雷公藤甲素对HCN4基因修饰大鼠MSCs同种异体心脏移植后细胞存活影响的研究
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摘要
背景与目的电子心脏起搏器是目前临床上治疗缓慢性心律失常（病态窦房结综合征及完全性房室传导阻滞等）的有效方法。虽然电子心脏起搏器治疗已挽救了数百万患者的生命，但因电子心脏起搏器植入术存在诸多问题（如缺乏对机体神经递质变化的自动反应性、电极位置不稳定、电池寿命有限、严重感染及电磁干扰等）而并非最佳选择。鉴于上述原因，基于细胞和基因治疗的“生物起搏器”已成为当前缓慢性心律失常治疗领域研究的热点之一。
骨髓间充质干细胞（mesenchymal stem cells, MSCs）作为具有多向分化潜能的成体干细胞，目前已成为最具前景的“种子细胞”。其不仅能够容易从骨髓中获取，且通过注射方式移植至心肌后能够分化成心肌样细胞。本课题组近期的实验研究显示携带HCN4基因的MSCs（HCN4-MSCs）同种异体移植到犬完全性房室传导阻滞模型心脏后能产生源自注射部位的自发频率，但因其频率明显低于体外诱导培养的心脏起搏样细胞的自发频率，推测移植细胞在宿主体内大量死亡可能是其频率低下的原因之一。
在体动物实验研究已证实大多数移植至心脏的MSCs均在移植后的最初两天内死亡，急性炎症反应可能是移植细胞死亡的重要原因。另有研究显示MSCs体内移植后存活率为20–30%，移植细胞死亡的主要原因可能为缺血、凋亡、炎症或免疫排斥反应。由此可见，MSCs移植治疗效果因移植细胞低生存率而受到影响，而寻找防治移植细胞死亡、提高移植细胞在宿主体内存活率新方法，则有助于提高细胞移植效果。
雷公藤甲素是从中药雷公藤中提取出的小分子活性物质，目前研究显示雷公藤甲素具有抗炎和免疫抑制作用，将其用于MSCs移植治疗，可能有助于抑制急性炎症反应和免疫排斥，减少移植细胞死亡、提高移植细胞在宿主体内的存活率。
因此，本研究拟在行HCN4-MSCs同种异体心脏移植时，用雷公藤甲素进行干预，以探讨雷公藤甲素对移植细胞存活率的影响及可能机制。
方法
1.无菌条件下取健康雄性SD大鼠双侧股骨及胫骨的骨髓，经密度梯度离心法结合贴壁培养法获得MSCs，然后通过频繁的换液体外进行扩增及纯化。
2.以mHCN4慢病毒载体（pMSCV-mHCN4-EGFP）转染大鼠MSCs，转染后应用杀稻瘟菌素药物加压筛选。免疫荧光法检测mHCN4及EGFP目的基因的表达情况。CCK-8法检测HCN4-MSCs的细胞增值能力。
3.应用性别错配的细胞移植模型，即将雄性来源的HCN4-MSCs同种异体移植到雌性SD大鼠心脏，通过定量RT-PCR法检测雌性大鼠心脏中雄性基因Sry的表达量而得出移植细胞的存活数量。首先将已知数量且经过倍比稀释的（2.0×10~2，2.0×10~3，2.0×10~4，2.0×10~5，2.0×10~6）HCN4-MSCs体外注射入雌性大鼠心脏中通过检测雄性基因Sry的表达量得出移植细胞数量与Sry的表达量之间关系的标准曲线。
4.将不同数量的HCN4-MSCs通过开胸心外膜直视下注射法以单点心肌注射方式同种异体移植到雌性SD大鼠心脏，用定量RT-PCR法检测心脏标本中Sry的表达量，根据标准曲线得出移植细胞的最佳移植数量。
5.将按照最佳移植数量进行细胞移植术的雌性SD大鼠随机分为三组（每组30只大鼠）：
（1）雷公藤甲素组（Tri）：大鼠腹腔内注射雷公藤甲素200μg/kg/d，共10天(术前3天至术后7天)；
（2）地塞米松组（Dex）：大鼠腹腔内注射地塞米松400μg/kg/d，共10天(术前3天至术后7天)；
（3）对照组（Con）：大鼠腹腔内注射无菌生理盐水(2ml/次)，共10天(术前3天至术后7天)。
6.各组动物按术后观察时间点不同（术后24小时、72小时、7天、14天、28天）分为5各亚组（每个亚组6只大鼠）。于细胞移植后不同时间点采集血标本或处死动物、取出心脏，进行标本处理及以下检测。
（1）对各时间点心脏标本用定量RT-PCR法检测雌性大鼠心脏标本中雄性基因Sry的表达量，根据标准曲线得出移植细胞的存活数量。
（2）于术后24小时、72小时取血，以ELISA法检测血清中核转录因子NF-κB和炎症因子IL-1β, IL-6, TNF-α的蛋白表达水平。同时用RT-PCR法测定包含细胞移植区心肌组织上述相关因子mRNA的表达。
（3）TUNEL法检测细胞移植区移植细胞的凋亡发生情况，免疫印迹法检测细胞移植区心肌组织凋亡相关蛋白Bax和Bcl-2的表达，膜片钳检测细胞移植区域移植细胞的起搏电流(If)。
（4）细胞移植术后28天，麻醉后处死大鼠，细胞移植区心肌组织应用4%多聚甲醛固定、石蜡包埋后切片，利用免疫组化的方法检测心肌特异性标志物肌钙蛋白T（cardiac troponin T，cTnT）及缝隙连接蛋白43（connexin43, CX43）的表达。
结果
1.分离培养出的第二代MSCs紧贴于培养瓶壁上，外观呈纺锤形或多角形。流式检测结果显示骨髓间充质干细胞抗原CD29和CD90呈阳性表达，抗原CD45呈阴性表达。
