Survivin、Bax基因与人晶状体上皮细胞增殖凋亡的实验研究
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摘要
前言
后发性白内障又称晶状体后囊膜混浊(posterior capsule opacification,PCO),是目前白内障术后导致视力下降的最主要并发症。研究指出PCO的发生率从5%至50%不等;儿童白内障患者若术中保留后囊膜,PCO的发生率几乎100%。
激光后囊膜切开术是目前治疗PCO的主要方法,但该方法有可能引起其他并发症,如视网膜脱离、黄斑囊样水肿、继发性青光眼、人工晶状体(intraocular lens,IOL)激光损伤及IOL移位等。对于儿童,由于其无法配合,这一方法也难以适用。而现有的其他预防PCO发生方法,如白内障手术技术改进、IOL设计,以及各种抗增殖药物的应用,虽然在一定程度上降低了PCO的发生率,但都没有从根本上解决这一问题。因此,探索新的PCO防治途径具有重要意义。
近年来研究表明PCO形成的病理过程主要为白内障术后赤道部的晶状体上皮细胞(lens epithelial cells,LECs)过度增殖并移行至晶状体后囊膜,其中大量细胞发生上皮-间质转化(epithelial-mesenchymal transition,EMT)并分化为成纤维细胞。而白内障术后房水中细胞因子如转化生长因子β(transforming growthfactorβ,TGF-β)、成纤维生长因子(fibroblast growth factor,FGF)等浓度改变是造成LECs的增殖,移行,分化的重要因素。
目前PCO基因治疗的研究主要集中于自杀基因、细胞周期调控基因和细胞因子相关基因。这些方法为PCO的治疗提供了新思路,但仍存在一些缺陷。而本研究拟通过进行有关凋亡基因的转导来实现对LECs增殖的抑制。细胞凋亡(apoptosis)又称程序性细胞死亡(programmed cell death,PCD),是受基因调控的细胞主动自杀过程。Bax/Bcl-2以及Survivin是细胞凋亡通路上的重要调控基因,TGF-β与FGF信号通路中也可以观察到上述基因表达的改变。研究表明抑制Bcl-2、Survivin基因或者增强Bax基因表达均可有效抑制肿瘤细胞的生长,明显增强放疗或化疗对肿瘤细胞的杀伤作用。
本研究首先对TGF-β与FGF作用下体外培养的人晶状体上皮细胞(humanlens epithelial cells,HLECs)的增殖情况进行观察,检测Bax、Bcl2、Survivin基因表达的变化;并通过脂质体Bax基因转染以及siRNA技术沉默Survivin表达,从而诱导对HLECs细胞的凋亡,比较不同方法对HLECs凋亡的作用,为基因治疗PCO提供新思路。
第一部分Survivin、Bax与生长因子诱导下人晶状体上皮细胞增殖凋亡的关系
目的比较TGF-β与FGF作用下HLECs增殖过程中Survivin与Bcl-2/Bax基因的表达变化。
方法体外培养HLECs系SRA01/04,并分别加入不同浓度的TGF-β与FGF。通过相差显微镜观察HLECs的增殖情况,同时采用CCK-8方法检测细胞增殖率。应用Western Blot与RT-PCR技术检测HLECs中Bcl-2、Bax、Survivin基因的表达情况。
结果随着浓度的增加,TGF-β能抑制HLECs的增殖,而FGF能够促进HLECs的增殖,且TGF-β对HLECs的抑制作用能被FGF所阻断。TGF-β作用下Survivin与Bcl-2基因表达下调,Bax基因表达上调。而FGF作用下,可检测到Survivin与Bcl-2的表达上调,Bax表达下调。
结论在细胞因子促进或抑制晶状体上皮细胞增殖过程中,细胞内凋亡调控因子如Bcl-2,Bax,Survivin表达水平也发生变化,参与细胞增殖的调控。
第二部分Bax基因转染人晶状体上皮细胞诱导凋亡
目的观察EGFP-N1-Bax质粒转染后HLECs,Bax基因的表达情况及其对细胞凋亡的影响。
方法对体外培养的HLECs采用脂质体技术对进行Bax基因转染。在转染后48小时,采用相差显微镜与流式细胞仪观察Bax基因转染情况。以RT-PCR的方法检测Bax基因mRNA表达,Western Blot法检测Bax基因蛋白表达蛋白的表达。流式细胞仪联合Annexin V染色法检测转染成功的HLECs的凋亡情况。
结果采用流式细胞仪检测的EGFP-N1-Bax的转染效率为45.47±1.30%。转染后48hr,EGFP-N1-Bax组细胞Bax蛋白与mRNA的表达量较对照组均表达上升,流式细胞仪检测细胞凋亡率为23.98±1.88%。转染后细胞内survivin基因表达下调,而Bcl-2 mRNA表达也发生下调。
结论EGFP-N1-Bax质粒转染HLECs后成功表达Bax基因,诱导细胞凋亡。细胞内Bcl-2/Bax比值,Survivin表达也发生相应变化。
第三部分Survivin siRNA转染对人晶状体上皮细胞凋亡的影响
目的通过siRNA转染抑制Survivin基因表达,诱导HLECs凋亡;并联合EGFP-N1-Bax质粒转染诱导细胞凋亡。
方法经脂质体法将siRNA转染至HLECs。转染后48小时,采用相差显微镜与流式细胞仪观察siRNA转染情况。并分别以Western Blot、RT-PCR方法检测三对Survivin siRNA对HLECs中Survivin基因蛋白表达、mRNA表达的影响。流式细胞仪联合Annexin V染色法检测转染成功的HLECs的凋亡效率。选择诱导凋亡效率最好的一对Survivin siRNA联合EGFP-N1-Bax共转染HLECs,采用同样方法观察细胞凋亡情况。
结果采用流式细胞仪检测siRNA转染HLECs的效率为56.58±2.67%。转染后48小时,Survivin siRNA-51组细胞的Survivin蛋白与mRNA的表达量较其余各组明显下调。流式细胞仪联合Annexin V方法检测显示该组细胞凋亡率为24.49±2.42%。联合EGFP-N1-Bax共转染HLECs后,细胞内Bax表达上调,Survivin与Bcl-2表达下调。HLECs凋亡率为33.28±2.69%。
结论通过RNA干扰方法抑制Survivin mRNA表达翻译可成功诱导HLECs发生凋亡。联合EGFP-N1-Bax共转染HLECs可以明显提高凋亡效率。
