HCN2基因转染脂肪干细胞自体移植治疗完全性房室传导阻滞的实验研究
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摘要
目的
本课题将细胞移植与基因治疗相结合,构建HCN2重组腺病毒载体Ad.HCN2并体外转染猪脂肪干细胞(adipose-derived stem cells, ADSCs)以构建起搏细胞,并自体移植以有效起搏心室,从而治疗完全性房室传导阻滞。
方法
(一)猪ADSCs的分离、培养与鉴定
获取实验猪颈部脂肪组织,0.075%Ⅱ型胶原酶分离得到ADSCs,将ADSCs培养于完全DMEM培养基中,每72h换液1次,细胞融合达90%传代培养；观察培养细胞的形态学特点,绘制细胞生长曲线并检测细胞周期；通过CD29、CD34、CD44、CD45、HLA-DR和c-kit的流式细胞仪检测和免疫荧光染色检测细胞表面标志；将ADSCs置入成脂肪、成骨、成软骨、成肝细胞的诱导培养基进行诱导分化并鉴定分化细胞的性质。
(二)Ad.HCN2的构建及其检测
扩增目的基因HCN2,构建重组质粒pShuttleCMV. HCN2并行PCR和基因测序检测；应用阳离子脂质体转染法制备重组腺病毒Ad.HCN;将Ad.HCN2扩增、纯化并行滴度测定；HeLa细胞检测确定其使用安全性；Ad.GFP以不同感染倍数(multiplication of infection, MOI)转染ADSCs后检测转染效率,确定最佳MOI；检测Ad.GFP转染ADSCs表达强度并绘制时间曲线；检测Ad.HCN2转染ADSCs后细胞活力与细胞周期变化；绘制转染Ad.HCN2、Ad.Null以及未转染ADSCs的生长曲线,对比明确Ad.HCN2的安全性。
(三)Ad.HCN2转染猪ADSCs构建起搏细胞
Ad.HCN2转染ADSCs后, RT-PCR检测HCN2 mRNA表达；免疫荧光检测HCN2通道蛋白表达；流式细胞仪检测HCN2表达阳性细胞百分比；Western-blot检测HCN2蛋白；全细胞膜片钳技术行电生理学检测；分离培养乳鼠心肌细胞(neonatal rat's cardiomyocytes, NRCs)并行免疫组织化学鉴定；联合培养ADSCs和NRCs,观察细胞搏动情况并记录收缩频率；加入异丙肾上腺素后观察细胞收缩频率变化；将Ad.HCN2转染的ADSCs与NRCs联合培养后,行抗缝隙连接蛋白43(Connexin 43, Cx43)免疫荧光染色。
(四)起搏细胞自体移植治疗完全性房室传导阻滞
实验猪植入心脏电子起搏器后,射频消融希氏束建立完全性房室传导阻滞动物模型；于右心室前壁自体移植ADSCs;术后常规行体表心电图和24h动态心电图检查；分别注射异丙肾上腺素和阿托品以观察实验猪心率对神经递质的反应性；获取细胞移植部位心室肌,分别行HE染色观察和免疫荧光检测。结果
(一)猪ADSCs的分离、培养与鉴定
消化分离得到的ADSCs活细胞百分率为93.5%。刚接种的原代细胞呈圆形或类圆形,3h后开始有细胞贴壁,16h贴壁细胞明显增多,48h后大部分细胞已贴壁,并开始伸展。72h换液后红细胞等杂质被去除,可见贴壁细胞几乎均伸展为多角形或梭形。6-7d后细胞融合成单层,局部排列具有一定方向性,细胞变得细长,呈典型的成纤维细胞样。传代的ADSCsl h开始贴壁,5-10h后基本贴壁,24h内完全贴壁,3-4d后细胞完全融合。连续传9、10代,细胞的形态无明显变化。ADSCs第3、5、10代,细胞生长曲线基本相同。细胞传代后第1-2d,细胞种群生长处于停滞期,3-6d为细胞对数生长期,6-7d后出现平台期。第10代细胞增殖速度较第1、5代慢。培养细胞G0/G1期的细胞比例为89.3%。流式细胞仪和免疫荧光检测结果显示第3代ADSCs对CD29和CD44的表达均为阳性,CD34、CD45、c-kit、HLA-DR表达弱阳性或不表达。ADSCs在不同的诱导环境下,可分化成脂肪细胞、骨细胞、软骨细胞和肝细胞。
(二)Ad.HCN2的构建及其检测
PCR扩增后可见特异性和基因测序证实成功构建pShuttleCMV.HCN2。PCR确定成功构建pAd.HCN2。应用脂质体转染方法,将重组腺病毒质粒pAd.HCN2导入HEK293细胞,产生重组腺病毒。PCR结果表明重组腺病毒已携带目的基因HCN2。滴度测定测得第四代Ad.HCN2滴度为1.5×109pfu/ml。Ad.HCN2以MOI=50值转染HeLa细胞后,观察7d,细胞未出现致细胞病变效应(cytopathic effect, CPE),各时间点细胞计数,结果显示实验组和对照组之间统计学差异不显著。Ad.GFP转染ADSCs后,48-72h后开始表达绿色荧光蛋白,荧光显微镜下可见细胞呈绿色荧光。不同的MOI值得到不同的转染效率。随着MOI值的增加,GFP阳性细胞比率也增加,显微镜下细胞表达绿色荧光更强。MOI=50时,ADSCs转染率>95%,同时细胞状态明显优于MOI=100、200的细胞状态。Ad.GFP转染ADSCs 72 h后观察可见各组细胞均开始表达绿色荧光蛋白,表达强度随时间变化。Ad.HCN2转染ADSCs后,转染组与对照组在3d和10d时细胞活力统计学差异不显著。细胞周期转染组与对照组在3d和10d时两组统计学差异不显著,同一组细胞在3d和10d之间统计学差异不显著。转染Ad.HCN2、Ad.Null以及未转染ADSCs各组之间增殖速度统计学差异不显著。
(三)Ad.HCN2转染猪ADSCs构建起搏细胞
RT-PCR后所得两条泳道28S和18S条带明亮、清晰、锐利,28S的亮度在18S条带的两倍以上。保温试验显示两样本18S、28S条带无差异。PCR扩增得到的目的条带与和携带HCN2基因的重组质粒所得条带相同。免疫荧光检测可见细胞呈红色荧光。流式细胞仪检测显示,Ad.HCN2转染组阳性细胞百分比明显高于Ad.Null转染组与未转染组,与空白对照和阴性对照阳性细胞百分比统计学存在明显差异,而Ad.Null转染组与未转染组、空白对照和阳性对照之间统计学差异不显著。Western-blot检测结果,Ad.HCN2转染组中得到特异性电泳条带,其分子量为96kd。全细胞膜片钳行电生理学检测,实验组可得到超极化激活的内向电流(If),其激活电位约为-60 mV,完全激活电位约为-140 mV,呈电压依赖性。实验组细胞外液中加入4 mmol/L的CsCl2后,If几乎完全被抑制。洗脱CsCl2后,If重新出现。ADSCs与NRCs联合培养,实验组2d即可看到细胞协调同步收缩,对照组均未出现细胞收缩。5d后阳性对照组可见到部分同步收缩的NRCs,收缩频率较实验组慢。观察21d,统计各组细胞收缩频率,结果提示实验组和阳性对照组之间统计学差异显著,而阴性对照和空白对照组未见细胞收缩。培养5d的各组细胞中加入异丙肾上腺素,实验组与阳性对照组细胞收缩频率增快,实验组与阳性对照组之间心率增加次数统计学差异显著。抗Cx43免疫荧光染色,可见细胞之间有绿色荧光。
