慢病毒介导hHCN1和hHCN4基因不同比例体外转染骨髓间充质干细胞的表达及意义
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摘要
研究背景：
病态窦房结综合征是临床常见的疾病，其具体发病机理目前尚不十分明确，以现在的医疗条件，其治疗的主要措施是植入永久性电子起搏器。但这种治疗方法存在一些缺陷与不足，如起搏器电池寿命有限、永久性的植入电极、不受神经-体液因素调节等。从心脏的生理功能和人体适应性的角度出发，近些年提出了“生物起搏”的概念。相关的起搏电流If，其编码基因为超极化激活的环核苷酸门控离子通道（Hyperpolarization-activated cyclic-nucleotide gated，HCN），在窦房结舒张期自动去极化过程中起着重要作用。故我们提出将骨髓间充质干细胞(mesenchymal stemcells,MSCs)作为转染HCN的细胞平台，构建“生物起搏器”，为将来替代电子起搏器用于临床治疗提供实验依据。
研究目的：
将带有绿色荧光的慢病毒（Lentiv-GFP)作为载体，研究不同比例慢病毒hHCN1(Lentiv-GFP-hHCN1)和慢病毒hHCN4(Lentiv-GFP-hHCN4)共同转染大鼠MSCs后的起搏电流特点，同时对转染阳性的细胞进行相关的生化检测，旨在为体外建立可能的细胞-基因生物起搏治疗奠定实验基础。
研究方法：
1.SD大鼠骨髓间充质干细胞的提取、培养及纯化。
2. Lentiv-GFP-hHCN1、Lentiv-GFP-hHCN4分别转染和按照不同比例共同转染MSCs。
将Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4按照不同比例转染第3代MSCs，同时设置hHCN1和hHCN4单独转染MSCs及不含hHCN基因的慢病毒做为对照组。
3.检测Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4转染MSCs情况
①荧光显微镜观察绿色免疫荧光蛋白的表达情况。
②实时定量聚合酶链反应（RT-PCR）检测hHCN1和hHCN4的基因表达。
③Western blot免疫印迹检测hHCN1和hHCN4通道蛋白
4.全细胞膜片钳技术记录转染阳性的MSCs的起搏电流。
研究结果：
1．通过普通倒置显微镜可见原代培养的MSCs形态大多呈梭形，分散排列；
2．成功将Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4按照不同比例转染MSCs，同时分别各自转染MSCs，荧光显微镜下可见各组均有绿色荧光蛋白表达，转染效率约为50-70％。
3．RT-PCR证实转染阳性的MSCs中存在hHCN1和hHCN4mRNA的表达；Western blot免疫印迹检测可见在99kDa和150kDa附近有hHCN1和hHCN4的条带。而GFP组、对照组和空白组则没有。
4．全细胞膜片钳技术记录转染阳性的MSCs的电流，可记录到时间和电压依赖性的超极化激活的内向电流，其激活电压为-70mV～-84mV。GFP组、对照组和空白组的MSCs细胞均不能记录到起搏电流。Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4共转染的3组电流密度较hHCN1、hHCN4单独转染组的大。
结论：
1．骨髓贴壁细胞分离法可以成功分离、纯化大鼠骨髓间充质干细胞。
2．hHCN1和hHCN4通过慢病毒载体可成功转染MSCs，并表达相应通道蛋白及产生相应的电流，这为心律失常的基因治疗奠定了基础。
3．转染阳性的MSCs能够表达HCN通道特点并产生If，这为体外构建生物起搏奠定了基础。
Background:Sick sinus syndrome is a common clinical disease,but the mechanism is yet to bedefined. In the present medical condition, we mainly use the permanent electronic pacemaker. Butwith the advantages come with the disadvantages, such as limited battery life, the need for permanentimplanation of electrodes into the heart and lack of response to nerve-humoral factors regulation.
Lately, considering the physical function of heart and adaptability of human body, we bring upthe concept of the biological pacemaker. The related current, If,encoded by thehyperpolarization-activated cyclic-nucleotide gated, plays an important role in theprocess of spontaneous diastolic depolarization of sinoatrial cells. Therefore, we putforward bone marrow mesenchymal stem cells(MSCs) transfected with HCN as cellplatform to construct a “biological pacemaker” which provides experimental basis for thealternative to electronic pacemaker for clinical treatment in the future.
Objective:
We adopt the lentiv-GFP, carrying green fluorescent, as a vector to study thecharacteristics of the pacemaker current in the MSCs co-transfected with differentproportions of hHCN1and hHCN4. Meanwhile, we do some chemical tests on the cellstransfected positive aimed at the establishment of cell–genetically modified organismpacing therapy experimental basis in vitro.
