慢病毒介导hHCN4基因体外转染骨髓间充质干细胞构建生物起搏器的实验研究
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摘要
实验背景:
在过去的70年中,电子起搏器在心脏传导阻滞性疾病(病窦综合征、房室传导阻滞等)及顽固性心力衰竭、心室颤动等的治疗方面拯救了大量患者的生命。但是它也逐渐暴露出一些缺点:电池寿命有限(8～10年)、非生理性顺序激动、一些难以避免的并发症(感染、出血、心肌穿孔、电极脱位)等等。从心脏的生理功能和人体适应性的角度,理想的起搏器应该是“生物起搏器”。现有的生物起搏方法主要包括基因生物起搏及细胞生物起搏。前者主要集中在3个基因治疗策略上:①上调心肌细胞膜上的β2肾上腺素能受体表达;②抑制Kir2基因家族编码内向整流电流Ik1通道;③上调心肌细胞起搏电流的α(HCN2)和β(MiRP1)亚单位基因。但目前这些研究尚未取得突破性进展,且上述生物起搏靶基因也不是窦房结细胞自动去极化的特异性调控基因。
大量研究表明,由超极化激活的环核苷酸门控通道基因超家族编码的起搏电流(If),在窦房结起搏细胞舒张期自动除极化过程中发挥着至关重要的作用。If通道家族共有4个成员,即HCN1～HCN4,其中HCN4在窦房结中表达最高,是调控If电流及维持窦房结起搏细胞电生理功能的特异性基因,是一种理想的生物起搏靶基因。骨髓间充质干细胞(MSCs)具有多向分化潜能及其对外源基因的高转染效率和高效稳定表达,不少学者利用MSCs的上述特性将其作为基因转染的细胞平台,导入目的片段基因,把细胞工程和基因治疗进行有机结合。例如,将mHCN2转染到骨髓间充质干细胞,发现MSCs能产生高水平的类似If的电流;将转染了mHCN2起搏基因的MSCs进行体内移植,可明显提高心室的“自律性”。但是HCN2亚型的生理功能在于稳定静息状态下起搏细胞的舒张期膜电位,从而起到稳定起搏节律的作用,而对于运动和交感刺激状态下的节律稳定性则无明显影响;而HCN4不仅对产生正常起搏电位和基础心率至关重要,而且也参与交感神经对心率的调节作用。
研究目的:
我们设想以hHCN4为生物起搏的靶基因,应用猪骨髓间充质干细胞(MSCs)作为细胞生物起搏的平台,通过慢病毒载体转染以获取稳定高表达hHCN4基因的MSCs,并对转染细胞的起搏通道蛋白的表达及If电流动力学特性进行检测,为建立新型有效的细胞生物起搏治疗奠定实验基础。
研究方法:
1.猪间充质干细胞的分离与纯化
采用密度梯度离心法分离纯化猪骨髓间充质干细胞,培养采用直接贴壁法培养;用倒置显微镜进行活细胞形态学的动态观察。
2.慢病毒hHCN4基因转染MSCs
hHCN4慢病毒载体(Lentiv-EGFP-hHCN4)和不含hHCN4的慢病毒对照载体(Lentiv-EGFP)对第2代MSCs进行转染。荧光显微镜观察绿色免疫荧光蛋白表达情况;免疫组化染色检测hHCN4蛋白表达情况;
3. Western blot印迹检测hHCN4转染MSCs各组的hHCN4通道蛋白表达情况;
4.全细胞膜片钳技术对重组hHCN4通道进行动力学测定。
研究结果:
1.倒置显微镜可见,经原代培养的MSCs多数为成纤维样的梭形细胞,呈克隆样生长;
2.成功将hHCN4慢病毒载体(Lentiv-EGFP-hHCN4)和不含hHCN4的慢病毒对照载体(Lentiv-EGFP)转染至猪MSCs,获得了稳定表达hHCN4基因的MSCs。
3.荧光显微镜可见,两组绿色荧光蛋白表达及分布无明显差异,转染效率约为90%。
4.转染后免疫细胞化学染色可见,hHCN4组细胞hHCN4抗体染色为阳性,GFP组及对照组细胞则呈弱阳性;
5. Western blot印迹检测可见hHCN4组有一条约170kDa的条带,而GFP组及对照组条带不明显;
6.全细胞膜片钳技术记录到IHCN4,得到电流密度-电压曲线,其激活电压约为-70±5mV,此电流可以被4mmol/L CsCl阻断。GFP组和对照组的MSCs细胞均无IHCN4。
结论:
1.密度梯度离心法可以成功分离纯化猪骨髓间充质干细胞。
2.以hHCN4作为生物起搏的靶基因,通过慢病毒载体可以成功转染MSCs,并表达通道蛋白。
3.转染后的MSCs表达的hHCN4通道与生理性起搏离子流通道具有相同的动力学特性。
Background:In the past 70 years, artificial ectrioc ardiac pacemaker has rescued many patients who sufferred with the heart block disease(sick sinus syndrome,atrial ventricular block) and intractable heart failure,ventricular fibrillation.However,gradually it snagged some shortage,such as limited batery life(8-10 years), the need for permanent catheter implantation into the heart, non-physiological excited procedure,lack of response to autonomic neurohumors and complications(infection,bleeding,myocardial perforation,loose electrode placement),and so on.Ideal pacemaker should be the“biological pacemaker”,considenring phusiologic function of heart and adaptability of human body.Biological Pacemakers include gene therapy and cell therapy.Gene therapy has 3 kinds of strategies: (1) overexpression ofβ2-adrenergic receptors. (2) Inhibition of Kir2 gene family encoding inward rectifier current Ik1. (3) overexpression ofα(HCN2) andβ(MiRP1) subunit genes of inward depolarizing curent.There search in gene therapy have not get breakthrough.And the target genes of gene therapy are not pivotal moduIation gene of spontaneous depolarization of sinoatrial cells.