(四)起搏细胞自体移植治疗完全性房室传导阻滞
完全性房室传导阻滞动物模型建立后,体表心电图可见P波与QRS波完全分离,心房率快于心室率,室性心率<50次/分(beat per minute, bpm)。ADSCs自体移植后48h内,各组动物心率之间统计学差异不显著。72h后实验组动物自主室性心率明显增快。移植后7d心率趋于稳定并固定在峰值。72h后实验组与对照组之间心率统计学差异显著。而阴性对照组和空白对照组动物之间心率统计学差异不显著。实验组动物体表心电图QRS波和起搏标测QRS波的形态和方向相似。24 h Holter检查显示,实验组与对照组心率相比统计学差异显著。实验组动物心率在移植2w后为70.2±2.9 bpm,移植6w后降为54.9±3.4 bpm。注射异丙肾上腺素后,实验组和各对照组动物心率均增加,统计学差异显著。实验组与阴性对照和空白对照组心率增加次数对比,统计学差异显著。注射阿托品后,实验组和各对照组动物心率均增加,但实验组前后心率统计学差异不显著,阴性对照和空白对照组前后心率统计学差异显著。实验组与阴性对照和空白对照组心率增加次数对比,统计学差异显著。取细胞注射部位心室肌行HE染色,移植组可见ADSCs细胞核深染,实验组和对照组注射部位未发现明显纤维组织增生。免疫荧光检测可见2w后所得标本细胞注射部位可见红色荧光。
结论
(一)猪ADSCs的分离、培养与鉴定
ADSCs来源广泛,脂肪组织抽取后残留于供区的脂肪组织仍可扩增,容易大量获得,对供区损伤小。ADSCs的分离效率较其他干细胞高,扩增能力强,传代培养易于获得大量有分化能力的细胞。ADSCs表达CD29和CD44,显示其具有间充质干细胞特性；ADSCs不表达CD34、CD45、c-kit,证明其为非造血类细胞。而其MHCⅡ相关蛋白HLA-DR近不表达,说明它们具有极少量与移植排斥相关的抗原分子,提示ADSCs具有免疫逃逸特性,具备异体移植的可能性。成脂、成骨、成软骨及成肝细胞诱导结果证明ADSCs是具有多向分化能力的干细胞。
(二)Ad.HCN2的构建及其检测
构建的重组腺病毒携带目的基因HCN2。HeLa细胞未出现CPE,说明制备的重组腺病毒中未混有野生型病毒,体外转染安全。MOI=50的Ad.GFP转染率较高,且细胞状态好,为了达到最佳量效比,后期实验均选择MOI=50作为标准的转染倍数。Ad.GFP转染ADSCs后,目的基因表达随时间改变逐渐增强,在7d左右表达强度最高,之后随时间延长逐渐下降。Ad.HCN2转染的ADSCs细胞活力和细胞周期无变化,Ad.HCN2对ADSCs无毒副作用。
(三)Ad.HCN2转染猪ADSCs构建起搏细胞
RT-PCR证实Ad.HCN2转染ADSCs后,细胞内有HCN2 mRNA合成。保温试验证实我们提取的RNA样品纯度高,可以满足进一步实验要求。Ad.HCN2转染的ADSCs可表达HCN2基因。免疫荧光提示转染细胞有HCN2蛋白表达,其表达后功能区域位于细胞膜。流式细胞仪检测提示Ad.HCN2转染的ADSCs细胞膜上HCN2通道蛋白的高表达。Western-blot检测结果说明Ad.HCN2转染的ADSCs后可高效表达特异性目的蛋白HCN2。全细胞膜片钳证实转染细胞能产生If。ADSCs与NRCs联合培养,Ad.HCN2转染的ADSCs能有效起搏NRCs,并且对神经递质调节有良好的反应性。联合培养后,ADSCs和NRCs之间可形成Cx43。
(四)起搏细胞自体移植治疗完全性房室传导阻滞
ADSCs自体移植后,起搏细胞可有效起搏心室。对比实验组动物体表心电图QRS波和起搏标测QRS波的形态和方向,表明实验组室性心律起源于细胞移植区。24 h Holter结果提示,心率呈先上升后下降趋势。实验组动物对异丙肾上腺素调节反应良好,对阿托品调节无反应。HE染色说明ADSCs移植后能在心肌组织内有效成活,移植部位炎症反应轻微。免疫荧光检测提示自体移植的ADSCs在心肌组织内能够有效表达HCN2通道蛋白。
Objective
In this study, we aim to develop an alternative treatment of complete atrioventricular block (AVB). To do this we use combined cell and gene therapy to construct a biological pacemaker based on porcine adipose derived stem cells (ADSCs) adenovirally transduced with HCN2. Sustained ventricular pacing could indeed be demonstrated in a porcine model of complete heart block
Methods
1. Isolation, cultivation and identification of porcine ADSCs
Adipose tissue was obtained from the neck of swine and isolated by 0.075%Ⅱcollagenase to obtain ADSCs. ADSCs were cultured in DMEM medium with 10% fetal bovine serum, and the medium was changed every 72 h. The cells were split after they reached 90% confluence. The morphological characteristics of the cultured cells were observed, and the growth curve and cycle of the cells were detected. Flow Cytometry and immunofluorescence detected the cell surface markers CD29、CD34、CD44、CD45、HLA-DR and c-kit. After induced differentiation of ADSCs in adipogenic, osteogenic, chondrogenic and hepatic cell lines, and then the characters of the differentiated cells were identified.