2.成功地将携带mHCN4基因的慢病毒载体（pMSCV-mHCN4-EGFP）转染至大鼠骨髓MSCs。在MOI=10的条件下，转染效率约为67%，抗生素筛选后阳性率可达90%以上。免疫荧光染色证实转染后的细胞稳定表达mHCN4及EGFP蛋白。CCK-8法检测结果显示转染mHCN4后的骨髓MSCs呈现较强的增值能力。
3.体外将雄性来源且倍比稀释(2.0×10~2,2.0×10~3,2.0×10~4,2.0×10~5及2.0×10~6)的HCN4-MSCs分别移植入雌性大鼠心肌中，定量RT-PCR技术检测标本中Sry的表达量，得出移植细胞数量与Sry基因表达量的间关系的标准曲线。
4.应用性别错配的移植模型将不同数量的HCN4-MSCs同种异体移植到雌性大鼠心脏中，移植后72小时定量RT-PCR法检测心脏标本中Sry的表达量，根据标准曲线得出移植细胞的存活数量。结果显示1×10~6为细胞同种异体心脏移植单点心肌注射的最佳移植数量。
5.选取1×10~6为移植细胞数量，雷公藤甲素治疗组HCN4-MSCs心肌内移植后24小时、72小时、7天、14天及28天，移植细胞的存活率分别为29.27±0.93%、17.55±1.24%、7.37±0.34%、4.15±0.38%及3.40±0.14%；均显著高于相同时间段对照组的细胞存活率14.74±0.94%、6.90±0.19%、3.17±0.16%、1.41±0.40%和0.76±0.36%（各时间段P＜0.01）。地塞米松干预组在相同时间段细胞的存活率分别为28.17±1.76%、15.63±0.36%、6.08±0.26%、3.38±0.32%和2.55±0.12%；也显著高于对照组的细胞存活率（n=4/组/时间点，各时间段P＜0.01）。
6.直接心肌注射可引起机械性损伤随后产生急性炎症反应，引起移植细胞的死亡。雷公藤甲素具有抗炎作用，能够降低核转录因子NF-κB和IL-1β, IL-6及TNF-α三种炎症因子的表达水平。短期应用雷公藤甲素干预治疗能够提高HCN4-MSCs心肌内移植后的存活率。
7.雷公藤甲素干预治疗可减少移植细胞的凋亡发生率，降低移植区域促凋亡蛋白Bax的表达但升高抗凋亡蛋白Bcl-2的表达水平。
8.膜片钳检测结果示HCN4-MSCs心肌内移植后在宿主心脏中存活4周以上并可记录到被CsCl可逆性阻断的超极化激活的起搏电流(If)。雷公藤甲素干预治疗对移植细胞的功能无不良影响。
9.免疫组化检测结果显示部分HCN4-MSCs心肌内移植后4周既表达EGFP又表达心肌特异性标志物cTnT或缝隙连接蛋白CX43，提示HCN4-MSCs心肌内移植后分化为新的心肌样细胞。
结论
1.不同数量雄性来源的HCN4-MSCs应用性别错配的移植模型同种异体移植到雌性大鼠心脏中，定量RT-PCR检测Sry基因表达量，根据标准曲线得出各组移植细胞的存活数量，结果示1×10~6为细胞同种异体心脏移植单点心肌注射的最佳细胞移植数量。
2.雷公藤甲素干预治疗可提高HCN4-MSCs心脏移植后的存活率。其机制可能为直接心肌注射引起机械性损伤并产生急性炎症反应引起移植细胞的死亡，雷公藤甲素具有抗炎作用，能有效地抑制核转录因子NF-κB及炎症因子IL-1β, IL-6及TNF-α的释放，减轻炎症反应。
3.雷公藤甲素对移植细胞的功能无不良影响，HCN4-MSCs心肌内移植后可记录到起搏电流(If)；部分HCN4-MSCs心脏移植后4周表达EGFP及心肌特异性标志物cTnT或缝隙连接蛋白CX43，支持移植入心肌的HCN4-MSCs分化为新的心肌样细胞。
Background and objection
Electronic pacemakers are currently the primary treatment to sustain the heart rate forsick sinus syndrome or complete atrioventricular block. Although electronic pacemakers areeffective to treat these diseases and have already saved millions of lives, they are notoptimal because such devices have many shortcomings, such as lack of biologicalresponsiveness to autonomic neurotransmitter changes, unstable electrode position, limitedbattery life, severe infection, electronic and magnetic interference. For these reasons,biological pacemaker based on cell and gene therapy is becoming an important candidate totreat bradyarrhythmias.