全文小结
1.FGF与TGF-β调控HLECs细胞增殖过程中,凋亡相关蛋白Bax、Survivin也参与其中,各自表达都发生一定变化。
2.EGFP-N1-Bax质粒转染HLECs后可上调Bax表达,下调Bcl-2/Bax比值与Survivin表达并诱导细胞凋亡。
3.RNA干扰技术可成功下调HLECs内Survivin表达,诱导细胞凋亡。
4.Survivin siRNA联合EGFP-N1-Bax共转染HLECs可以明显提高凋亡效率
Preface
Posterior capsule opacification(PCO) is a common complication of cataract surgery,which incidence is approximately 50%in adults and 100%in children.PCO develops in a significant proportion of patients to such an extant that a secondary vision loss,which consequently requires further corrective laser surgery.The proliferation,migration and abnormal differentiation of residual lens epithelial cells (LECs) and fiber cells in the capsular bag have been implicated in the pathogenesis of PCO.Growth factors such as TGF-beta(TGFβ) and FGF appear to play a key role in this process.
At present PCO is treated by YAG capsulotomy,which carries a some risk of sight threatening complications such as cystoid macular edema or retinal detachment Several attempts have been made to find an appropriate therapeutic concept to significantly lower the rate of PCO,which include mechanical strategies by striving to remove all LECs during surgery,certain IOL materials and sharp-edged IOL designs. Different pharmacological studies have been conducted in vitro and in vivo,using immunotoxins,cytotoxins,and antimetabolic agents.Such approaches have shown promising results regarding the reduction of the PCO rate,but are limited in clinical application by the possible side effects affecting the surrounding normal structures inside the eye.Therefore,the development of an alternative therapy for preventing PCO is of critical importance.
Apoptosis is the physiological,energy-dependent form of cell death.It is,in contrast to necrosis,controlled and,under certain conditions,even initiated by the cell itself.Apoptosis causes no inflammatory side effects.The programmed cell death is initiated either extracellularly by death ligands binding specifically to death receptors or cellular stress factors such as ionizing radiation,heat or hypoxia,thereby initiating an intracellular signalling cascade leading finally to the death of the cell.
The IAPs(inhibitor of apoptosis proteins) and the Bcl-2 families are among the most important regulators of the cell suicide process.Bcl-2 and Bax are,respectively, anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family mainly involved in the regulation of the intrinsic apoptosis pathway.And Survivin is a recently identified member of the IAPs,with an intriguing bifunctional role:it facilitates cell cycle progression while counteracting a large variety of stimuli involved both in the extrinsic and in the intrinsic apoptotic pathways.