2. Construction and examination of Ad.HCN2
Previously constructed pShuttleCMV.HCN2 used to generate a first seeding stock (using cationic transduction) of adenovirus. Subsequently, Ad.HCN2 was amplified, purified, and titrated. To measure Ad.HCN2 vector cytotoxicity, Hele-cells were transduced and the minimal multiplicity of infection (MOI) that induces cytotoxicity was detected. Thereafter, a similar experiment was performed to determine cytotoxicity of both Ad.GFP and Ad.HCN2 in ADSCs. To quantify cell cycle activity and cell viability we respectively constructed growth curves and performed flow cytometry.
3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCs
Ad.HCN2-transduced ADSCs were collected and tested for the expression of HCN2 mRNA (RT-PCR), and protein (immunofluorescence and Western-blot). The percentage of HCN2-expressing cells was calculated by flow cytometry. The electrophysiology of Ad.HCN2-transduced ADSCs was detected by patch clamp. To test functionality of the HCN2 loaded ADSCs we performed a coculture experiment with cultured neonatalrat's cardiomyocytes (NRCs). First, we isolated NRCs and characterized the obtained cells using immunohistochemistry. Subsequently, NRCs were cocultured with HCN2 loaded ADSCs and beating frequencies, both at baseline and in the presence of isoproterenol, were counted. In addition, Cx43 immunofluorescence was detected between Ad.HCN2-transduced ADSCs and NRCs.
4. Transplantation of autologous pacemaker cells in treatment of complete atrioventricular block
To establish in vivo function of HCN2 loaded ADSCs, we performed autologous transplantation studies in porcine with complete AVB. First, we implanted a right ventricular endocardial pacemaker and created AVB using His-bundle procedures. Two days later, we exposed the heart through a right lateral thoracotomy and injected pacemaker cells in a single site of the right anterior ventricular wall. After implantation, ECG and 24h Holter was regularly performed. In addition we tested autonomic response using isoproterenol and atropine. After termination, HE staining and immunofluorescence were performed on the injection site.
Results
1. Isolation, cultivation and identification of porcine ADSCs
Cell viability of ADSCs after isolation was 93.5%. And they looked round or in round-like shape.3 h later, some cells attached to the plate, and the number of adherent cells dramatically increased after 16 h. Most of the cells got attached after 48 h, and then started to stretch. Media were changed with the removal of red blood cells and some other debris after 72 h. Cells had polygon or spindle-like shape.6-7 d later, after cells got confluent into a single layer, local arrangement had a certain direction, and the cells became elongated, showing a typical fibroblast-like shape.1 h after the passage of ADSCs, they started to adhere to the plates.5-10 h later, the adherence increased.24 hours later, cells completely became adherent.3-4 d later, the cells got fully confluent. There were no significant changes in cell morphology after continuous passage of 9 to 10 generations. Cell growth curves of the 3rd,5th, and 10th generation of ADSCs were similar.1-2 d after passage, cell growth was at a standstill period.3-6 d later, cells entered the logarithmic growth phase.6-7 d later, cell growth stayed in plateau phase. The proliferation rate of the 10th generation was much slower than the 1st and 5th generation. 89.3% of cells were in G0/G1 phase. The expression of CD29 and CD44 were both positive in the 3rd generation of ADSCs shown by flow cytometry and immunofluorescence assay. The expressions of CD34, CD45, c-kit, HLA-DR were weakly positive or not expressed. ADSCs induced in different environments could be differentiated into adipose cells, osteocytes, chondrocytes and hepatocytes.
2. Construction and examination of Ad.HCN2
Construction of the pShuttleCMV.HCN2 was confirmed by PCR and gene sequencing. Construction of the pAd.HCN2 was confirmed by PCR. By cationic transduction, recombinant adenovirus plasmid pAd.HCN2 was introduced into HEK293 cells to produce recombinant adenovirus. PCR results confirmed that the recombinant gene carries HCN2 gene. The titer of the fourth generation of Ad.HCN2 was 1.5×109 pfu/ml. HeLa cells transduced with Ad.HCN2 at MOI= 50 did not appear cytopathic effect (CPE) after 7 days, since cell numbers at different time points showed no statistical difference between the experimental group and control group.48-72 h after ADSCs were transduced with Ad.GFP, green fluorescent cells were visible under fluorescence microscopy. Different MOI values correspond to different rates of transduction efficiency. With an increase in MOI values, the ratio of GFP-positive cells was also increased, together with a higher green fluorescence under fluorescence microscope. When MOI=50, the transduced rate of ADSCs was over 95%, and meanwhile cells looked much better than cells at the conditions of MOI=100 or 200.72 h after ADSCs were transduced with Ad.GFP, GFP protein expression was observed and the intensity of green fluorescence increased over time. There was no statistical significance of cell viability between transduced group and control groups 3 d and 10 d after ADSCs were transduced by Ad.HCN2. Neither was the cell cycle between the transduction group and the control group, and even among the same group of cells. No statistical significance in proliferation rate was observed between the Ad.HCN2-transduced group, Ad.Null-transduced group and non-transduced ADSCs groups.