As one type of adult pluripotent stem cell, mesenchymal stem cells (MSCs) may be themost promising candidate. They can easily be acquired from bone marrow. The injectedMSCs can differentiate into cardiac myocytes after being implanted directly into themyocardium. In our recent experiments, transplanted canine MSCs carryinghyperpolarization-activated cyclic nucleotide-gated4(HCN4) genes could inducespontaneous ventricular rhythms in models of complete atrioventricular block byallografting into the host heart. However, the beating rate ranged from40to50bpm, whichwas markedly lower than that of MSCs transfected with HCN4co-cultured withcardiomyocytes in vitro. A high level of donor cells death may be one of the adversefactors.
Some in vivo studies have demonstrated that the majority of cells implanted into theheart die within the initial2days following implantation and that acute inflammation mayplay an important role in donor cell death. Other studies have also shown that the number ofsurviving engrafted cells was limited to20–30%, possibly due to ischemia, apoptosis,inflammation, or immunological rejection. The therapeutic effects of stem celltransplantation into the heart are hindered by the poor survival of the implanted cells. So it is very important and necessary to find ways to promote the therapeutic effects byimproving the survival rate of grafted cells within this arduous microenvironment.
Triptolide is a small molecule extracted from the traditional Chinese herbal plantTripterygium wilfordii Hook F (TWHF). Currently, triptolide has been shown to possessboth anti-inflammatory and immunosuppressive properties. Maybe triptolide can prolongthe survival of donor cells by its anti-inflammatory and immunosuppressive roles in vivo.This study was designed to investigate the role of the inflammatory response in acute celldeath after implantation into the myocardium and the effects of triptolide.
Methods
1. Under sterile conditions, male rat mesenchymal stem cells were isolated from bonemarrow of the bilateral femurs and tibias by gradient centrifugation and the character ofadherence to culture plates, and further purified and amplified by changing the mediumfrequently in vitro.
2. The mHCN4lentiviral vector pLenti6.3-mHCN4-IRES2-EGFP was used totransfect rat MSCs. The infected rat MSCs were selected by using blasticidin and theexpression of mHCN4and EGFP protein were detected by using immunohistochemicalmethod and fluorescence microscope. Cell proliferation was assessed by the Cell-CountingKit-8(CCK-8) assay.
3. Sex-mismatched cell transplantation models were used in our experiments. MSCswere isolated from male Sprague-Dawley (SD) rats and transduced with mHCN4(HCN4-MSCs) and then injected into syngeneic female rats. The total number of maleHCN4-MSCs grafted into the female heart was analyzed by quantitative reversetranscription polymerase chain reaction (RT-PCR) for the male-specific Y chromosome(Sry). Intact female hearts were collected and injected ex vivo with known cell numbers ofHCN4-MSCs (2.0×10~2，2.0×10~3，2.0×104，2.0×10~5and2.0×10~6). The samples wereanalyzed for the amount of Sry gene to calculate the numbers of HCN4-MSCs and to obtaina standard curve.
4. According to the standard curve, different cell numbers of HCN4-MSCs wereinjected directly into the myocardium at one site to explore the optimal transplanted numberby quantitative RT-PCR for the male-specific Y chromosome (Sry).
5. All of the female rats were given intramyocardial injections containing the optimaltransplanted number of HCN4-MSCs, and the rats were randomly divided into three groups(n=30in each group):
i) The triptolide group (Tri), in which rats were injected intraperitoneally (i.p.) with200μg/kg/d triptolide for10d (from day-3to7);
ii) The dexamethasone group (Dex), in which the rats were injected i.p. with400μg/kg/d dexamethasone for10d (from day-3to7);
iii) The control group (Con), in which rats were injected i.p. with sterile normal salinefor10d (from day-3to7).
6. The rats in each group were divided into five subgroups according to different timepoints (24h,72h,7d,14d and28d) after cell transplantation (n=6in each subgroup). Theanimals were sacrificed at the indicated time points, the blood and the hearts werecollected.
(1) The surviving number of male donor HCN4-MSCs in the female host heart wasestimated by the amount of Sry gene in the implanted area of each sample by referring tothe standard curve.
