AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-compleme...AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.展开更多
目的:研究SLP-2(stomatin like protein2)基因在结肠癌及正常结肠黏膜中的表达情况.方法:应用RT-PCR法及免疫组织化学法分别检测40例和50例结肠癌及其癌旁正常结肠黏膜中SLP-2mRNA和蛋白的表达,并结合免疫组织化学结果及结肠癌患者的临...目的:研究SLP-2(stomatin like protein2)基因在结肠癌及正常结肠黏膜中的表达情况.方法:应用RT-PCR法及免疫组织化学法分别检测40例和50例结肠癌及其癌旁正常结肠黏膜中SLP-2mRNA和蛋白的表达,并结合免疫组织化学结果及结肠癌患者的临床病理资料进行分析.结果:RT-PCR结果显示,结肠癌及配对癌旁正常组织中SLP-2mRNA的表达水平分别为1.31±0.28和0.74±0.16,前者较后者明显升高(P<0.05).免疫组织化学结果显示,SLP-2蛋白在结肠癌及配对癌旁正常组织中的高表达率分别为70.0%(35/50)和22%(11/50),两者差异有统计学意义(P<0.05).且SLP-2蛋白的表达与有无淋巴结转移及TNM分期相关(均P<0.05).结论:SLP-2基因在结肠癌组织中高表达,可能参与结肠癌的发生发展和转移.展开更多
目的:研究stomatin样蛋白2(stomatin-like protein 2,SLP-2)对人子宫内膜基质细胞系T-HESC的增殖和分化的调控作用。方法:培养子宫内膜基质细胞系T-HESC,体外诱导蜕膜化,RT-q PCR、Western blot和免疫荧光方法检测SLP-2在体外诱导蜕膜...目的:研究stomatin样蛋白2(stomatin-like protein 2,SLP-2)对人子宫内膜基质细胞系T-HESC的增殖和分化的调控作用。方法:培养子宫内膜基质细胞系T-HESC,体外诱导蜕膜化,RT-q PCR、Western blot和免疫荧光方法检测SLP-2在体外诱导蜕膜化模型中的表达变化以及蜕膜化标志分子催乳素(prolactin,PRL)、胰岛素样生长因子结合蛋白1(insulin like growth factor binding protein 1,IGFBP1)和Desmin的表达。转染特异性SLP-2干扰小RNA(si RNA),敲低SLP-2的表达,分析敲低SLP-2表达对基质细胞增殖和分化(蜕膜化)的影响。结果:体外诱导蜕膜化条件下,诱导蜕膜化标志物IGFBP1和PRL的表达明显增加;Desmin显著表达于在体外诱导蜕膜化的细胞中;SLP-2表达明显升高。在蜕膜化细胞中转染SLP-2 si RNA敲低SLP-2表达,蜕膜化行为受到显著抑制;细胞增殖标志分子CCND3表达明显下降,细胞增殖降低。结论:SLP-2的表达可影响人子宫内膜基质细胞细胞系T-HESC的增殖和分化。展开更多
目的:探讨红细胞膜整合蛋白SLP-2(stomatin-like protein 2)在胃癌中的表达及其与临床病理学指标及预后的关系。方法:选取中山大学肿瘤防治中心病理科有完整临床资料的胃癌手术标本190例,应用免疫组织化学方法检测胃癌中SLP-2蛋白的表达...目的:探讨红细胞膜整合蛋白SLP-2(stomatin-like protein 2)在胃癌中的表达及其与临床病理学指标及预后的关系。方法:选取中山大学肿瘤防治中心病理科有完整临床资料的胃癌手术标本190例,应用免疫组织化学方法检测胃癌中SLP-2蛋白的表达,并分析其与临床病理特征及预后的关系。结果:(1)胃癌组织中SLP-2蛋白表达的阳性率为63.2%(120/190)。SLP-2表达与胃癌浸润深度、TNM分期及淋巴结转移有关(P<0.05),而与患者性别、年龄、肿瘤分化程度、肿瘤直径和远处转移均无关(P>0.05);(2)Kaplan-Meier生存曲线分析结果显示:SLP-2阳性表达患者的累积生存率明显低于阴性表达患者(P<0.01);(3)单因素分析结果显示:SLP-2的表达、淋巴结转移、肿瘤分化程度、肿瘤直径、TNM分期、浸润深度及远处转移均影响胃癌预后;多因素分析表明,只有肿瘤直径和TNM分期是独立的预后指标。结论:(1)SLP-2在胃腺癌组织中高表达,可能参与胃腺癌的发生发展和转移;(2)SLP-2在一定程度上影响胃癌的预后,其过度表达提示胃癌预后差。展开更多
Post-translational modifications of cellular proteins with ubiquitin or ubiquitin-like proteins regulate many cellular processes,such as cell proliferation,differentiation,apoptosis, signal transduction,intercellular ...Post-translational modifications of cellular proteins with ubiquitin or ubiquitin-like proteins regulate many cellular processes,such as cell proliferation,differentiation,apoptosis, signal transduction,intercellular immune recognition,inflammatory response,stress response,and DNA repair.Nice4/UBAP2L is an important member in the family of ubiquitin-like proteins,and its biological function remains unknown.This study aimed to investigate the effect of UBAP2L on spinal cord injury (SCI).At first,rat bone marrow mesenchymal stem cells (BMSCs)were infected with adeno-associated virus to induce over-expression of Nice4.Subsequently,the infected BMSCs were transplanted into rats suffering from semi-sectioned SCI.The results showed that the over-expression of Nice4 significantly promoted the proliferation and differentiation of BMSCs. In addition,the transplantation of infected BMSCs into the injured area of SCI rats