Lots of studys indicated that,Funny Current (If), encoded by the hyperpolarization- activated cyclic-nucleotide-modulated (HCN1～4) channel gene family,plays a significant role in the process of spontaneous diastolic depolarization of sinoatrial cells. Especially,expression of HCN4 in sinoatrial node(SAN) is highest.Therefore, HCN4 is a optimal bioIogical pacemaker target gene because it is essential for modulation If and maintenance of electri-physiologic function of spontaneous cell in SAN.The bone mesenchymal stem cells not only have pluripotent differentiation potential,but aslo show high transfection and high stability under inducement of foreign gene.Many scholars used those characteres of MSCs as a platform for delievery of genes.The strategy take profits of cell engieneer and gene therapy.Obvious If-like current occur in mesenchymal stem cells(MSCs) transfected with mHCN2,and the vebtricular spontaneous rhythms got evident improvement after implantation of this MSCs in vivo.the physiological function of subtype HCN2 is to stabilize membrane potential in diastolic phase under quiescent condition,but fails to response to exercise and adrenergic nerve.While,the HCN4 not only play an important role in normal pacemaker potential and basal heart rate,but also participates in regulation of heart rate by adrenergic nerve.
Objective:We adopted the hHCN4 as the target gene of biological pacemaker,utilized the platform of MSCs and obtained the MSCs that had stably high expression of hHCN4 through lentivirus vector transfection.Then we detected the expression of channel protein, and determine the kinetics characters of If channal in this MSCs transfected.The aim of this study was to establish a research base of the neotype and effective cell pacemaker therapy.
Methods :
1. Isolation and purification of swine mesenchymal stem cells were established by the density gradient centrifugation and the adherent method.Living cell morphology was observed by the invert microscope dynamically.
2. hHCN4 lentivirus vector transfects MSCs: The hHCN4 lentivirus vector (Lentiv-EGFP- hHCN4) and the control vector(Lentiv-EGFP) respectively transfected the second filial generation MSCs.After transfected, the green fluorescent protein(GFP) was observed by fluorescence microscope and the expression of hHCN4 channel protein was detected by immunocytochemical stain.
3. The expression of hHCN4 channel protein was detected by Western blot.
4. Whole-cell patch clamp detected ionic currents kinetics of transfected hHCN4 and got density-voltage curves.
Results:
1. Under the invert microscope,most MSCs in primary culture were fibroblast-like spindle cells ,cloning growth.
2. The hHCN4 lentivirus vector (Lentiv-EGFP- hHCN4) and the control vector (Lentiv-EGFP) respectively transfected MSCs successfully.and the cells stably express the gene of hHCN4.
3. The expression and distribution of the Green fluorescent protein(GFP) were without the difference in the MSCs of two groups,observed by fluorescence microscope.Both vectors got 90% successful ratio of transfection on MSCs.
4. After transfected, the HCN4 antibody stain was positive in group HCN4 cells, while weakly positive in group GFP and the control, in immunocytochemical stain.
5. After transfected, the MSCs in group HCN4 were highly expressed hHCN4 channel protein (a strap about 170kDa), while weakly in group GFP and the control, in the Western blot.
6. Whole-cell patch clamp recorded ionic currents of transfected hHCN4 and got density-voltage curves.The threshold for activation of IhHCN4 was -70±5mV. IHCN4 is blocked by 4mmol/L CsCl.The group GFP and the control has not IHCN4.
Conclusion:
1. The method of density gradient centrifugation Isolated and purificated swine mesenchymal stem cells.
2. hHCN4 as a target gene of biological pacemaker,successfuly transfected MSCs by retrovirus vector, expressing hHCN4 channel protein.