(2) At different time points (24h and72h) after cell injection, enzyme linkedimmunosorbent assay (ELISA) and RT-PCR were used to measure the protein and mRNAlevels, respectively, of nuclear factor (NF)-κB and pro-inflammatory cytokines (IL-1β, IL-6and TNF-α).
(3) The apoptosis of grafted HCN4-MSCs was tested by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay and apoptosis-regulatingproteins (Bax and Bcl-2) were analyzed by western blotting. The pacemaker current (If) ofthe donor cells was detected by using patch-clamp technique.
(4) The rats were sacrificed28days after the transplantation of HCN4-MSCs. Theheart tissue was fixed in4%paraformaldehyde, and embedded in paraffin. Serial sectionswere cut at4μm. Immunohistochemical staining was done for cardiac troponin T (cTnT)and connexin43(CX43).
Results
1. The second passage of MSCs were attached to the culture flasks tightly and spread in the shape of spindles or polygons. Purified MSCs were positive for CD29and CD90, butnegative for CD45with the use of flow cytometry analysis.
2. Rat MSCs were transfected with pLenti6.3-mHCN4-IRES2-EGFP vectorsuccessfully and the transfection efficiency were about67%at multiplicities of infection(MOIs) of10and over90%after antibiotic selection. The cells stably expressed both EGFPand HCN4protein by immunofluorescent staining. The cell proliferation assay by CCK-8showed that HCN4-MSCs exhibited significantly increased cell viability beforetransplantation.
3. Intact female hearts were collected and injected ex vivo with known cell numbers(2.0×10~2,2.0×10~3,2.0×10~4,2.0×10~5and2.0×10~6) of the mHCN4-infected male MSCs,respectively. The samples were analyzed for the amount of Sry gene by quantitativeRT-PCR and obtained a standard curve.
4. Sex-mismatched cell transplantation models were used in our experiments. Differentcell numbers of HCN4-MSCs were injected directly into the myocardium of syngeneicfemale rats. Grafted cell survival was analyzed by RT-PCR for the Sry gene from heartsharvested after72hours of cell transplantation by referring to the standard curve. We foundthat1×10~6may be the optimal transplanted number of engrafted HCN4-MSCs at one site.
5. All of the rats were given intramyocardial injections containing1×10~6HCN4-MSCs.The numbers of surviving HCN4-MSCs injected were29.27±0.93%,17.55±1.24%,7.37±0.34%,4.15±0.38%and3.40±0.14%at24h,72h,7d,14d and28d in the Tri group,respectively. Which were much higher than those found in the controls by RT-PCR(14.74±0.94%,6.90±0.19%,3.17±0.16%,1.41±0.40%and0.76±0.36%) at each time point(P＜0.01). The numbers of surviving cells injected in Dex group were28.17±1.76%,15.63±0.36%,6.08±0.26%,3.38±0.32%and2.55±0.12%at the same time point,respectively (n=4in each point). Which were also much higher than those found in thecontrols (P＜0.01).
6. Direct intramyocardial injection caused mechanical injury and acute inflammation,which caused the engrafted cells to die. Triptolide exhibited anti-inflammatory activity andsignificantly decreased the expression levels of IL-1β, IL-6and TNF-α, as well as NF-κB.The short-term administration of triptolide can promote the early graft survival of HCN4-MSCs in myocardium after transplantation.
7. Pretreatment with triptolide significantly reduced the apoptosis of donor cells. Inaddition, triptolide downregulated the Bax level but upregulated the Bcl-2level in theinjected region.
8. The results of patch-clamp experiments suggested that the allografted HCN4-MSCssurvived for over4weeks and expressed funny current (If) in the host heart. The activity ofHCN4-MSCs was unaffected by triptolide in vivo.
9. Immunohistochemical results showed that some donor HCN4-MSCs were positivefor both EGFP and troponin T (cTnT) or connexin43(CX43) in the host heart at4weeksafter transplantation, which indicated that the implanted HCN4-MSCs differentiated intonew cardiomyocytes.
Conclusions
1. Sex-mismatched cell transplantation models were used in our experiments. Differentcell numbers of male HCN4-MSCs were injected directly into the myocardium of syngeneicfemale rats. The number of engrafted HCN4-MSCs was estimated by using the amount ofSry gene of each sample by quantitative RT-PCR. We found that1×10~6may be the optimalnumber of engrafted HCN4-MSCs at one site by referring to the standard curve.
2. The short-term administration of triptolide can promote the survival ofHCN4-MSCs in myocardium after transplantation. Direct intramyocardial injection causedmechanical injury and acute inflammation, which caused the engrafted cells to die.Triptolide exhibited anti-inflammatory activity and significantly decreased the expressionlevels of IL-1β, IL-6and TNF-α, as well as NF-κB.