3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCs
After RT-PCR, two sharp bands were observed as 28S and 18S bands. The brightness of 28S was twice more than that of the 18S band. There was no difference of 28S and 18S between two different groups after incubation test. The amplified band in the experimental group was the same with the band derived from the recombinant plasmid carrying HCN2 gene. Red fluorescent cells were visible by immunofluorescence detection. Flow cytometry results showed that, the percentage of Ad.HCN2-positive cells was significantly higher in Ad.HCN2-transduced group than that in Ad.Null-transduced group and non-transduced group, with significant statistical differences compared with the blank control and positive control. However, no statistically significant difference was observed between Ad.Null-transduced group and non-transduced group, or between the blank control and positive control, suggesting the high expression of HCN2 channel protein in the membrane of Ad.HCN2-transduced ADSCs. Western blot result indicated that, a specific electrophoretic band with molecular weight of 96 kd was observed in Ad.HCN2-transduced group. By patch-clamp, a hyperpolarization-activated inward current (If) was detected in the experimental group, with the activation potential of about-60 mV, and fully activation potential of-140 mV, which was voltage-dependent. If was almost completely suppressed when the media was added with 4 mmol/L of CsCl2 in experimental group. After elution of CsCl2, If reappeared. When ADSCs were cocultured with the NRCs, after 2 d, synchronized contraction of cells was only observed in the experimental group, but not in the control group.5 d later, some of the NRCs in positive control group showed synchronized contraction, but the contraction frequency was slower than that of the experimental group.21 d later, there was significant statistical difference in cell contraction frequency between the experimental group and the positive control group, while no cell contraction was observed between the negative control and blank control group. The cells in each group were added isoprenaline, cell contraction frequency in both experimental group and the positive control group was increased, with an even higher heart rate in the experimental group compared with the positive control group. Anti-Cx43 immunofluorescence staining could be observed under fluorescence microscope.
4. Transplantation of autologous pacemaker cells in treatment of complete atrioventricular block
The surface ECG showed P wave and QRS wave was totally separated in the animal mode of complete atrioventricular block. The rate of the ventricular, which was under 50 bpm, was slower than the rate of atrium.48 h after implantation, there was no statistical significance of heart rate among each group. After 72 h, the heart rate of experimental group became faster significantly, and the rate was stable at maximum after 7 d. There were significant statistical differences compared with the experimental group and the control groups after 7 d, but no statistically significant difference was observed between the negative control and the blank control. The shape and direction of the QRS was the same between the ECG and pace mapping of the experimental group. The result of 24 h Holter showed that there were significant statistical differences compared with the experimental group and the control groups. The heart rate of the experimental group was decreased from 70.2±2.9 bpm after 2 w to 54.9±3.4 bpm after 6 w. Added isoprenaline, the rate of all animals increased, and there was significantly statistical difference among all groups. And there were significant statistical differences in increased heart rates compared with the experimental group and the control groups. Added atropine, the rate of all animals increased as well. No statistically significant difference of changed heart rate in the experimental group was observed, but there was significant statistical difference of the control groups. And there were significant statistical differences in increased heart rates compared with the experimental group and the control groups. HE staining showed there was no significant fibroplasia in injection region and immunochemistry showed red fluorescent cells were visible.
Conclusion
1. Isolation, cultivation and identification of porcine ADSCs
The resource of ADSCs is widespread. The residue adipose tissue at donor site still has the ability to amplify and is easy to obtain in a large number, without severe damage. Comparing to other stem cells, ADSCs have higher isolation efficiency and stronger amplification ability, which render them easy to passage and obtain in large quantities. ADSCs express CD29 and CD44, which indicates theirs characteristics of mesenchymal stem cells; ADSCs have no expression of CD34, CD45 and c-kit, which demonstrates that they are non-hematopoietic. MHC II associated protein HLA-DR is rarely expressed in ADSCs, which suggests they have only very few antigen molecules related to immunological rejection and they have the potential of xenograft due to their possible ability of immune escape. Adipogenic, osteogenic, osteogenic and heptogenic differentiation demonstrates that ADSCs are multipotent.
2. Construction and examination of Ad.HCN2
Recombinant adenovirus carrier Ad.HCN2 was constructed by cloning HCN2 into adenovirus vector. CPE didn't show in infected Hela cells, which demonstrates there is no wild type virus in the recombinant adenovirus and it is safe to infect cells in vitro. In order to get the best cost-effectiveness ratio, MOI=50 is taken as the standard because of the high infection efficiency of Ad.GFP with MOI=50 and good condition of cells. The intensity of objective gene increased over time after transduced, which reached the top after 7 d, and then decreased. The cell viability and cell cycle of ADSCs didn't change after transduced by Ad.HCN2, which showed Ad.HCN2 was no harm to ADSCs. Recombinant adenovirus carrier Ad.HCN2 was constructed by cloning HCN2 into adenovirus vector. CPE was not observed in infected Hela cells, which demonstrates absence of wild type virus which indicates that the prep is safe to infect cells in vitro. In order to get the best maximal protein expression and minimal cytotoxicity a MOI=50 is used. The GFP fluorescence intensity increased over time, reached peak at day 7, and then decreased. Cell viability and cell cycle of ADSCs didn't change after Ad.HCN2 transduction, which demonstrated absence of negative effects from Ad.HCN2 on these parameters.
3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCs
RT-PCR demonstrated the expression of HCN2 mRNA in Ad.HCN2 transduced ADSCs. The incubation test further showed that the RNA samples were of high purity. In addition, we demonstrated HCN2 immunofluorescence on the membrane of Ad.HCN2-transduced ADSCs. Expression of HCN2 channel protein was most quantity in the Ad.HCN2 transduced-ADSCs with FCM and Western blot. The patch clamp showed that Ad.HCN2 transduced-ADSCs could produce If. Ad.HCN2 transduced-ADSCs could pace the NRCs effectually after cocultured them, and they showed positive to isoprenaline. Cx43 could be detected between ADSCs and NRCs after cocultured.
4. Transplantation of autologous pace cells in treatment of complete atrioventricular block
The pacemaker cells could pace the ventricle effectively after its autotransplantation. The ECG showed the ventricular heart rhythm of experimental group was from the injection region of cells. The result of 24 h Holter showed that the heart rate increased first and then decreased. The heart rate of experimental group showed positive to isoprenaline and negative to. HE staining showed ADSCs survived after implantation and there was no significant inflammatory reaction in injection region. Immunochemistry showed Ad.GFP transduced-ADSCs could express HCN2 channel protein in myocardium.