3. hHCN4 channel in transfected cell has the same kinetic characters as physiologic If current.
在过去的70年中,电子起搏器在心脏传导阻滞性疾病(病窦综合征、房室传导阻滞等)及顽固性心力衰竭、心室颤动等的治疗方面拯救了大量患者的生命。但是它也逐渐暴露出一些缺点:电池寿命有限(8～10年)、非生理性顺序激动、一些难以避免的并发症(感染、出血、心肌穿孔、电极脱位)等等。从心脏的生理功能和人体适应性的角度,理想的起搏器应该是“生物起搏器”。现有的生物起搏方法主要包括基因生物起搏及细胞生物起搏。前者主要集中在3个基因治疗策略上:①上调心肌细胞膜上的β2肾上腺素能受体表达;②抑制Kir2基因家族编码内向整流电流Ik1通道;③上调心肌细胞起搏电流的α(HCN2)和β(MiRP1)亚单位基因。但目前这些研究尚未取得突破性进展,且上述生物起搏靶基因也不是窦房结细胞自动去极化的特异性调控基因。
大量研究表明,由超极化激活的环核苷酸门控通道基因超家族编码的起搏电流(If),在窦房结起搏细胞舒张期自动除极化过程中发挥着至关重要的作用。If通道家族共有4个成员,即HCN1～HCN4,其中HCN4在窦房结中表达最高,是调控If电流及维持窦房结起搏细胞电生理功能的特异性基因,是一种理想的生物起搏靶基因。骨髓间充质干细胞(MSCs)具有多向分化潜能及其对外源基因的高转染效率和高效稳定表达,不少学者利用MSCs的上述特性将其作为基因转染的细胞平台,导入目的片段基因,把细胞工程和基因治疗进行有机结合。例如,将mHCN2转染到骨髓间充质干细胞,发现MSCs能产生高水平的类似If的电流;将转染了mHCN2起搏基因的MSCs进行体内移植,可明显提高心室的“自律性”。但是HCN2亚型的生理功能在于稳定静息状态下起搏细胞的舒张期膜电位,从而起到稳定起搏节律的作用,而对于运动和交感刺激状态下的节律稳定性则无明显影响;而HCN4不仅对产生正常起搏电位和基础心率至关重要,而且也参与交感神经对心率的调节作用。
研究目的:
我们设想以hHCN4为生物起搏的靶基因,应用猪骨髓间充质干细胞(MSCs)作为细胞生物起搏的平台,通过慢病毒载体转染以获取稳定高表达hHCN4基因的MSCs,并对转染细胞的起搏通道蛋白的表达及If电流动力学特性进行检测,为建立新型有效的细胞生物起搏治疗奠定实验基础。
研究方法:
1.猪间充质干细胞的分离与纯化
采用密度梯度离心法分离纯化猪骨髓间充质干细胞,培养采用直接贴壁法培养;用倒置显微镜进行活细胞形态学的动态观察。
2.慢病毒hHCN4基因转染MSCs
hHCN4慢病毒载体(Lentiv-EGFP-hHCN4)和不含hHCN4的慢病毒对照载体(Lentiv-EGFP)对第2代MSCs进行转染。荧光显微镜观察绿色免疫荧光蛋白表达情况;免疫组化染色检测hHCN4蛋白表达情况;
3. Western blot印迹检测hHCN4转染MSCs各组的hHCN4通道蛋白表达情况;
4.全细胞膜片钳技术对重组hHCN4通道进行动力学测定。
研究结果:
1.倒置显微镜可见,经原代培养的MSCs多数为成纤维样的梭形细胞,呈克隆样生长;
2.成功将hHCN4慢病毒载体(Lentiv-EGFP-hHCN4)和不含hHCN4的慢病毒对照载体(Lentiv-EGFP)转染至猪MSCs,获得了稳定表达hHCN4基因的MSCs。
3.荧光显微镜可见,两组绿色荧光蛋白表达及分布无明显差异,转染效率约为90%。
4.转染后免疫细胞化学染色可见,hHCN4组细胞hHCN4抗体染色为阳性,GFP组及对照组细胞则呈弱阳性;
5. Western blot印迹检测可见hHCN4组有一条约170kDa的条带,而GFP组及对照组条带不明显;
6.全细胞膜片钳技术记录到IHCN4,得到电流密度-电压曲线,其激活电压约为-70±5mV,此电流可以被4mmol/L CsCl阻断。GFP组和对照组的MSCs细胞均无IHCN4。
结论:
1.密度梯度离心法可以成功分离纯化猪骨髓间充质干细胞。
2.以hHCN4作为生物起搏的靶基因,通过慢病毒载体可以成功转染MSCs,并表达通道蛋白。
3.转染后的MSCs表达的hHCN4通道与生理性起搏离子流通道具有相同的动力学特性。
Background:In the past 70 years, artificial ectrioc ardiac pacemaker has rescued many patients who sufferred with the heart block disease(sick sinus syndrome,atrial ventricular block) and intractable heart failure,ventricular fibrillation.However,gradually it snagged some shortage,such as limited batery life(8-10 years), the need for permanent catheter implantation into the heart, non-physiological excited procedure,lack of response to autonomic neurohumors and complications(infection,bleeding,myocardial perforation,loose electrode placement),and so on.Ideal pacemaker should be the“biological pacemaker”,considenring phusiologic function of heart and adaptability of human body.Biological Pacemakers include gene therapy and cell therapy.Gene therapy has 3 kinds of strategies: (1) overexpression ofβ2-adrenergic receptors. (2) Inhibition of Kir2 gene family encoding inward rectifier current Ik1. (3) overexpression ofα(HCN2) andβ(MiRP1) subunit genes of inward depolarizing curent.There search in gene therapy have not get breakthrough.And the target genes of gene therapy are not pivotal moduIation gene of spontaneous depolarization of sinoatrial cells.