3. The results of patch-clamp experiments suggested that HCN4-MSCs expressedfunny current (If) in vivo and the activity of donor cells was unaffected by triptolide. SomeHCN4-MSCs were positive for both EGFP and troponin T (cTnT) or connexin43(CX43)in the host heart at4weeks after transplantation, which indicated that the implantedHCN4-MSCs differentiated into new cardiomyocytes.
骨髓间充质干细胞（mesenchymal stem cells, MSCs）作为具有多向分化潜能的成体干细胞，目前已成为最具前景的“种子细胞”。其不仅能够容易从骨髓中获取，且通过注射方式移植至心肌后能够分化成心肌样细胞。本课题组近期的实验研究显示携带HCN4基因的MSCs（HCN4-MSCs）同种异体移植到犬完全性房室传导阻滞模型心脏后能产生源自注射部位的自发频率，但因其频率明显低于体外诱导培养的心脏起搏样细胞的自发频率，推测移植细胞在宿主体内大量死亡可能是其频率低下的原因之一。
在体动物实验研究已证实大多数移植至心脏的MSCs均在移植后的最初两天内死亡，急性炎症反应可能是移植细胞死亡的重要原因。另有研究显示MSCs体内移植后存活率为20–30%，移植细胞死亡的主要原因可能为缺血、凋亡、炎症或免疫排斥反应。由此可见，MSCs移植治疗效果因移植细胞低生存率而受到影响，而寻找防治移植细胞死亡、提高移植细胞在宿主体内存活率新方法，则有助于提高细胞移植效果。
雷公藤甲素是从中药雷公藤中提取出的小分子活性物质，目前研究显示雷公藤甲素具有抗炎和免疫抑制作用，将其用于MSCs移植治疗，可能有助于抑制急性炎症反应和免疫排斥，减少移植细胞死亡、提高移植细胞在宿主体内的存活率。
因此，本研究拟在行HCN4-MSCs同种异体心脏移植时，用雷公藤甲素进行干预，以探讨雷公藤甲素对移植细胞存活率的影响及可能机制。
方法
1.无菌条件下取健康雄性SD大鼠双侧股骨及胫骨的骨髓，经密度梯度离心法结合贴壁培养法获得MSCs，然后通过频繁的换液体外进行扩增及纯化。
2.以mHCN4慢病毒载体（pMSCV-mHCN4-EGFP）转染大鼠MSCs，转染后应用杀稻瘟菌素药物加压筛选。免疫荧光法检测mHCN4及EGFP目的基因的表达情况。CCK-8法检测HCN4-MSCs的细胞增值能力。
3.应用性别错配的细胞移植模型，即将雄性来源的HCN4-MSCs同种异体移植到雌性SD大鼠心脏，通过定量RT-PCR法检测雌性大鼠心脏中雄性基因Sry的表达量而得出移植细胞的存活数量。首先将已知数量且经过倍比稀释的（2.0×10~2，2.0×10~3，2.0×10~4，2.0×10~5，2.0×10~6）HCN4-MSCs体外注射入雌性大鼠心脏中通过检测雄性基因Sry的表达量得出移植细胞数量与Sry的表达量之间关系的标准曲线。
4.将不同数量的HCN4-MSCs通过开胸心外膜直视下注射法以单点心肌注射方式同种异体移植到雌性SD大鼠心脏，用定量RT-PCR法检测心脏标本中Sry的表达量，根据标准曲线得出移植细胞的最佳移植数量。
5.将按照最佳移植数量进行细胞移植术的雌性SD大鼠随机分为三组（每组30只大鼠）：
（1）雷公藤甲素组（Tri）：大鼠腹腔内注射雷公藤甲素200μg/kg/d，共10天(术前3天至术后7天)；
（2）地塞米松组（Dex）：大鼠腹腔内注射地塞米松400μg/kg/d，共10天(术前3天至术后7天)；
（3）对照组（Con）：大鼠腹腔内注射无菌生理盐水(2ml/次)，共10天(术前3天至术后7天)。
6.各组动物按术后观察时间点不同（术后24小时、72小时、7天、14天、28天）分为5各亚组（每个亚组6只大鼠）。于细胞移植后不同时间点采集血标本或处死动物、取出心脏，进行标本处理及以下检测。
（1）对各时间点心脏标本用定量RT-PCR法检测雌性大鼠心脏标本中雄性基因Sry的表达量，根据标准曲线得出移植细胞的存活数量。
（2）于术后24小时、72小时取血，以ELISA法检测血清中核转录因子NF-κB和炎症因子IL-1β, IL-6, TNF-α的蛋白表达水平。同时用RT-PCR法测定包含细胞移植区心肌组织上述相关因子mRNA的表达。
（3）TUNEL法检测细胞移植区移植细胞的凋亡发生情况，免疫印迹法检测细胞移植区心肌组织凋亡相关蛋白Bax和Bcl-2的表达，膜片钳检测细胞移植区域移植细胞的起搏电流(If)。
（4）细胞移植术后28天，麻醉后处死大鼠，细胞移植区心肌组织应用4%多聚甲醛固定、石蜡包埋后切片，利用免疫组化的方法检测心肌特异性标志物肌钙蛋白T（cardiac troponin T，cTnT）及缝隙连接蛋白43（connexin43, CX43）的表达。
结果
1.分离培养出的第二代MSCs紧贴于培养瓶壁上，外观呈纺锤形或多角形。流式检测结果显示骨髓间充质干细胞抗原CD29和CD90呈阳性表达，抗原CD45呈阴性表达。