本课题将细胞移植与基因治疗相结合,构建HCN2重组腺病毒载体Ad.HCN2并体外转染猪脂肪干细胞(adipose-derived stem cells, ADSCs)以构建起搏细胞,并自体移植以有效起搏心室,从而治疗完全性房室传导阻滞。
方法
(一)猪ADSCs的分离、培养与鉴定
获取实验猪颈部脂肪组织,0.075%Ⅱ型胶原酶分离得到ADSCs,将ADSCs培养于完全DMEM培养基中,每72h换液1次,细胞融合达90%传代培养；观察培养细胞的形态学特点,绘制细胞生长曲线并检测细胞周期；通过CD29、CD34、CD44、CD45、HLA-DR和c-kit的流式细胞仪检测和免疫荧光染色检测细胞表面标志；将ADSCs置入成脂肪、成骨、成软骨、成肝细胞的诱导培养基进行诱导分化并鉴定分化细胞的性质。
(二)Ad.HCN2的构建及其检测
扩增目的基因HCN2,构建重组质粒pShuttleCMV. HCN2并行PCR和基因测序检测；应用阳离子脂质体转染法制备重组腺病毒Ad.HCN;将Ad.HCN2扩增、纯化并行滴度测定；HeLa细胞检测确定其使用安全性；Ad.GFP以不同感染倍数(multiplication of infection, MOI)转染ADSCs后检测转染效率,确定最佳MOI；检测Ad.GFP转染ADSCs表达强度并绘制时间曲线；检测Ad.HCN2转染ADSCs后细胞活力与细胞周期变化；绘制转染Ad.HCN2、Ad.Null以及未转染ADSCs的生长曲线,对比明确Ad.HCN2的安全性。
(三)Ad.HCN2转染猪ADSCs构建起搏细胞
Ad.HCN2转染ADSCs后, RT-PCR检测HCN2 mRNA表达；免疫荧光检测HCN2通道蛋白表达；流式细胞仪检测HCN2表达阳性细胞百分比；Western-blot检测HCN2蛋白；全细胞膜片钳技术行电生理学检测；分离培养乳鼠心肌细胞(neonatal rat's cardiomyocytes, NRCs)并行免疫组织化学鉴定；联合培养ADSCs和NRCs,观察细胞搏动情况并记录收缩频率；加入异丙肾上腺素后观察细胞收缩频率变化；将Ad.HCN2转染的ADSCs与NRCs联合培养后,行抗缝隙连接蛋白43(Connexin 43, Cx43)免疫荧光染色。
(四)起搏细胞自体移植治疗完全性房室传导阻滞
实验猪植入心脏电子起搏器后,射频消融希氏束建立完全性房室传导阻滞动物模型；于右心室前壁自体移植ADSCs;术后常规行体表心电图和24h动态心电图检查；分别注射异丙肾上腺素和阿托品以观察实验猪心率对神经递质的反应性；获取细胞移植部位心室肌,分别行HE染色观察和免疫荧光检测。结果
(一)猪ADSCs的分离、培养与鉴定
消化分离得到的ADSCs活细胞百分率为93.5%。刚接种的原代细胞呈圆形或类圆形,3h后开始有细胞贴壁,16h贴壁细胞明显增多,48h后大部分细胞已贴壁,并开始伸展。72h换液后红细胞等杂质被去除,可见贴壁细胞几乎均伸展为多角形或梭形。6-7d后细胞融合成单层,局部排列具有一定方向性,细胞变得细长,呈典型的成纤维细胞样。传代的ADSCsl h开始贴壁,5-10h后基本贴壁,24h内完全贴壁,3-4d后细胞完全融合。连续传9、10代,细胞的形态无明显变化。ADSCs第3、5、10代,细胞生长曲线基本相同。细胞传代后第1-2d,细胞种群生长处于停滞期,3-6d为细胞对数生长期,6-7d后出现平台期。第10代细胞增殖速度较第1、5代慢。培养细胞G0/G1期的细胞比例为89.3%。流式细胞仪和免疫荧光检测结果显示第3代ADSCs对CD29和CD44的表达均为阳性,CD34、CD45、c-kit、HLA-DR表达弱阳性或不表达。ADSCs在不同的诱导环境下,可分化成脂肪细胞、骨细胞、软骨细胞和肝细胞。
(二)Ad.HCN2的构建及其检测
PCR扩增后可见特异性和基因测序证实成功构建pShuttleCMV.HCN2。PCR确定成功构建pAd.HCN2。应用脂质体转染方法,将重组腺病毒质粒pAd.HCN2导入HEK293细胞,产生重组腺病毒。PCR结果表明重组腺病毒已携带目的基因HCN2。滴度测定测得第四代Ad.HCN2滴度为1.5×109pfu/ml。Ad.HCN2以MOI=50值转染HeLa细胞后,观察7d,细胞未出现致细胞病变效应(cytopathic effect, CPE),各时间点细胞计数,结果显示实验组和对照组之间统计学差异不显著。Ad.GFP转染ADSCs后,48-72h后开始表达绿色荧光蛋白,荧光显微镜下可见细胞呈绿色荧光。不同的MOI值得到不同的转染效率。随着MOI值的增加,GFP阳性细胞比率也增加,显微镜下细胞表达绿色荧光更强。MOI=50时,ADSCs转染率>95%,同时细胞状态明显优于MOI=100、200的细胞状态。Ad.GFP转染ADSCs 72 h后观察可见各组细胞均开始表达绿色荧光蛋白,表达强度随时间变化。Ad.HCN2转染ADSCs后,转染组与对照组在3d和10d时细胞活力统计学差异不显著。细胞周期转染组与对照组在3d和10d时两组统计学差异不显著,同一组细胞在3d和10d之间统计学差异不显著。转染Ad.HCN2、Ad.Null以及未转染ADSCs各组之间增殖速度统计学差异不显著。
(三)Ad.HCN2转染猪ADSCs构建起搏细胞
RT-PCR后所得两条泳道28S和18S条带明亮、清晰、锐利,28S的亮度在18S条带的两倍以上。保温试验显示两样本18S、28S条带无差异。PCR扩增得到的目的条带与和携带HCN2基因的重组质粒所得条带相同。免疫荧光检测可见细胞呈红色荧光。流式细胞仪检测显示,Ad.HCN2转染组阳性细胞百分比明显高于Ad.Null转染组与未转染组,与空白对照和阴性对照阳性细胞百分比统计学存在明显差异,而Ad.Null转染组与未转染组、空白对照和阳性对照之间统计学差异不显著。Western-blot检测结果,Ad.HCN2转染组中得到特异性电泳条带,其分子量为96kd。全细胞膜片钳行电生理学检测,实验组可得到超极化激活的内向电流(If),其激活电位约为-60 mV,完全激活电位约为-140 mV,呈电压依赖性。实验组细胞外液中加入4 mmol/L的CsCl2后,If几乎完全被抑制。洗脱CsCl2后,If重新出现。ADSCs与NRCs联合培养,实验组2d即可看到细胞协调同步收缩,对照组均未出现细胞收缩。5d后阳性对照组可见到部分同步收缩的NRCs,收缩频率较实验组慢。观察21d,统计各组细胞收缩频率,结果提示实验组和阳性对照组之间统计学差异显著,而阴性对照和空白对照组未见细胞收缩。培养5d的各组细胞中加入异丙肾上腺素,实验组与阳性对照组细胞收缩频率增快,实验组与阳性对照组之间心率增加次数统计学差异显著。抗Cx43免疫荧光染色,可见细胞之间有绿色荧光。