Methods：
1. Extraction, culture and purification of SD rat MSCs.
2. Lentiv-hHCN1and Lentiv-hHCN4transfect and co-transfect the MSCs in differentproportions.
Lentiv-hHCN1and Lentiv-hHCN4in different proportions transfect the3rdgenerationof MCS, at the same time, we set hHCN1and hHCN4individually transfecting MSCs and lentiv without HCN gene as a control group.
3.Detection of Lentiv-GFP-hHCN1and Lentiv-GFP-hHCN4transfection.
Observe the expression of the green immumofluorescene with a fluorescenemicroscopy.
Real-time quantitative polymerase chain reaction(RT-PCR) detection of the hHCN1and hHCN4expression.
The expression of hHCN1and hHCN4protein detected by Western blot.4.We use whole cell patch clamp technique to record the pacemaker current in the cellstransfected with hHCN1and hHCN4.
Results:
1.Most primary cultured MSCs were fibroblast-like spindle cells in dispersedarrangement under ordinary invert microscope.
2.The hHCN1/hHCN4transfect and co-transfect the MSCs successfully and each groupexpresses green fluorescent protein under a fluorescence microscope with a transfectionefficiency of about50-70%.
3.RT-PCR confirmed the existence of mRNA in the positive transfected MSCs. We cansee bands around99kDa and150kDa in cells transfected with hHCN1and hHCN4inWestern blot.
4. The whole cell patch clamp technique was used to record positive transfected MSCscurrent, and we got the voltage-dependence of hyperpolarization-activated inwardcurrent which activated at the voltage-75mV. In contrast, there was no pacemakercurrent in GFP group and the control group of MSCs. The current density in groupsco-transfected with hHCN1and hHCN4were larger than that of transfected with hHCN1and hHCN4individually.
Conclusion:
1.rat bone marrow mesenchymal stem cells can be successfully extracted and purifiedwith bone marrow adherent cell separation method.
2.hHCN1and hHCN4by lentiviral vectors successfully transfected MSCs which expressed the corresponding channel protein and current which laid the foundation forgene therapy for arrhythmia.
3.Positive transfected MSCs were able to express the characteristics of HCN channelsand generate the If which builded the foundation for constructing the biologicalpacemaker in vitro.
病态窦房结综合征是临床常见的疾病，其具体发病机理目前尚不十分明确，以现在的医疗条件，其治疗的主要措施是植入永久性电子起搏器。但这种治疗方法存在一些缺陷与不足，如起搏器电池寿命有限、永久性的植入电极、不受神经-体液因素调节等。从心脏的生理功能和人体适应性的角度出发，近些年提出了“生物起搏”的概念。相关的起搏电流If，其编码基因为超极化激活的环核苷酸门控离子通道（Hyperpolarization-activated cyclic-nucleotide gated，HCN），在窦房结舒张期自动去极化过程中起着重要作用。故我们提出将骨髓间充质干细胞(mesenchymal stemcells,MSCs)作为转染HCN的细胞平台，构建“生物起搏器”，为将来替代电子起搏器用于临床治疗提供实验依据。
研究目的：
将带有绿色荧光的慢病毒（Lentiv-GFP)作为载体，研究不同比例慢病毒hHCN1(Lentiv-GFP-hHCN1)和慢病毒hHCN4(Lentiv-GFP-hHCN4)共同转染大鼠MSCs后的起搏电流特点，同时对转染阳性的细胞进行相关的生化检测，旨在为体外建立可能的细胞-基因生物起搏治疗奠定实验基础。
研究方法：
1.SD大鼠骨髓间充质干细胞的提取、培养及纯化。
2. Lentiv-GFP-hHCN1、Lentiv-GFP-hHCN4分别转染和按照不同比例共同转染MSCs。
将Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4按照不同比例转染第3代MSCs，同时设置hHCN1和hHCN4单独转染MSCs及不含hHCN基因的慢病毒做为对照组。
3.检测Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4转染MSCs情况
①荧光显微镜观察绿色免疫荧光蛋白的表达情况。
②实时定量聚合酶链反应（RT-PCR）检测hHCN1和hHCN4的基因表达。
③Western blot免疫印迹检测hHCN1和hHCN4通道蛋白
4.全细胞膜片钳技术记录转染阳性的MSCs的起搏电流。
研究结果：
1．通过普通倒置显微镜可见原代培养的MSCs形态大多呈梭形，分散排列；
2．成功将Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4按照不同比例转染MSCs，同时分别各自转染MSCs，荧光显微镜下可见各组均有绿色荧光蛋白表达，转染效率约为50-70％。
3．RT-PCR证实转染阳性的MSCs中存在hHCN1和hHCN4mRNA的表达；Western blot免疫印迹检测可见在99kDa和150kDa附近有hHCN1和hHCN4的条带。而GFP组、对照组和空白组则没有。
4．全细胞膜片钳技术记录转染阳性的MSCs的电流，可记录到时间和电压依赖性的超极化激活的内向电流，其激活电压为-70mV～-84mV。GFP组、对照组和空白组的MSCs细胞均不能记录到起搏电流。Lentiv-GFP-hHCN1和Lentiv-GFP-hHCN4共转染的3组电流密度较hHCN1、hHCN4单独转染组的大。
结论：
1．骨髓贴壁细胞分离法可以成功分离、纯化大鼠骨髓间充质干细胞。
2．hHCN1和hHCN4通过慢病毒载体可成功转染MSCs，并表达相应通道蛋白及产生相应的电流，这为心律失常的基因治疗奠定了基础。