Research on apoptosis,its intracellular signalling cascade has made apoptosis a central issue in the development of anti-proliferative chemotherapies.In turn,this information could provide new opportunities and targets for therapies to prevent PCO. In this study we focused on LECs proliferation inhibition strategy,reducing PCO by means of inducing Bax gene overexpression and silencing Survivin gene expression, characterizing the pathway of apoptosis in LECs.
PartⅠSurvivin and Bax gene expressions in LECs by FGF or TGF-βstimuli
Purpose To investigate the impact of FGF or TGF-βstimuli on Survivin and Bax gene expressions.
Method Human Lens cell line(SRA01/04) was cultured in vitro and treated with different concentrations of TGF-βor FGF respectively.LECs proliferation was monitored by phase contrast microscopy,and determined using CCK-8 regent.Protein expressions of Survivin,Bcl-2 and Bax were observed by western blot analysis.And mRNA expressions of target gene were investigated by means of RT-PCR using specific primers.
Results It was found that LEC proliferation was suppressed when exposed to the increasing concentration of TGF-β,whereas FGF enhanced proliferation. Proliferation suppression by TGF-βwas blocked by FGF.TGF-βcaused a significant down-regulated in expression of Survivin and Bcl-2,while increased Bax expression. FGF could up-regulate Survivin and Bcl-2 protein and mRNA expression while reduce Bax expression.
Conclusion Growth factor TGF-βor FGF regulated the proliferation of LECs.Survivin,Bcl-2 and Bax,the members of the apoptosis regulatory gene,might play a role in these processes.The findings suggest that these genes are good candidates for blocking proliferation of LEC.
PartⅡBax gene overexpression to induce LECs apoptosis
Purpose To investigate the influence of Bax overexpression on the apoptosis of LECs.
Methods The plasmid EGFP-N1-Bax,EGFP-N1 were transferred to LECs using lipofectamine 2000.48 hours after transfection,Bax gene mRNA expression was measured using RT-PCR and protein expression was determined by western blot analysis.The expression level of Bcl2 and Survivin gene was also examined using the same techniques.Flow cytometry was used to evaluate Bax transfection and the effect of Bax on apoptosis.
Results EGFP-N1-Bax transfection rate was 45.47±1.30%evaluated by cytometry.48 hours after Bax gene transfection,LECs exhibited a significant increase in the expression level of Bax gene mRNA and protein,compared to cells transferred with plasmid EGFP-N1.The apoptosis rate in EGFP-N1-Bax group was approximately 23.98±1.88%.The Bax transfection group also showed a significant decrease in the mRNA expression of Survivin and Bcl-2.
Conclusion After treatment with plasmid EGFP-N1-Bax,expression of Bax gene was greatly enhanced.Overexpression of Bax gene could trigger the apoptosis of LECs.
PartⅢInducing apoptosis of LECs by Silencing Survivin gene expression
Purpose To investigate the role of silencing Survivin gene expression in apoptosis pathway and the response of LECs death induced by silencing Survivin and over expressing Bax.
Methods Survivin siRNA was introduced into LECs using lipofectamine alone and combining with EGFP-N1-GFP.The transfection rates and apoptosis rates were assayed by flow cytometry.The change of mRNA and protein of the Survivin gene and Bax/Bcl-2 gene were detected by RT-PCR and Western blot,respectively.
Results Survivin siRNA transfection rate was 56.58±2.67%evaluated by cytometry. LECs showed a significant apoptosis by 24.49±2.42%,33.28±2.69%when they transferred with Survivin siRNA,and combining with EGFP-N1-Bax.The Survivin mRNA and protein were knocked down in LECs.Combined with Bax transfection, the cell presented a decrease in apoptosis index Bcl-2/Bax and Survivin expression.
Conclusion Small interfering RNA can exert a knockdown of Survivin gene expression in LECs,and induce apoptosis significantly.Combined with Bax transfection could enhanc cell apoptosis.
Summary
1.Survivin,Bcl-2 and Bax,the members of the apoptosis regulatory gene,might play a role in the process of LEC proliferation mediated by growth factor(FGF and TGF-β).
2.Overexpression of Bax gene by transfection EGFP-N1-Bax could trigger the apoptosis of LEC.
3.Small interfering RNA could exert a knockdown of Survivin gene expression in LECs,and effectively induce apoptosis.