improved the function repair of SCI.Importantly,the immunohistochemical and hematoxylin-eosin staining and RT-PCR results showed that the number of neuronal cells,oligodendrocytes,and astrocytes was significantly increased in the injured area,along with significantly upregulated expression ofcyclin D1 and p38 mitogen-activated protein kinase (MAPK).Meanwhile,the expression of caspase 3 protein was significantly down-regulated.In conclusion,the over-expression of Nice4 gene can promote the functional recovery in SCI rats by promoting cell proliferation and inhibiting apoptosis. The results of this study indicate an alternative option for the clinical treatment of SCI.展开更多
Aim:Development of multi drug resistance and dose limiting cardiotoxicity are hindering the use of Doxorubicin(Dox)in clinical settings.Augmented dox efflux induced by lung resistance protein(LRP)over expression has b...Aim:Development of multi drug resistance and dose limiting cardiotoxicity are hindering the use of Doxorubicin(Dox)in clinical settings.Augmented dox efflux induced by lung resistance protein(LRP)over expression has been related to multi drug resistance phenotype in various cancers.An alkaloid from lotus,Neferine(Nef)shows both anticancer and cardioprotective effects.Here,we have investigated the interconnection between nuclear factor erythroid-derived 2-like 2(NRF2)and LRP in Dox resistance and how Nef can overcome Dox resistance in lung cancer cells by altering this signaling.Methods:Anti-proliferative and apoptotic-inducing effects of Nef and Dox combination in Parental and Dox resistant lung cancer cells were determined in monolayers and 3D spheroids.Intracellular Dox was analyzed using flow cytometry,siRNA knockdown and western blot analysis were used to elucidate NRF2-LRP crosstalk mechanism.展开更多
在口腔、食管和结直肠癌病灶中,TATA序列结合蛋白相关因子1(TATA-box binding protein associated factor 1 like,TAF1L)、stomatin家族蛋白1(stomatin-like protein 1,STOML1)和stomatin家族蛋白2(stomatin-like protein 2,STOML2)存...在口腔、食管和结直肠癌病灶中,TATA序列结合蛋白相关因子1(TATA-box binding protein associated factor 1 like,TAF1L)、stomatin家族蛋白1(stomatin-like protein 1,STOML1)和stomatin家族蛋白2(stomatin-like protein 2,STOML2)存在异常表达.为探究它们在消化系统癌变过程中的作用机理,利用6种消化道肿瘤(食道、胃、结肠、直肠、肝脏和胰腺)及癌旁组织的微陈列芯片,通过多重免疫组织化学染色,比对分析TAF1L、STOML1和STOML2这3种蛋白质在癌与癌旁组织的差异表达及与癌变的关联.结果发现,STOML2在食道、胃、结肠、直肠、肝脏和胰腺这6种消化系统癌组织中均呈显著性高表达(P<0.001),而同一家族成员STOML1则仅在食管癌和肝癌组织中表达明显增高(P<0.005).另外,除胰腺癌灶外,TAF1L在其余5种消化系统癌组织中也呈现高表达(P<0.005).同时,TAF1L、STOML1和STOML2这3种蛋白质均与上皮间充质转化的关键蛋白质MMP9有共定位现象.由此表明,3种蛋白质TAF1L、STOML1和STOML2在多种癌组织中可能具有广泛的促癌作用.由于3种蛋白质在多种癌组织的表达水平不同,说明它们各自存在着组织特异性,且具有不同的功能使命,需进一步研究TAF1L、STOML1和STOML2这3种蛋白质在消化系统恶性肿瘤发生发展过程中各自的分子病理学潜能,如能将它们打造成用于精准诊疗和预后判断的新型分子标志物,此研究结果将有重要的临床意义.展开更多
基金Supported by the National Natural Science Foundation of China(No.81900829,No.82070963)the Xiamen Medical and Health Guiding Project Fund Project(No.3502Z20214ZD1214)+1 种基金the Guangdong Basic and Applied Basic Research Foundation(No.2019A1515011234)the Science and Technology Innovation Committee of Shenzhen(No.JCYJ20210324125614039)。
文摘AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.