3．转染阳性的MSCs能够表达HCN通道特点并产生If，这为体外构建生物起搏奠定了基础。
Background:Sick sinus syndrome is a common clinical disease,but the mechanism is yet to bedefined. In the present medical condition, we mainly use the permanent electronic pacemaker. Butwith the advantages come with the disadvantages, such as limited battery life, the need for permanentimplanation of electrodes into the heart and lack of response to nerve-humoral factors regulation.
Lately, considering the physical function of heart and adaptability of human body, we bring upthe concept of the biological pacemaker. The related current, If,encoded by thehyperpolarization-activated cyclic-nucleotide gated, plays an important role in theprocess of spontaneous diastolic depolarization of sinoatrial cells. Therefore, we putforward bone marrow mesenchymal stem cells(MSCs) transfected with HCN as cellplatform to construct a “biological pacemaker” which provides experimental basis for thealternative to electronic pacemaker for clinical treatment in the future.
Objective:
We adopt the lentiv-GFP, carrying green fluorescent, as a vector to study thecharacteristics of the pacemaker current in the MSCs co-transfected with differentproportions of hHCN1and hHCN4. Meanwhile, we do some chemical tests on the cellstransfected positive aimed at the establishment of cell–genetically modified organismpacing therapy experimental basis in vitro.
Methods：
1. Extraction, culture and purification of SD rat MSCs.
2. Lentiv-hHCN1and Lentiv-hHCN4transfect and co-transfect the MSCs in differentproportions.
Lentiv-hHCN1and Lentiv-hHCN4in different proportions transfect the3rdgenerationof MCS, at the same time, we set hHCN1and hHCN4individually transfecting MSCs and lentiv without HCN gene as a control group.
3.Detection of Lentiv-GFP-hHCN1and Lentiv-GFP-hHCN4transfection.
Observe the expression of the green immumofluorescene with a fluorescenemicroscopy.
Real-time quantitative polymerase chain reaction(RT-PCR) detection of the hHCN1and hHCN4expression.
The expression of hHCN1and hHCN4protein detected by Western blot.4.We use whole cell patch clamp technique to record the pacemaker current in the cellstransfected with hHCN1and hHCN4.
Results:
1.Most primary cultured MSCs were fibroblast-like spindle cells in dispersedarrangement under ordinary invert microscope.
2.The hHCN1/hHCN4transfect and co-transfect the MSCs successfully and each groupexpresses green fluorescent protein under a fluorescence microscope with a transfectionefficiency of about50-70%.
3.RT-PCR confirmed the existence of mRNA in the positive transfected MSCs. We cansee bands around99kDa and150kDa in cells transfected with hHCN1and hHCN4inWestern blot.
4. The whole cell patch clamp technique was used to record positive transfected MSCscurrent, and we got the voltage-dependence of hyperpolarization-activated inwardcurrent which activated at the voltage-75mV. In contrast, there was no pacemakercurrent in GFP group and the control group of MSCs. The current density in groupsco-transfected with hHCN1and hHCN4were larger than that of transfected with hHCN1and hHCN4individually.
Conclusion:
1.rat bone marrow mesenchymal stem cells can be successfully extracted and purifiedwith bone marrow adherent cell separation method.
2.hHCN1and hHCN4by lentiviral vectors successfully transfected MSCs which expressed the corresponding channel protein and current which laid the foundation forgene therapy for arrhythmia.
3.Positive transfected MSCs were able to express the characteristics of HCN channelsand generate the If which builded the foundation for constructing the biologicalpacemaker in vitro.
引文
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