(四)起搏细胞自体移植治疗完全性房室传导阻滞
完全性房室传导阻滞动物模型建立后,体表心电图可见P波与QRS波完全分离,心房率快于心室率,室性心率<50次/分(beat per minute, bpm)。ADSCs自体移植后48h内,各组动物心率之间统计学差异不显著。72h后实验组动物自主室性心率明显增快。移植后7d心率趋于稳定并固定在峰值。72h后实验组与对照组之间心率统计学差异显著。而阴性对照组和空白对照组动物之间心率统计学差异不显著。实验组动物体表心电图QRS波和起搏标测QRS波的形态和方向相似。24 h Holter检查显示,实验组与对照组心率相比统计学差异显著。实验组动物心率在移植2w后为70.2±2.9 bpm,移植6w后降为54.9±3.4 bpm。注射异丙肾上腺素后,实验组和各对照组动物心率均增加,统计学差异显著。实验组与阴性对照和空白对照组心率增加次数对比,统计学差异显著。注射阿托品后,实验组和各对照组动物心率均增加,但实验组前后心率统计学差异不显著,阴性对照和空白对照组前后心率统计学差异显著。实验组与阴性对照和空白对照组心率增加次数对比,统计学差异显著。取细胞注射部位心室肌行HE染色,移植组可见ADSCs细胞核深染,实验组和对照组注射部位未发现明显纤维组织增生。免疫荧光检测可见2w后所得标本细胞注射部位可见红色荧光。
结论
(一)猪ADSCs的分离、培养与鉴定
ADSCs来源广泛,脂肪组织抽取后残留于供区的脂肪组织仍可扩增,容易大量获得,对供区损伤小。ADSCs的分离效率较其他干细胞高,扩增能力强,传代培养易于获得大量有分化能力的细胞。ADSCs表达CD29和CD44,显示其具有间充质干细胞特性；ADSCs不表达CD34、CD45、c-kit,证明其为非造血类细胞。而其MHCⅡ相关蛋白HLA-DR近不表达,说明它们具有极少量与移植排斥相关的抗原分子,提示ADSCs具有免疫逃逸特性,具备异体移植的可能性。成脂、成骨、成软骨及成肝细胞诱导结果证明ADSCs是具有多向分化能力的干细胞。
(二)Ad.HCN2的构建及其检测
构建的重组腺病毒携带目的基因HCN2。HeLa细胞未出现CPE,说明制备的重组腺病毒中未混有野生型病毒,体外转染安全。MOI=50的Ad.GFP转染率较高,且细胞状态好,为了达到最佳量效比,后期实验均选择MOI=50作为标准的转染倍数。Ad.GFP转染ADSCs后,目的基因表达随时间改变逐渐增强,在7d左右表达强度最高,之后随时间延长逐渐下降。Ad.HCN2转染的ADSCs细胞活力和细胞周期无变化,Ad.HCN2对ADSCs无毒副作用。
(三)Ad.HCN2转染猪ADSCs构建起搏细胞
RT-PCR证实Ad.HCN2转染ADSCs后,细胞内有HCN2 mRNA合成。保温试验证实我们提取的RNA样品纯度高,可以满足进一步实验要求。Ad.HCN2转染的ADSCs可表达HCN2基因。免疫荧光提示转染细胞有HCN2蛋白表达,其表达后功能区域位于细胞膜。流式细胞仪检测提示Ad.HCN2转染的ADSCs细胞膜上HCN2通道蛋白的高表达。Western-blot检测结果说明Ad.HCN2转染的ADSCs后可高效表达特异性目的蛋白HCN2。全细胞膜片钳证实转染细胞能产生If。ADSCs与NRCs联合培养,Ad.HCN2转染的ADSCs能有效起搏NRCs,并且对神经递质调节有良好的反应性。联合培养后,ADSCs和NRCs之间可形成Cx43。
(四)起搏细胞自体移植治疗完全性房室传导阻滞
ADSCs自体移植后,起搏细胞可有效起搏心室。对比实验组动物体表心电图QRS波和起搏标测QRS波的形态和方向,表明实验组室性心律起源于细胞移植区。24 h Holter结果提示,心率呈先上升后下降趋势。实验组动物对异丙肾上腺素调节反应良好,对阿托品调节无反应。HE染色说明ADSCs移植后能在心肌组织内有效成活,移植部位炎症反应轻微。免疫荧光检测提示自体移植的ADSCs在心肌组织内能够有效表达HCN2通道蛋白。
Objective
In this study, we aim to develop an alternative treatment of complete atrioventricular block (AVB). To do this we use combined cell and gene therapy to construct a biological pacemaker based on porcine adipose derived stem cells (ADSCs) adenovirally transduced with HCN2. Sustained ventricular pacing could indeed be demonstrated in a porcine model of complete heart block
Methods
1. Isolation, cultivation and identification of porcine ADSCs
Adipose tissue was obtained from the neck of swine and isolated by 0.075%Ⅱcollagenase to obtain ADSCs. ADSCs were cultured in DMEM medium with 10% fetal bovine serum, and the medium was changed every 72 h. The cells were split after they reached 90% confluence. The morphological characteristics of the cultured cells were observed, and the growth curve and cycle of the cells were detected. Flow Cytometry and immunofluorescence detected the cell surface markers CD29、CD34、CD44、CD45、HLA-DR and c-kit. After induced differentiation of ADSCs in adipogenic, osteogenic, chondrogenic and hepatic cell lines, and then the characters of the differentiated cells were identified.