文摘目的:研究SLP-2(stomatin like protein2)基因在结肠癌及正常结肠黏膜中的表达情况.方法:应用RT-PCR法及免疫组织化学法分别检测40例和50例结肠癌及其癌旁正常结肠黏膜中SLP-2mRNA和蛋白的表达,并结合免疫组织化学结果及结肠癌患者的临床病理资料进行分析.结果:RT-PCR结果显示,结肠癌及配对癌旁正常组织中SLP-2mRNA的表达水平分别为1.31±0.28和0.74±0.16,前者较后者明显升高(P<0.05).免疫组织化学结果显示,SLP-2蛋白在结肠癌及配对癌旁正常组织中的高表达率分别为70.0%(35/50)和22%(11/50),两者差异有统计学意义(P<0.05).且SLP-2蛋白的表达与有无淋巴结转移及TNM分期相关(均P<0.05).结论:SLP-2基因在结肠癌组织中高表达,可能参与结肠癌的发生发展和转移.
文摘目的:研究stomatin样蛋白2(stomatin-like protein 2,SLP-2)对人子宫内膜基质细胞系T-HESC的增殖和分化的调控作用。方法:培养子宫内膜基质细胞系T-HESC,体外诱导蜕膜化,RT-q PCR、Western blot和免疫荧光方法检测SLP-2在体外诱导蜕膜化模型中的表达变化以及蜕膜化标志分子催乳素(prolactin,PRL)、胰岛素样生长因子结合蛋白1(insulin like growth factor binding protein 1,IGFBP1)和Desmin的表达。转染特异性SLP-2干扰小RNA(si RNA),敲低SLP-2的表达,分析敲低SLP-2表达对基质细胞增殖和分化(蜕膜化)的影响。结果:体外诱导蜕膜化条件下,诱导蜕膜化标志物IGFBP1和PRL的表达明显增加;Desmin显著表达于在体外诱导蜕膜化的细胞中;SLP-2表达明显升高。在蜕膜化细胞中转染SLP-2 si RNA敲低SLP-2表达,蜕膜化行为受到显著抑制;细胞增殖标志分子CCND3表达明显下降,细胞增殖降低。结论:SLP-2的表达可影响人子宫内膜基质细胞细胞系T-HESC的增殖和分化。
文摘目的:探讨红细胞膜整合蛋白SLP-2(stomatin-like protein 2)在胃癌中的表达及其与临床病理学指标及预后的关系。方法:选取中山大学肿瘤防治中心病理科有完整临床资料的胃癌手术标本190例,应用免疫组织化学方法检测胃癌中SLP-2蛋白的表达,并分析其与临床病理特征及预后的关系。结果:(1)胃癌组织中SLP-2蛋白表达的阳性率为63.2%(120/190)。SLP-2表达与胃癌浸润深度、TNM分期及淋巴结转移有关(P<0.05),而与患者性别、年龄、肿瘤分化程度、肿瘤直径和远处转移均无关(P>0.05);(2)Kaplan-Meier生存曲线分析结果显示:SLP-2阳性表达患者的累积生存率明显低于阴性表达患者(P<0.01);(3)单因素分析结果显示:SLP-2的表达、淋巴结转移、肿瘤分化程度、肿瘤直径、TNM分期、浸润深度及远处转移均影响胃癌预后;多因素分析表明,只有肿瘤直径和TNM分期是独立的预后指标。结论:(1)SLP-2在胃腺癌组织中高表达,可能参与胃腺癌的发生发展和转移;(2)SLP-2在一定程度上影响胃癌的预后,其过度表达提示胃癌预后差。
基金the National Natural Science Foundation of China(No.81271347).