4.Combined Survivin gene inhibition with Bax overexpression may enhance LEC apoptosis.
后发性白内障又称晶状体后囊膜混浊(posterior capsule opacification,PCO),是目前白内障术后导致视力下降的最主要并发症。研究指出PCO的发生率从5%至50%不等;儿童白内障患者若术中保留后囊膜,PCO的发生率几乎100%。
激光后囊膜切开术是目前治疗PCO的主要方法,但该方法有可能引起其他并发症,如视网膜脱离、黄斑囊样水肿、继发性青光眼、人工晶状体(intraocular lens,IOL)激光损伤及IOL移位等。对于儿童,由于其无法配合,这一方法也难以适用。而现有的其他预防PCO发生方法,如白内障手术技术改进、IOL设计,以及各种抗增殖药物的应用,虽然在一定程度上降低了PCO的发生率,但都没有从根本上解决这一问题。因此,探索新的PCO防治途径具有重要意义。
近年来研究表明PCO形成的病理过程主要为白内障术后赤道部的晶状体上皮细胞(lens epithelial cells,LECs)过度增殖并移行至晶状体后囊膜,其中大量细胞发生上皮-间质转化(epithelial-mesenchymal transition,EMT)并分化为成纤维细胞。而白内障术后房水中细胞因子如转化生长因子β(transforming growthfactorβ,TGF-β)、成纤维生长因子(fibroblast growth factor,FGF)等浓度改变是造成LECs的增殖,移行,分化的重要因素。
目前PCO基因治疗的研究主要集中于自杀基因、细胞周期调控基因和细胞因子相关基因。这些方法为PCO的治疗提供了新思路,但仍存在一些缺陷。而本研究拟通过进行有关凋亡基因的转导来实现对LECs增殖的抑制。细胞凋亡(apoptosis)又称程序性细胞死亡(programmed cell death,PCD),是受基因调控的细胞主动自杀过程。Bax/Bcl-2以及Survivin是细胞凋亡通路上的重要调控基因,TGF-β与FGF信号通路中也可以观察到上述基因表达的改变。研究表明抑制Bcl-2、Survivin基因或者增强Bax基因表达均可有效抑制肿瘤细胞的生长,明显增强放疗或化疗对肿瘤细胞的杀伤作用。
本研究首先对TGF-β与FGF作用下体外培养的人晶状体上皮细胞(humanlens epithelial cells,HLECs)的增殖情况进行观察,检测Bax、Bcl2、Survivin基因表达的变化;并通过脂质体Bax基因转染以及siRNA技术沉默Survivin表达,从而诱导对HLECs细胞的凋亡,比较不同方法对HLECs凋亡的作用,为基因治疗PCO提供新思路。
第一部分Survivin、Bax与生长因子诱导下人晶状体上皮细胞增殖凋亡的关系
目的比较TGF-β与FGF作用下HLECs增殖过程中Survivin与Bcl-2/Bax基因的表达变化。
方法体外培养HLECs系SRA01/04,并分别加入不同浓度的TGF-β与FGF。通过相差显微镜观察HLECs的增殖情况,同时采用CCK-8方法检测细胞增殖率。应用Western Blot与RT-PCR技术检测HLECs中Bcl-2、Bax、Survivin基因的表达情况。
结果随着浓度的增加,TGF-β能抑制HLECs的增殖,而FGF能够促进HLECs的增殖,且TGF-β对HLECs的抑制作用能被FGF所阻断。TGF-β作用下Survivin与Bcl-2基因表达下调,Bax基因表达上调。而FGF作用下,可检测到Survivin与Bcl-2的表达上调,Bax表达下调。
结论在细胞因子促进或抑制晶状体上皮细胞增殖过程中,细胞内凋亡调控因子如Bcl-2,Bax,Survivin表达水平也发生变化,参与细胞增殖的调控。
第二部分Bax基因转染人晶状体上皮细胞诱导凋亡
目的观察EGFP-N1-Bax质粒转染后HLECs,Bax基因的表达情况及其对细胞凋亡的影响。
方法对体外培养的HLECs采用脂质体技术对进行Bax基因转染。在转染后48小时,采用相差显微镜与流式细胞仪观察Bax基因转染情况。以RT-PCR的方法检测Bax基因mRNA表达,Western Blot法检测Bax基因蛋白表达蛋白的表达。流式细胞仪联合Annexin V染色法检测转染成功的HLECs的凋亡情况。
结果采用流式细胞仪检测的EGFP-N1-Bax的转染效率为45.47±1.30%。转染后48hr,EGFP-N1-Bax组细胞Bax蛋白与mRNA的表达量较对照组均表达上升,流式细胞仪检测细胞凋亡率为23.98±1.88%。转染后细胞内survivin基因表达下调,而Bcl-2 mRNA表达也发生下调。
结论EGFP-N1-Bax质粒转染HLECs后成功表达Bax基因,诱导细胞凋亡。细胞内Bcl-2/Bax比值,Survivin表达也发生相应变化。
第三部分Survivin siRNA转染对人晶状体上皮细胞凋亡的影响
目的通过siRNA转染抑制Survivin基因表达,诱导HLECs凋亡;并联合EGFP-N1-Bax质粒转染诱导细胞凋亡。
方法经脂质体法将siRNA转染至HLECs。转染后48小时,采用相差显微镜与流式细胞仪观察siRNA转染情况。并分别以Western Blot、RT-PCR方法检测三对Survivin siRNA对HLECs中Survivin基因蛋白表达、mRNA表达的影响。流式细胞仪联合Annexin V染色法检测转染成功的HLECs的凋亡效率。选择诱导凋亡效率最好的一对Survivin siRNA联合EGFP-N1-Bax共转染HLECs,采用同样方法观察细胞凋亡情况。
结果采用流式细胞仪检测siRNA转染HLECs的效率为56.58±2.67%。转染后48小时,Survivin siRNA-51组细胞的Survivin蛋白与mRNA的表达量较其余各组明显下调。流式细胞仪联合Annexin V方法检测显示该组细胞凋亡率为24.49±2.42%。联合EGFP-N1-Bax共转染HLECs后,细胞内Bax表达上调,Survivin与Bcl-2表达下调。HLECs凋亡率为33.28±2.69%。
结论通过RNA干扰方法抑制Survivin mRNA表达翻译可成功诱导HLECs发生凋亡。联合EGFP-N1-Bax共转染HLECs可以明显提高凋亡效率。