2. Construction and examination of Ad.HCN2
Previously constructed pShuttleCMV.HCN2 used to generate a first seeding stock (using cationic transduction) of adenovirus. Subsequently, Ad.HCN2 was amplified, purified, and titrated. To measure Ad.HCN2 vector cytotoxicity, Hele-cells were transduced and the minimal multiplicity of infection (MOI) that induces cytotoxicity was detected. Thereafter, a similar experiment was performed to determine cytotoxicity of both Ad.GFP and Ad.HCN2 in ADSCs. To quantify cell cycle activity and cell viability we respectively constructed growth curves and performed flow cytometry.
3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCs
Ad.HCN2-transduced ADSCs were collected and tested for the expression of HCN2 mRNA (RT-PCR), and protein (immunofluorescence and Western-blot). The percentage of HCN2-expressing cells was calculated by flow cytometry. The electrophysiology of Ad.HCN2-transduced ADSCs was detected by patch clamp. To test functionality of the HCN2 loaded ADSCs we performed a coculture experiment with cultured neonatalrat's cardiomyocytes (NRCs). First, we isolated NRCs and characterized the obtained cells using immunohistochemistry. Subsequently, NRCs were cocultured with HCN2 loaded ADSCs and beating frequencies, both at baseline and in the presence of isoproterenol, were counted. In addition, Cx43 immunofluorescence was detected between Ad.HCN2-transduced ADSCs and NRCs.
4. Transplantation of autologous pacemaker cells in treatment of complete atrioventricular block
To establish in vivo function of HCN2 loaded ADSCs, we performed autologous transplantation studies in porcine with complete AVB. First, we implanted a right ventricular endocardial pacemaker and created AVB using His-bundle procedures. Two days later, we exposed the heart through a right lateral thoracotomy and injected pacemaker cells in a single site of the right anterior ventricular wall. After implantation, ECG and 24h Holter was regularly performed. In addition we tested autonomic response using isoproterenol and atropine. After termination, HE staining and immunofluorescence were performed on the injection site.
Results
1. Isolation, cultivation and identification of porcine ADSCs
Cell viability of ADSCs after isolation was 93.5%. And they looked round or in round-like shape.3 h later, some cells attached to the plate, and the number of adherent cells dramatically increased after 16 h. Most of the cells got attached after 48 h, and then started to stretch. Media were changed with the removal of red blood cells and some other debris after 72 h. Cells had polygon or spindle-like shape.6-7 d later, after cells got confluent into a single layer, local arrangement had a certain direction, and the cells became elongated, showing a typical fibroblast-like shape.1 h after the passage of ADSCs, they started to adhere to the plates.5-10 h later, the adherence increased.24 hours later, cells completely became adherent.3-4 d later, the cells got fully confluent. There were no significant changes in cell morphology after continuous passage of 9 to 10 generations. Cell growth curves of the 3rd,5th, and 10th generation of ADSCs were similar.1-2 d after passage, cell growth was at a standstill period.3-6 d later, cells entered the logarithmic growth phase.6-7 d later, cell growth stayed in plateau phase. The proliferation rate of the 10th generation was much slower than the 1st and 5th generation. 89.3% of cells were in G0/G1 phase. The expression of CD29 and CD44 were both positive in the 3rd generation of ADSCs shown by flow cytometry and immunofluorescence assay. The expressions of CD34, CD45, c-kit, HLA-DR were weakly positive or not expressed. ADSCs induced in different environments could be differentiated into adipose cells, osteocytes, chondrocytes and hepatocytes.
2. Construction and examination of Ad.HCN2
Construction of the pShuttleCMV.HCN2 was confirmed by PCR and gene sequencing. Construction of the pAd.HCN2 was confirmed by PCR. By cationic transduction, recombinant adenovirus plasmid pAd.HCN2 was introduced into HEK293 cells to produce recombinant adenovirus. PCR results confirmed that the recombinant gene carries HCN2 gene. The titer of the fourth generation of Ad.HCN2 was 1.5×109 pfu/ml. HeLa cells transduced with Ad.HCN2 at MOI= 50 did not appear cytopathic effect (CPE) after 7 days, since cell numbers at different time points showed no statistical difference between the experimental group and control group.48-72 h after ADSCs were transduced with Ad.GFP, green fluorescent cells were visible under fluorescence microscopy. Different MOI values correspond to different rates of transduction efficiency. With an increase in MOI values, the ratio of GFP-positive cells was also increased, together with a higher green fluorescence under fluorescence microscope. When MOI=50, the transduced rate of ADSCs was over 95%, and meanwhile cells looked much better than cells at the conditions of MOI=100 or 200.72 h after ADSCs were transduced with Ad.GFP, GFP protein expression was observed and the intensity of green fluorescence increased over time. There was no statistical significance of cell viability between transduced group and control groups 3 d and 10 d after ADSCs were transduced by Ad.HCN2. Neither was the cell cycle between the transduction group and the control group, and even among the same group of cells. No statistical significance in proliferation rate was observed between the Ad.HCN2-transduced group, Ad.Null-transduced group and non-transduced ADSCs groups.