文摘Post-translational modifications of cellular proteins with ubiquitin or ubiquitin-like proteins regulate many cellular processes,such as cell proliferation,differentiation,apoptosis, signal transduction,intercellular immune recognition,inflammatory response,stress response,and DNA repair.Nice4/UBAP2L is an important member in the family of ubiquitin-like proteins,and its biological function remains unknown.This study aimed to investigate the effect of UBAP2L on spinal cord injury (SCI).At first,rat bone marrow mesenchymal stem cells (BMSCs)were infected with adeno-associated virus to induce over-expression of Nice4.Subsequently,the infected BMSCs were transplanted into rats suffering from semi-sectioned SCI.The results showed that the over-expression of Nice4 significantly promoted the proliferation and differentiation of BMSCs. In addition,the transplantation of infected BMSCs into the injured area of SCI rats improved the function repair of SCI.Importantly,the immunohistochemical and hematoxylin-eosin staining and RT-PCR results showed that the number of neuronal cells,oligodendrocytes,and astrocytes was significantly increased in the injured area,along with significantly upregulated expression ofcyclin D1 and p38 mitogen-activated protein kinase (MAPK).Meanwhile,the expression of caspase 3 protein was significantly down-regulated.In conclusion,the over-expression of Nice4 gene can promote the functional recovery in SCI rats by promoting cell proliferation and inhibiting apoptosis. The results of this study indicate an alternative option for the clinical treatment of SCI.
文摘Aim:Development of multi drug resistance and dose limiting cardiotoxicity are hindering the use of Doxorubicin(Dox)in clinical settings.Augmented dox efflux induced by lung resistance protein(LRP)over expression has been related to multi drug resistance phenotype in various cancers.An alkaloid from lotus,Neferine(Nef)shows both anticancer and cardioprotective effects.Here,we have investigated the interconnection between nuclear factor erythroid-derived 2-like 2(NRF2)and LRP in Dox resistance and how Nef can overcome Dox resistance in lung cancer cells by altering this signaling.Methods:Anti-proliferative and apoptotic-inducing effects of Nef and Dox combination in Parental and Dox resistant lung cancer cells were determined in monolayers and 3D spheroids.Intracellular Dox was analyzed using flow cytometry,siRNA knockdown and western blot analysis were used to elucidate NRF2-LRP crosstalk mechanism.
文摘在口腔、食管和结直肠癌病灶中,TATA序列结合蛋白相关因子1(TATA-box binding protein associated factor 1 like,TAF1L)、stomatin家族蛋白1(stomatin-like protein 1,STOML1)和stomatin家族蛋白2(stomatin-like protein 2,STOML2)存在异常表达.为探究它们在消化系统癌变过程中的作用机理,利用6种消化道肿瘤(食道、胃、结肠、直肠、肝脏和胰腺)及癌旁组织的微陈列芯片,通过多重免疫组织化学染色,比对分析TAF1L、STOML1和STOML2这3种蛋白质在癌与癌旁组织的差异表达及与癌变的关联.结果发现,STOML2在食道、胃、结肠、直肠、肝脏和胰腺这6种消化系统癌组织中均呈显著性高表达(P<0.001),而同一家族成员STOML1则仅在食管癌和肝癌组织中表达明显增高(P<0.005).另外,除胰腺癌灶外,TAF1L在其余5种消化系统癌组织中也呈现高表达(P<0.005).同时,TAF1L、STOML1和STOML2这3种蛋白质均与上皮间充质转化的关键蛋白质MMP9有共定位现象.由此表明,3种蛋白质TAF1L、STOML1和STOML2在多种癌组织中可能具有广泛的促癌作用.由于3种蛋白质在多种癌组织的表达水平不同,说明它们各自存在着组织特异性,且具有不同的功能使命,需进一步研究TAF1L、STOML1和STOML2这3种蛋白质在消化系统恶性肿瘤发生发展过程中各自的分子病理学潜能,如能将它们打造成用于精准诊疗和预后判断的新型分子标志物,此研究结果将有重要的临床意义.