2.成功地将携带mHCN4基因的慢病毒载体（pMSCV-mHCN4-EGFP）转染至大鼠骨髓MSCs。在MOI=10的条件下，转染效率约为67%，抗生素筛选后阳性率可达90%以上。免疫荧光染色证实转染后的细胞稳定表达mHCN4及EGFP蛋白。CCK-8法检测结果显示转染mHCN4后的骨髓MSCs呈现较强的增值能力。
3.体外将雄性来源且倍比稀释(2.0×10~2,2.0×10~3,2.0×10~4,2.0×10~5及2.0×10~6)的HCN4-MSCs分别移植入雌性大鼠心肌中，定量RT-PCR技术检测标本中Sry的表达量，得出移植细胞数量与Sry基因表达量的间关系的标准曲线。
4.应用性别错配的移植模型将不同数量的HCN4-MSCs同种异体移植到雌性大鼠心脏中，移植后72小时定量RT-PCR法检测心脏标本中Sry的表达量，根据标准曲线得出移植细胞的存活数量。结果显示1×10~6为细胞同种异体心脏移植单点心肌注射的最佳移植数量。
5.选取1×10~6为移植细胞数量，雷公藤甲素治疗组HCN4-MSCs心肌内移植后24小时、72小时、7天、14天及28天，移植细胞的存活率分别为29.27±0.93%、17.55±1.24%、7.37±0.34%、4.15±0.38%及3.40±0.14%；均显著高于相同时间段对照组的细胞存活率14.74±0.94%、6.90±0.19%、3.17±0.16%、1.41±0.40%和0.76±0.36%（各时间段P＜0.01）。地塞米松干预组在相同时间段细胞的存活率分别为28.17±1.76%、15.63±0.36%、6.08±0.26%、3.38±0.32%和2.55±0.12%；也显著高于对照组的细胞存活率（n=4/组/时间点，各时间段P＜0.01）。
6.直接心肌注射可引起机械性损伤随后产生急性炎症反应，引起移植细胞的死亡。雷公藤甲素具有抗炎作用，能够降低核转录因子NF-κB和IL-1β, IL-6及TNF-α三种炎症因子的表达水平。短期应用雷公藤甲素干预治疗能够提高HCN4-MSCs心肌内移植后的存活率。
7.雷公藤甲素干预治疗可减少移植细胞的凋亡发生率，降低移植区域促凋亡蛋白Bax的表达但升高抗凋亡蛋白Bcl-2的表达水平。
8.膜片钳检测结果示HCN4-MSCs心肌内移植后在宿主心脏中存活4周以上并可记录到被CsCl可逆性阻断的超极化激活的起搏电流(If)。雷公藤甲素干预治疗对移植细胞的功能无不良影响。
9.免疫组化检测结果显示部分HCN4-MSCs心肌内移植后4周既表达EGFP又表达心肌特异性标志物cTnT或缝隙连接蛋白CX43，提示HCN4-MSCs心肌内移植后分化为新的心肌样细胞。
结论
1.不同数量雄性来源的HCN4-MSCs应用性别错配的移植模型同种异体移植到雌性大鼠心脏中，定量RT-PCR检测Sry基因表达量，根据标准曲线得出各组移植细胞的存活数量，结果示1×10~6为细胞同种异体心脏移植单点心肌注射的最佳细胞移植数量。
2.雷公藤甲素干预治疗可提高HCN4-MSCs心脏移植后的存活率。其机制可能为直接心肌注射引起机械性损伤并产生急性炎症反应引起移植细胞的死亡，雷公藤甲素具有抗炎作用，能有效地抑制核转录因子NF-κB及炎症因子IL-1β, IL-6及TNF-α的释放，减轻炎症反应。
3.雷公藤甲素对移植细胞的功能无不良影响，HCN4-MSCs心肌内移植后可记录到起搏电流(If)；部分HCN4-MSCs心脏移植后4周表达EGFP及心肌特异性标志物cTnT或缝隙连接蛋白CX43，支持移植入心肌的HCN4-MSCs分化为新的心肌样细胞。
Background and objection
Electronic pacemakers are currently the primary treatment to sustain the heart rate forsick sinus syndrome or complete atrioventricular block. Although electronic pacemakers areeffective to treat these diseases and have already saved millions of lives, they are notoptimal because such devices have many shortcomings, such as lack of biologicalresponsiveness to autonomic neurotransmitter changes, unstable electrode position, limitedbattery life, severe infection, electronic and magnetic interference. For these reasons,biological pacemaker based on cell and gene therapy is becoming an important candidate totreat bradyarrhythmias.