3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCs
After RT-PCR, two sharp bands were observed as 28S and 18S bands. The brightness of 28S was twice more than that of the 18S band. There was no difference of 28S and 18S between two different groups after incubation test. The amplified band in the experimental group was the same with the band derived from the recombinant plasmid carrying HCN2 gene. Red fluorescent cells were visible by immunofluorescence detection. Flow cytometry results showed that, the percentage of Ad.HCN2-positive cells was significantly higher in Ad.HCN2-transduced group than that in Ad.Null-transduced group and non-transduced group, with significant statistical differences compared with the blank control and positive control. However, no statistically significant difference was observed between Ad.Null-transduced group and non-transduced group, or between the blank control and positive control, suggesting the high expression of HCN2 channel protein in the membrane of Ad.HCN2-transduced ADSCs. Western blot result indicated that, a specific electrophoretic band with molecular weight of 96 kd was observed in Ad.HCN2-transduced group. By patch-clamp, a hyperpolarization-activated inward current (If) was detected in the experimental group, with the activation potential of about-60 mV, and fully activation potential of-140 mV, which was voltage-dependent. If was almost completely suppressed when the media was added with 4 mmol/L of CsCl2 in experimental group. After elution of CsCl2, If reappeared. When ADSCs were cocultured with the NRCs, after 2 d, synchronized contraction of cells was only observed in the experimental group, but not in the control group.5 d later, some of the NRCs in positive control group showed synchronized contraction, but the contraction frequency was slower than that of the experimental group.21 d later, there was significant statistical difference in cell contraction frequency between the experimental group and the positive control group, while no cell contraction was observed between the negative control and blank control group. The cells in each group were added isoprenaline, cell contraction frequency in both experimental group and the positive control group was increased, with an even higher heart rate in the experimental group compared with the positive control group. Anti-Cx43 immunofluorescence staining could be observed under fluorescence microscope.
4. Transplantation of autologous pacemaker cells in treatment of complete atrioventricular block
The surface ECG showed P wave and QRS wave was totally separated in the animal mode of complete atrioventricular block. The rate of the ventricular, which was under 50 bpm, was slower than the rate of atrium.48 h after implantation, there was no statistical significance of heart rate among each group. After 72 h, the heart rate of experimental group became faster significantly, and the rate was stable at maximum after 7 d. There were significant statistical differences compared with the experimental group and the control groups after 7 d, but no statistically significant difference was observed between the negative control and the blank control. The shape and direction of the QRS was the same between the ECG and pace mapping of the experimental group. The result of 24 h Holter showed that there were significant statistical differences compared with the experimental group and the control groups. The heart rate of the experimental group was decreased from 70.2±2.9 bpm after 2 w to 54.9±3.4 bpm after 6 w. Added isoprenaline, the rate of all animals increased, and there was significantly statistical difference among all groups. And there were significant statistical differences in increased heart rates compared with the experimental group and the control groups. Added atropine, the rate of all animals increased as well. No statistically significant difference of changed heart rate in the experimental group was observed, but there was significant statistical difference of the control groups. And there were significant statistical differences in increased heart rates compared with the experimental group and the control groups. HE staining showed there was no significant fibroplasia in injection region and immunochemistry showed red fluorescent cells were visible.
Conclusion
1. Isolation, cultivation and identification of porcine ADSCs
The resource of ADSCs is widespread. The residue adipose tissue at donor site still has the ability to amplify and is easy to obtain in a large number, without severe damage. Comparing to other stem cells, ADSCs have higher isolation efficiency and stronger amplification ability, which render them easy to passage and obtain in large quantities. ADSCs express CD29 and CD44, which indicates theirs characteristics of mesenchymal stem cells; ADSCs have no expression of CD34, CD45 and c-kit, which demonstrates that they are non-hematopoietic. MHC II associated protein HLA-DR is rarely expressed in ADSCs, which suggests they have only very few antigen molecules related to immunological rejection and they have the potential of xenograft due to their possible ability of immune escape. Adipogenic, osteogenic, osteogenic and heptogenic differentiation demonstrates that ADSCs are multipotent.
2. Construction and examination of Ad.HCN2
Recombinant adenovirus carrier Ad.HCN2 was constructed by cloning HCN2 into adenovirus vector. CPE didn't show in infected Hela cells, which demonstrates there is no wild type virus in the recombinant adenovirus and it is safe to infect cells in vitro. In order to get the best cost-effectiveness ratio, MOI=50 is taken as the standard because of the high infection efficiency of Ad.GFP with MOI=50 and good condition of cells. The intensity of objective gene increased over time after transduced, which reached the top after 7 d, and then decreased. The cell viability and cell cycle of ADSCs didn't change after transduced by Ad.HCN2, which showed Ad.HCN2 was no harm to ADSCs. Recombinant adenovirus carrier Ad.HCN2 was constructed by cloning HCN2 into adenovirus vector. CPE was not observed in infected Hela cells, which demonstrates absence of wild type virus which indicates that the prep is safe to infect cells in vitro. In order to get the best maximal protein expression and minimal cytotoxicity a MOI=50 is used. The GFP fluorescence intensity increased over time, reached peak at day 7, and then decreased. Cell viability and cell cycle of ADSCs didn't change after Ad.HCN2 transduction, which demonstrated absence of negative effects from Ad.HCN2 on these parameters.
3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCs
RT-PCR demonstrated the expression of HCN2 mRNA in Ad.HCN2 transduced ADSCs. The incubation test further showed that the RNA samples were of high purity. In addition, we demonstrated HCN2 immunofluorescence on the membrane of Ad.HCN2-transduced ADSCs. Expression of HCN2 channel protein was most quantity in the Ad.HCN2 transduced-ADSCs with FCM and Western blot. The patch clamp showed that Ad.HCN2 transduced-ADSCs could produce If. Ad.HCN2 transduced-ADSCs could pace the NRCs effectually after cocultured them, and they showed positive to isoprenaline. Cx43 could be detected between ADSCs and NRCs after cocultured.
4. Transplantation of autologous pace cells in treatment of complete atrioventricular block
The pacemaker cells could pace the ventricle effectively after its autotransplantation. The ECG showed the ventricular heart rhythm of experimental group was from the injection region of cells. The result of 24 h Holter showed that the heart rate increased first and then decreased. The heart rate of experimental group showed positive to isoprenaline and negative to. HE staining showed ADSCs survived after implantation and there was no significant inflammatory reaction in injection region. Immunochemistry showed Ad.GFP transduced-ADSCs could express HCN2 channel protein in myocardium.
引文
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