Lots of studys indicated that,Funny Current (If), encoded by the hyperpolarization- activated cyclic-nucleotide-modulated (HCN1～4) channel gene family,plays a significant role in the process of spontaneous diastolic depolarization of sinoatrial cells. Especially,expression of HCN4 in sinoatrial node(SAN) is highest.Therefore, HCN4 is a optimal bioIogical pacemaker target gene because it is essential for modulation If and maintenance of electri-physiologic function of spontaneous cell in SAN.The bone mesenchymal stem cells not only have pluripotent differentiation potential,but aslo show high transfection and high stability under inducement of foreign gene.Many scholars used those characteres of MSCs as a platform for delievery of genes.The strategy take profits of cell engieneer and gene therapy.Obvious If-like current occur in mesenchymal stem cells(MSCs) transfected with mHCN2,and the vebtricular spontaneous rhythms got evident improvement after implantation of this MSCs in vivo.the physiological function of subtype HCN2 is to stabilize membrane potential in diastolic phase under quiescent condition,but fails to response to exercise and adrenergic nerve.While,the HCN4 not only play an important role in normal pacemaker potential and basal heart rate,but also participates in regulation of heart rate by adrenergic nerve.
Objective:We adopted the hHCN4 as the target gene of biological pacemaker,utilized the platform of MSCs and obtained the MSCs that had stably high expression of hHCN4 through lentivirus vector transfection.Then we detected the expression of channel protein, and determine the kinetics characters of If channal in this MSCs transfected.The aim of this study was to establish a research base of the neotype and effective cell pacemaker therapy.
Methods :
1. Isolation and purification of swine mesenchymal stem cells were established by the density gradient centrifugation and the adherent method.Living cell morphology was observed by the invert microscope dynamically.
2. hHCN4 lentivirus vector transfects MSCs: The hHCN4 lentivirus vector (Lentiv-EGFP- hHCN4) and the control vector(Lentiv-EGFP) respectively transfected the second filial generation MSCs.After transfected, the green fluorescent protein(GFP) was observed by fluorescence microscope and the expression of hHCN4 channel protein was detected by immunocytochemical stain.
3. The expression of hHCN4 channel protein was detected by Western blot.
4. Whole-cell patch clamp detected ionic currents kinetics of transfected hHCN4 and got density-voltage curves.
Results:
1. Under the invert microscope,most MSCs in primary culture were fibroblast-like spindle cells ,cloning growth.
2. The hHCN4 lentivirus vector (Lentiv-EGFP- hHCN4) and the control vector (Lentiv-EGFP) respectively transfected MSCs successfully.and the cells stably express the gene of hHCN4.
3. The expression and distribution of the Green fluorescent protein(GFP) were without the difference in the MSCs of two groups,observed by fluorescence microscope.Both vectors got 90% successful ratio of transfection on MSCs.
4. After transfected, the HCN4 antibody stain was positive in group HCN4 cells, while weakly positive in group GFP and the control, in immunocytochemical stain.
5. After transfected, the MSCs in group HCN4 were highly expressed hHCN4 channel protein (a strap about 170kDa), while weakly in group GFP and the control, in the Western blot.
6. Whole-cell patch clamp recorded ionic currents of transfected hHCN4 and got density-voltage curves.The threshold for activation of IhHCN4 was -70±5mV. IHCN4 is blocked by 4mmol/L CsCl.The group GFP and the control has not IHCN4.
Conclusion:
1. The method of density gradient centrifugation Isolated and purificated swine mesenchymal stem cells.
2. hHCN4 as a target gene of biological pacemaker,successfuly transfected MSCs by retrovirus vector, expressing hHCN4 channel protein.
3. hHCN4 channel in transfected cell has the same kinetic characters as physiologic If current.
引文
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