全文小结
1.FGF与TGF-β调控HLECs细胞增殖过程中,凋亡相关蛋白Bax、Survivin也参与其中,各自表达都发生一定变化。
2.EGFP-N1-Bax质粒转染HLECs后可上调Bax表达,下调Bcl-2/Bax比值与Survivin表达并诱导细胞凋亡。
3.RNA干扰技术可成功下调HLECs内Survivin表达,诱导细胞凋亡。
4.Survivin siRNA联合EGFP-N1-Bax共转染HLECs可以明显提高凋亡效率
Preface
Posterior capsule opacification(PCO) is a common complication of cataract surgery,which incidence is approximately 50%in adults and 100%in children.PCO develops in a significant proportion of patients to such an extant that a secondary vision loss,which consequently requires further corrective laser surgery.The proliferation,migration and abnormal differentiation of residual lens epithelial cells (LECs) and fiber cells in the capsular bag have been implicated in the pathogenesis of PCO.Growth factors such as TGF-beta(TGFβ) and FGF appear to play a key role in this process.
At present PCO is treated by YAG capsulotomy,which carries a some risk of sight threatening complications such as cystoid macular edema or retinal detachment Several attempts have been made to find an appropriate therapeutic concept to significantly lower the rate of PCO,which include mechanical strategies by striving to remove all LECs during surgery,certain IOL materials and sharp-edged IOL designs. Different pharmacological studies have been conducted in vitro and in vivo,using immunotoxins,cytotoxins,and antimetabolic agents.Such approaches have shown promising results regarding the reduction of the PCO rate,but are limited in clinical application by the possible side effects affecting the surrounding normal structures inside the eye.Therefore,the development of an alternative therapy for preventing PCO is of critical importance.
Apoptosis is the physiological,energy-dependent form of cell death.It is,in contrast to necrosis,controlled and,under certain conditions,even initiated by the cell itself.Apoptosis causes no inflammatory side effects.The programmed cell death is initiated either extracellularly by death ligands binding specifically to death receptors or cellular stress factors such as ionizing radiation,heat or hypoxia,thereby initiating an intracellular signalling cascade leading finally to the death of the cell.
The IAPs(inhibitor of apoptosis proteins) and the Bcl-2 families are among the most important regulators of the cell suicide process.Bcl-2 and Bax are,respectively, anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family mainly involved in the regulation of the intrinsic apoptosis pathway.And Survivin is a recently identified member of the IAPs,with an intriguing bifunctional role:it facilitates cell cycle progression while counteracting a large variety of stimuli involved both in the extrinsic and in the intrinsic apoptotic pathways.