As one type of adult pluripotent stem cell, mesenchymal stem cells (MSCs) may be themost promising candidate. They can easily be acquired from bone marrow. The injectedMSCs can differentiate into cardiac myocytes after being implanted directly into themyocardium. In our recent experiments, transplanted canine MSCs carryinghyperpolarization-activated cyclic nucleotide-gated4(HCN4) genes could inducespontaneous ventricular rhythms in models of complete atrioventricular block byallografting into the host heart. However, the beating rate ranged from40to50bpm, whichwas markedly lower than that of MSCs transfected with HCN4co-cultured withcardiomyocytes in vitro. A high level of donor cells death may be one of the adversefactors.
Some in vivo studies have demonstrated that the majority of cells implanted into theheart die within the initial2days following implantation and that acute inflammation mayplay an important role in donor cell death. Other studies have also shown that the number ofsurviving engrafted cells was limited to20–30%, possibly due to ischemia, apoptosis,inflammation, or immunological rejection. The therapeutic effects of stem celltransplantation into the heart are hindered by the poor survival of the implanted cells. So it is very important and necessary to find ways to promote the therapeutic effects byimproving the survival rate of grafted cells within this arduous microenvironment.
Triptolide is a small molecule extracted from the traditional Chinese herbal plantTripterygium wilfordii Hook F (TWHF). Currently, triptolide has been shown to possessboth anti-inflammatory and immunosuppressive properties. Maybe triptolide can prolongthe survival of donor cells by its anti-inflammatory and immunosuppressive roles in vivo.This study was designed to investigate the role of the inflammatory response in acute celldeath after implantation into the myocardium and the effects of triptolide.
Methods
1. Under sterile conditions, male rat mesenchymal stem cells were isolated from bonemarrow of the bilateral femurs and tibias by gradient centrifugation and the character ofadherence to culture plates, and further purified and amplified by changing the mediumfrequently in vitro.
2. The mHCN4lentiviral vector pLenti6.3-mHCN4-IRES2-EGFP was used totransfect rat MSCs. The infected rat MSCs were selected by using blasticidin and theexpression of mHCN4and EGFP protein were detected by using immunohistochemicalmethod and fluorescence microscope. Cell proliferation was assessed by the Cell-CountingKit-8(CCK-8) assay.
3. Sex-mismatched cell transplantation models were used in our experiments. MSCswere isolated from male Sprague-Dawley (SD) rats and transduced with mHCN4(HCN4-MSCs) and then injected into syngeneic female rats. The total number of maleHCN4-MSCs grafted into the female heart was analyzed by quantitative reversetranscription polymerase chain reaction (RT-PCR) for the male-specific Y chromosome(Sry). Intact female hearts were collected and injected ex vivo with known cell numbers ofHCN4-MSCs (2.0×10~2，2.0×10~3，2.0×104，2.0×10~5and2.0×10~6). The samples wereanalyzed for the amount of Sry gene to calculate the numbers of HCN4-MSCs and to obtaina standard curve.
4. According to the standard curve, different cell numbers of HCN4-MSCs wereinjected directly into the myocardium at one site to explore the optimal transplanted numberby quantitative RT-PCR for the male-specific Y chromosome (Sry).
5. All of the female rats were given intramyocardial injections containing the optimaltransplanted number of HCN4-MSCs, and the rats were randomly divided into three groups(n=30in each group):
i) The triptolide group (Tri), in which rats were injected intraperitoneally (i.p.) with200μg/kg/d triptolide for10d (from day-3to7);
ii) The dexamethasone group (Dex), in which the rats were injected i.p. with400μg/kg/d dexamethasone for10d (from day-3to7);
iii) The control group (Con), in which rats were injected i.p. with sterile normal salinefor10d (from day-3to7).
6. The rats in each group were divided into five subgroups according to different timepoints (24h,72h,7d,14d and28d) after cell transplantation (n=6in each subgroup). Theanimals were sacrificed at the indicated time points, the blood and the hearts werecollected.
(1) The surviving number of male donor HCN4-MSCs in the female host heart wasestimated by the amount of Sry gene in the implanted area of each sample by referring tothe standard curve.
(2) At different time points (24h and72h) after cell injection, enzyme linkedimmunosorbent assay (ELISA) and RT-PCR were used to measure the protein and mRNAlevels, respectively, of nuclear factor (NF)-κB and pro-inflammatory cytokines (IL-1β, IL-6and TNF-α).
