【作者】 马金；

【导师】 张存泰；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 研究背景目前，治疗严重心动过缓的主要措施是植入电子起搏器。但是这种治疗方法仍存在一些缺陷与不足，如电池寿命有限、心脏永久性植入导管、不受神经体液因素调节等。为了进一步提高患者的生活质量，希望构建一种“生物起搏器”，使其可以象正常窦房结细胞一样在人体内表达起搏电流并通过缝隙连接传导，弥补窦房结或房室结功能不足，改善病人生存状况。研究表明，超极化激活环核苷酸门控离子通道（HCN）表达的起搏电流在窦房结舒张期自动去极化过程中起着重要作用。HCN基因家族有4个成员，即HCN1～HCN4，其中HCN2对cAMP的反应强烈，有较快速的动力学等优点，是一种理想的生物起搏靶基因。干细胞具有自我增殖、多向分化潜能等优点，其研究及应用是国内外医学和生物学研究的热点之一。近年研究发现，成体干细胞不仅仅可以分化为组织特异性细胞，还具有跨系、跨胚层分化能力，同样可以分化为其他细胞或组织，为干细胞的应用开创了更为广泛的空间。此外，成体干细胞还具有易于取材，避免组织配型及免疫排斥反应，易于基因的导入及表达等优点。我们选用成体干细胞中的骨髓间充质干细胞作为传递基因的载体，利用转基因技术，将HCN2基因转入其中，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。目的本研究以mHCN2为生物起搏的靶基因，应用大鼠的骨髓间充质干细胞（MSCs）作为生物起搏的平台，通过载体质粒转染获得mHCN2基因修饰的MSCs，并检测mHCN2基因在核酸、蛋白及电流水平的表达，为生物起搏技术的应用奠定试验基础并为其可行性提供依据。方法采用密度梯度离心法和贴壁法相结合分离获得MSCs并用流式细胞术鉴定。EcoRⅠ和BamHⅠ双酶切质粒pGH-mHCN2和pIRES2-EGFP，回收目的片断，T4DNA连接酶连接。转化筛选阳性菌落，酶切和测序鉴定mHCN2是否插入pIRES2-EGFP中。脂质体转染质粒pIRES2-EGFP-mHCN2至MSCs，24～48h后，荧光显微镜下通过观察EGFP表达情况进一步判断转染效率。通过RT-PCR技术及Western blot方法检测已导入基因mHCN2的MSCs中mHCN2 mRNA和蛋白的表达情况。利用膜片钳技术分别记录转染和未转染质粒mHCN2的MSCs的内向电流，并记录Cs⁺对电流的影响。结果流式细胞仪检测结果显示获得纯度为95％的MSCs。酶切鉴定和测序结果均证明mHCN2片断插入质粒pIRES2-EGFP。荧光显微镜下可见转染了质粒的MSCs发出绿色荧光。已转染质粒MSCs的mHCN2 mRNA是未转染质粒MSCs的5．31倍（P＜0．05），mHCN2蛋白是未转染的7．55倍（P＜0．05）。转染了mHCN2基因的细胞在超极化状态下记录到电压依赖型内向电流，未转染的MSCs未记录到该种电流。该电流在-140mV时被激活，阈电位为-60mV，半最大激活电位为（-95．1±0．9）mV。Cs+明显抑制该种电流。结论成功构建真核表达质粒pIRES2-EGFP-mHCN2并通过脂质体转染方法使其在MSCs中表达。外源性mHCN2基因在MSCs中核酸和蛋白水平上均有表达，并且也成功表达了具有生理性起搏电流特征的I_HCN2。mHCN2基因修饰的MSCs有可能替代窦房结起搏细胞在自动除极过程中发挥重要作用。更多还原

【Abstract】 Background Abnormalities of impulse generation and propagation induce cardiacarrhythmias. Although electronic pacemakers are currently the mainstay of therapy for heartblock and other electrophysiological abnormalities, they are not optimal. Among theirshortcomings are limited battery life, the need for permantent catherter implantation intothe heart, and lack of response to autonomic neurohumors. For these reasons, several genetherapy approaches have been explored as potential alternatives.Hyperpolarization-activated cyclic nucleotide-gated channel （HCN） played animportant role in the automatic depolarization of the diastolic sinus node. HCN gene familyhave 4 members, HCN1-HCN4. HCN2 is an optimal biological pacemaker target genebecause it is essential for modulation funny current and maintenance of electric-physiologicfunction of pacemaker cells in sinus node. Self-proliferation and multipotency, two majoradvantages of stem cells, makes its research and application one of the hot spots in themedical and biological research activities at home and abroad. Where, embryonic stem cellsof the capacity to differentiate into all the tissues in the organism have drawn the mostattention. In spit of the tremendous potential in the medical applications, ethic controversiesarose due to the constraints its material sources. In recent years, studies found that morethan the capacities to differentiate into tissue-specific cells, adult stem cells can also have multi-lineage, multi-layer differentiation of embryos. In addition, they can differentiate