Research on apoptosis,its intracellular signalling cascade has made apoptosis a central issue in the development of anti-proliferative chemotherapies.In turn,this information could provide new opportunities and targets for therapies to prevent PCO. In this study we focused on LECs proliferation inhibition strategy,reducing PCO by means of inducing Bax gene overexpression and silencing Survivin gene expression, characterizing the pathway of apoptosis in LECs.
PartⅠSurvivin and Bax gene expressions in LECs by FGF or TGF-βstimuli
Purpose To investigate the impact of FGF or TGF-βstimuli on Survivin and Bax gene expressions.
Method Human Lens cell line(SRA01/04) was cultured in vitro and treated with different concentrations of TGF-βor FGF respectively.LECs proliferation was monitored by phase contrast microscopy,and determined using CCK-8 regent.Protein expressions of Survivin,Bcl-2 and Bax were observed by western blot analysis.And mRNA expressions of target gene were investigated by means of RT-PCR using specific primers.
Results It was found that LEC proliferation was suppressed when exposed to the increasing concentration of TGF-β,whereas FGF enhanced proliferation. Proliferation suppression by TGF-βwas blocked by FGF.TGF-βcaused a significant down-regulated in expression of Survivin and Bcl-2,while increased Bax expression. FGF could up-regulate Survivin and Bcl-2 protein and mRNA expression while reduce Bax expression.
Conclusion Growth factor TGF-βor FGF regulated the proliferation of LECs.Survivin,Bcl-2 and Bax,the members of the apoptosis regulatory gene,might play a role in these processes.The findings suggest that these genes are good candidates for blocking proliferation of LEC.
PartⅡBax gene overexpression to induce LECs apoptosis
Purpose To investigate the influence of Bax overexpression on the apoptosis of LECs.
Methods The plasmid EGFP-N1-Bax,EGFP-N1 were transferred to LECs using lipofectamine 2000.48 hours after transfection,Bax gene mRNA expression was measured using RT-PCR and protein expression was determined by western blot analysis.The expression level of Bcl2 and Survivin gene was also examined using the same techniques.Flow cytometry was used to evaluate Bax transfection and the effect of Bax on apoptosis.
Results EGFP-N1-Bax transfection rate was 45.47±1.30%evaluated by cytometry.48 hours after Bax gene transfection,LECs exhibited a significant increase in the expression level of Bax gene mRNA and protein,compared to cells transferred with plasmid EGFP-N1.The apoptosis rate in EGFP-N1-Bax group was approximately 23.98±1.88%.The Bax transfection group also showed a significant decrease in the mRNA expression of Survivin and Bcl-2.
Conclusion After treatment with plasmid EGFP-N1-Bax,expression of Bax gene was greatly enhanced.Overexpression of Bax gene could trigger the apoptosis of LECs.
PartⅢInducing apoptosis of LECs by Silencing Survivin gene expression
Purpose To investigate the role of silencing Survivin gene expression in apoptosis pathway and the response of LECs death induced by silencing Survivin and over expressing Bax.
Methods Survivin siRNA was introduced into LECs using lipofectamine alone and combining with EGFP-N1-GFP.The transfection rates and apoptosis rates were assayed by flow cytometry.The change of mRNA and protein of the Survivin gene and Bax/Bcl-2 gene were detected by RT-PCR and Western blot,respectively.
Results Survivin siRNA transfection rate was 56.58±2.67%evaluated by cytometry. LECs showed a significant apoptosis by 24.49±2.42%,33.28±2.69%when they transferred with Survivin siRNA,and combining with EGFP-N1-Bax.The Survivin mRNA and protein were knocked down in LECs.Combined with Bax transfection, the cell presented a decrease in apoptosis index Bcl-2/Bax and Survivin expression.
Conclusion Small interfering RNA can exert a knockdown of Survivin gene expression in LECs,and induce apoptosis significantly.Combined with Bax transfection could enhanc cell apoptosis.
Summary
1.Survivin,Bcl-2 and Bax,the members of the apoptosis regulatory gene,might play a role in the process of LEC proliferation mediated by growth factor(FGF and TGF-β).
2.Overexpression of Bax gene by transfection EGFP-N1-Bax could trigger the apoptosis of LEC.
3.Small interfering RNA could exert a knockdown of Survivin gene expression in LECs,and effectively induce apoptosis.
4.Combined Survivin gene inhibition with Bax overexpression may enhance LEC apoptosis.
引文
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