(3) The apoptosis of grafted HCN4-MSCs was tested by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay and apoptosis-regulatingproteins (Bax and Bcl-2) were analyzed by western blotting. The pacemaker current (If) ofthe donor cells was detected by using patch-clamp technique.
(4) The rats were sacrificed28days after the transplantation of HCN4-MSCs. Theheart tissue was fixed in4%paraformaldehyde, and embedded in paraffin. Serial sectionswere cut at4μm. Immunohistochemical staining was done for cardiac troponin T (cTnT)and connexin43(CX43).
Results
1. The second passage of MSCs were attached to the culture flasks tightly and spread in the shape of spindles or polygons. Purified MSCs were positive for CD29and CD90, butnegative for CD45with the use of flow cytometry analysis.
2. Rat MSCs were transfected with pLenti6.3-mHCN4-IRES2-EGFP vectorsuccessfully and the transfection efficiency were about67%at multiplicities of infection(MOIs) of10and over90%after antibiotic selection. The cells stably expressed both EGFPand HCN4protein by immunofluorescent staining. The cell proliferation assay by CCK-8showed that HCN4-MSCs exhibited significantly increased cell viability beforetransplantation.
3. Intact female hearts were collected and injected ex vivo with known cell numbers(2.0×10~2,2.0×10~3,2.0×10~4,2.0×10~5and2.0×10~6) of the mHCN4-infected male MSCs,respectively. The samples were analyzed for the amount of Sry gene by quantitativeRT-PCR and obtained a standard curve.
4. Sex-mismatched cell transplantation models were used in our experiments. Differentcell numbers of HCN4-MSCs were injected directly into the myocardium of syngeneicfemale rats. Grafted cell survival was analyzed by RT-PCR for the Sry gene from heartsharvested after72hours of cell transplantation by referring to the standard curve. We foundthat1×10~6may be the optimal transplanted number of engrafted HCN4-MSCs at one site.
5. All of the rats were given intramyocardial injections containing1×10~6HCN4-MSCs.The numbers of surviving HCN4-MSCs injected were29.27±0.93%,17.55±1.24%,7.37±0.34%,4.15±0.38%and3.40±0.14%at24h,72h,7d,14d and28d in the Tri group,respectively. Which were much higher than those found in the controls by RT-PCR(14.74±0.94%,6.90±0.19%,3.17±0.16%,1.41±0.40%and0.76±0.36%) at each time point(P＜0.01). The numbers of surviving cells injected in Dex group were28.17±1.76%,15.63±0.36%,6.08±0.26%,3.38±0.32%and2.55±0.12%at the same time point,respectively (n=4in each point). Which were also much higher than those found in thecontrols (P＜0.01).
6. Direct intramyocardial injection caused mechanical injury and acute inflammation,which caused the engrafted cells to die. Triptolide exhibited anti-inflammatory activity andsignificantly decreased the expression levels of IL-1β, IL-6and TNF-α, as well as NF-κB.The short-term administration of triptolide can promote the early graft survival of HCN4-MSCs in myocardium after transplantation.
7. Pretreatment with triptolide significantly reduced the apoptosis of donor cells. Inaddition, triptolide downregulated the Bax level but upregulated the Bcl-2level in theinjected region.
8. The results of patch-clamp experiments suggested that the allografted HCN4-MSCssurvived for over4weeks and expressed funny current (If) in the host heart. The activity ofHCN4-MSCs was unaffected by triptolide in vivo.
9. Immunohistochemical results showed that some donor HCN4-MSCs were positivefor both EGFP and troponin T (cTnT) or connexin43(CX43) in the host heart at4weeksafter transplantation, which indicated that the implanted HCN4-MSCs differentiated intonew cardiomyocytes.
Conclusions
1. Sex-mismatched cell transplantation models were used in our experiments. Differentcell numbers of male HCN4-MSCs were injected directly into the myocardium of syngeneicfemale rats. The number of engrafted HCN4-MSCs was estimated by using the amount ofSry gene of each sample by quantitative RT-PCR. We found that1×10~6may be the optimalnumber of engrafted HCN4-MSCs at one site by referring to the standard curve.
2. The short-term administration of triptolide can promote the survival ofHCN4-MSCs in myocardium after transplantation. Direct intramyocardial injection causedmechanical injury and acute inflammation, which caused the engrafted cells to die.Triptolide exhibited anti-inflammatory activity and significantly decreased the expressionlevels of IL-1β, IL-6and TNF-α, as well as NF-κB.
3. The results of patch-clamp experiments suggested that HCN4-MSCs expressedfunny current (If) in vivo and the activity of donor cells was unaffected by triptolide. SomeHCN4-MSCs were positive for both EGFP and troponin T (cTnT) or connexin43(CX43)in the host heart at4weeks after transplantation, which indicated that the implantedHCN4-MSCs differentiated into new cardiomyocytes.
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