intoother cells or tissues, creating broader applications of stem cells. Adult stem cells also canget easy access to material to avoid tissue typing and immunity rejection, easy for importand expression of gene. We selected rats mesenchymal stem cells, one kind of the adultstem cells, as the vector of genetic transmission, and tried to use transgenic technology toestablish the biological pacemaker, in attempt to replace electronic pacemakers in theapplication of clinical treatment.Objective This experiment was executed using selected the rats mesenchymal stemcells modified by gene HCN2 as the target. The pacing current expressed by us constructthe plasmid pIRES2-EGFP-mHCN2 carrying a gene HCN2 and markers, and transferredthe plasmid to MSCs by liposome. Gene HCN2 was detected by the expression of nucleicacid, protein, and the current level, providing the evidences on the feasibility of testing forbiological pacemaker and mesenchymal stem cells as gene transfer vectors.Methods MSCs were obtained by density gradient centrifugation method andadherence separation then identified by flow cytometer. The plasmid pGH-mHCN2 andplasmid pIRES2-EGFP were digested by EcoRⅠand BamHⅠ. The objective fragmentswere reclaimed and linked by T4 DNA Ligase. The recombinant plasmid was transformedto the competent cells and chose the masculine colony on the next day. Restriction enzymeand sequencing method were used to proof that mHCN2 was insert to pIRES2-EGFP. Theobjective gene was transfected with Lipofectamine 2000 into MSCs and the transfectingresults were observed by fluorescence microscope. The expression of mHCN2 mRNA andprotein in the transfected cells were identified by RT-PCR and Western blot. I_HCN2 wasrecorded by whole-cell patch clamp. The effect of Cs⁺ which is the specificity blocker ofpacemaker current on I_HCN2 was detected.Results MSCs were proved correctly by flow cytometer and the purity was 95%above. The identification using restriction enzyme and sequencing indicated that themHCN2 was inserted to the pIRES2-EGFP. The green fluorescence can be seen intransfected MSCs after 24 to 48 hours under fluorescence microscope. The mHCN2 mRNAin transfected MSCs is 5.31 times of the MSCs by RT-PCR （P＜0.05）. The mHCN2 protein in transfected MSCs is 7.55 times of the MSCs by Western blot （P＜0.05）. Non-transfectedMSCs demonstrated no significant voltage-dependent currents, mHCN2-transfected MSCsexpressed a large voltage-dependent inward current activating on hyperpolarizations. I_HCN2was fully activated around -140 mV with an activation threshold of -60 mV. The midpoint(V₅₀) was （-95.1±0.9） mV. Cs⁺ （4 mmol/L） obviously blocked the current.Conclusion We success construct the plasmid pIRES2-EGFP-mHCN2 and make itexpress in MSCs by Lipofectamine 2000.We demonstrate that mHCN2 gene can express inmRNA and protein levels in MSCs. mHCN2-transfected MSCs expressed the currents ofphysiological pacemaker current character. Its may substitute the sino-atrial nodepacemaker cells and play important effects in depolarization.更多还原