAi)对大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖的影响并探讨相关的作用机制。方法:构建并制备Kindlin-2 siRNA慢病毒载体并感染大鼠VSMC,CCK-8和BrdU技术检测重组Wnt3a蛋白诱导的VSMC增殖情况,实时定量PCR测定VSMC中Kindlin-2、c-Myc和cyclin D1 mRNA的表达水平,免疫共沉淀了解Kindlin-2和β-catenin的关系,Western blot检测VSMC中Kindlin-2、β-catenin、磷酸化β-catenin(Ser675)、GSK-3β和磷酸化GSK-3β(Ser9)蛋白的表达。结果:Kindlin-2 siRNA慢病毒载体能有效感染大鼠VSMC。CCK-8和BrdU结果表明Kindlin-2 RNAi能够显著抑制Wnt3a诱导的VSMC增殖(1.12±0.14 vs. 2.25±0.15,P=0.000;0.162±0.017 vs. 0.288±0.019,P=0.000)。与阴性对照组相比,Kindlin-2 RNAi组和Kindlin-2 RNAi+Wnt3a组中Kindlin-2、c-Myc和cyclin D1 mRNA表达水平均明显下降(0.964±0.014 vs. 0.530±0.029,P=0.000;0.980±0.025 vs. 0.572±0.022,P=0.000;0.979±0.009 vs. 0.590±0.035,P=0.002和0.964±0.014 vs. 0.569±0.027,P=0.000;0.980±0.025 vs. 0.741±0.026,P=0.001;0.979±0.009 vs. 0.769±0.017,P=0.023)。免疫共沉淀证实VSMC中Kindlin-2可以与β-catenin结合,而且Kindlin-2 RNAi+Wnt3a组中Kindlin-2、磷酸化β-catenin(Ser675)和磷酸化GSK-3β(Ser9)蛋白的表达水平较阴性对照+Wnt3a组明显降低(0.468±0.029 vs. 0.725±0.033,P=0.001;1.058±0.109 vs. 1.478±0.045,P=0.001;0.624±0.048 vs. 0.809±0.067,P=0.020)。但各组中总β-catenin和总GSK-3β蛋白水平没有明显变化(F=0.638,P=0.647;F=0.781,P=0.563)。结论:Kindlin-2 RNAi可以通过Wnt信号通路发挥作用并抑制VSMC增殖。
文摘目的探讨Kindlin-2介导足细胞损伤在局灶节段肾小球硬化症模型小鼠发病中的作用。方法 8周龄雄性BALB/C小鼠分为正常对照组和模型组,各18只。模型组小鼠注射阿霉素(ADR,浓度2mg/m L,剂量10mg/kg鼠重)造模,正常对照组小鼠给予等量的生理盐水,于第4、8、12周每组各处死6只小鼠,检测24h尿蛋白定量、血清白蛋白(ALB)、总胆固醇(TC),光镜观察肾脏组织病理学改变,免疫组织化学技术检测足细胞数量,RT-q PCR技术检测肾脏组织Kindlin-2、肾病蛋白(Nephrin)及Podocin m RNA表达水平。结果与正常对照组比较,模型组4、8、12周24h尿蛋白定量、血总胆固醇(TC)均增加[24h尿蛋白定量:(5.66±1.06)mg/24h比(1.72±0.62)mg/24h、(4.63±1.16)mg/24h比(1.77±0.63)mg/24h、(4.78±0.87)mg/24h比(1.69±0.49)mg/24h;血TC:(8.84±1.30)mmol/L比(2.01±0.49)mmol/L、(7.45±1.27)mmol/L比(2.06±0.49)mmol/L、(6.79±1.38)mmol/L比(2.10±0.54)mmol/L,P均<0.05];血ALB水平降低[(11.98±1.33)g/L比(22.27±1.41)g/L、(12.78±1.47)g/L比(22.65±1.49)g/L、(12.99±1.38)g/L比(23.31±1.49)g/L,P均<0.05];模型组第4周可见部分肾小球出现局灶、节段硬化,伴有足细胞肿胀;第8周部分肾小球出现球性硬化,伴有明显的足细胞增生和肥大;第12周弥漫性肾小球出现球形硬化。模型组足细胞数量随着时间的进展逐渐减少[(77.40±7.56)个/肾小球比(104.00±10.05)个/肾小球,(63.27±8.51)个/肾小球比(103.80±12.23)个/肾小球,(53.15±8.23)个/肾小球比(98.86±8.89)个/肾小球,P均<0.05]。模型组第4周开始Kindlin-2 m RNA水平明显上调[(1.14±0.06)、(1.13±0.08)、(1.45±0.10)比(0.82±0.07),P均<0.05],Podocin m RNA水平明显下调[(1.79±0.08)、(1.64±0.06)、(1.45±0.07)比(2.05±0.05),P均<0.05],第8周开始Nephrin m RNA水平明显下调[(1.42±0.13)、(1.17±0.11)比(2.06±0.20),P均<0.05]。Kindlin-2 m RNA水平与足细胞数量(r=-0.02,P<0.01)、Nephrin m RNA水平(r=-0.8,P<0.01)、Podocin m RNA水平(r=-0.9,P<0.01)呈显著负相关。结论 Kindlin-2可能通过介导足细胞损伤参与小鼠局灶节段肾小球硬化症的发病过程。
基金supported by grants from the National Natural Science Foundation of China (No. 81372291).
文摘Objective: Recent studies have shown that tumor-associated macrophages(TAMs) play an important role in cancer invasion and metastasis. Our previous studies have reported that TAMs promote the invasion and metastasis of gastric cancer(GC) cells through the Kindlin-2 pathway. However, the mechanism needs to be clarified.Methods: THP-1 monocytes were induced by PMA/interleukin(IL)-4/IL-13 to establish an efficient TAM model in vitro and M2 macrophages were isolated via flow cytometry. A dual luciferase reporter system and chromatin immunoprecipitation(Ch IP) assay were used to investigate the mechanism of transforming growth factor β2(TGFβ2) regulating Kindlin-2 expression. Immunohistochemistry was used to study the relationships among TAM infiltration in human GC tissues, Kindlin-2 protein expression, clinicopathological parameters and prognosis in human GC tissues. A nude mouse oncogenesis model was used to verify the invasion and metastasis mechanisms in vivo.Results: We found that Kindlin-2 expression was upregulated at both m RNA and protein levels in GC cells cocultured with TAMs, associated with higher invasion rate. Kindlin-2 knockdown reduced the invasion rate of GC cells under coculture condition. TGFβ2 secreted by TAMs regulated the expression of Kindlin-2 through the transcription factor NF-кB. TAMs thus participated in the progression of GC through the TGFβ2/NF-κB/Kindlin-2 axis. Kindlin-2 expression and TAM infiltration were significantly positively correlated with TNM stage, and patients with high Kindlin-2 expression had significantly poorer overall survival than patients with low Kindlin-2 expression. Furthermore, Kindlin-2 promoted the invasion of GC cells in vivo.Conclusions: This study elucidates the mechanism of TAMs participating in GC cell invasion and metastasis through the TGFβ2/NF-κB/Kindlin-2 axis, providing a possibility for new treatment options and approaches.
