曲美他嗪联合骨髓间质干细胞移植治疗急性心肌梗死的实验研究

目的探讨曲美他嗪(TMZ)能否改善骨髓间质干细胞(MSCs)在体外缺氧模型及急性心肌梗死(AMI)大鼠心脏的存活。方法加入或未加入TMZ的MSCs在无血清培养基培养并缺氧暴露12h,采用透射电子显微镜和流式细胞仪检查第3代MSCs的活力和凋亡。30只Wistar大鼠随机分为AMI对照组、MSCs组及(MSCs+TMZ)组,结扎左冠状动脉前降支制备AMI模型。将MSCs注入梗死心肌边缘[(MSCs组和(MSCs+TMZ)组)]。(MSCs+TMZ)组的大鼠在AMI前3d开始至AMI后28d加喂TMZ。移植28d后,超声心动图评估心脏结构和功能。免疫荧光染色检测移植细胞在体内的存活和分化。TUNEL法检测细胞凋亡。收集TMZ治疗开始前和AMI后24、48h的血液样本,测量C反应蛋白(CRP)与肿瘤坏死因子-α(TNF-α)的变化。结果缺氧培养下,TMZ处理过的MSCs细胞凋亡降低了一半。在体内与AMI对照组相比,MSCs组和(MSCs+TMZ)组的心肌梗死面积显著缩小,心功能明显改善。与单纯MSCs移植相比,TMZ与MSCs移植的组合治疗表现出了更低的干细胞凋亡、更高的干细胞存活、更小的心肌梗死面积和进一步改善的心功能。各组之间CRP、TNF-α的基线水平并无显著差异,然而24h时(MSCs+TMZ)组的所有参数均低于MSCs组。结论 MSCs移植添加TMZ治疗AMI增加MSCs存活和心脏功能的恢复上优于单纯MSCs移植,抑制炎症因子表达可能是其机制之一。     

Effects of Tongxinluo-facilitated cellular cardiomyoplasty with autologous bone marrow-mesenchymal stem cells on postinfarct swine hearts

Background  Treatment of ischemic heart disease remains an important challenge, though there have been enormous progresses in cardiovascular therapeutics. This study was conducted to evaluate whether Tongxinluo (TXL) treatment around the transplantation of mesenchymal stem cells (MSCs) can improve survival and subsequent activities of implanted cells in swine hearts with acute myocardial infarction (AMI) and reperfusion.
Methods  Twenty-eight Chinese mini-pigs were divided into four groups including a control group (n=7); group 2, administration of low-dose TXL alone from the 3rd day prior to AMI to the 4th day post transplantation (n=7); group 3, MSCs alone (n=7) and group 4, TXL + MSCs (n=7). AMI models were made by occlusion of the left anterior descending coronary artery for 90 minutes. Autologous bone marrow-MSCs (3×10⁷ cells/animal) were then injected into the post-infarct myocardium immediately after AMI and reperfusion. The survival and differentiation of implanted cells in vivo were detected by immunofluorescent analysis. The data of cardiac function were obtained at baseline (1 week after transplantation) and endpoint (6 weeks after transplantation) by single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Apoptosis was detected by TUNEL assay and the oxidative stress level was investigated in the post-infarct myocardium at endpoint.
Results  At endpoint, there was less fibrosis and inflammatory cell infiltration with more surviving myocardium in group 4 than in the control group. In group 4 the survival and differentiation of implanted MSCs were significantly improved more than that seen in group 3 alone (P<0.0001); the capillary density was also significantly greater than in the control group, group 2 or 3 both in the infarcted zone (P<0.0001) and the peri-infarct zone (P<0.0001). MRI showed that parameters at baseline were not significantly different between the 4 groups. At endpoint, regional wall thickening and the left ventricular ejection fraction were increased while the left ventricular mass index, dyskinetic segments and infarcted size were decreased only in group 4 compared with control group (P<0.0001). SPECT showed that the area of perfusion defect was significantly decreased at endpoint only in group 4 compared with control group (P<0.0001). TUNEL assay indicated that TXL administration significantly decreased cell apoptosis in peri-infarct myocardium in groups 2 and 4. Furthermore, superoxide dismutase (SOD) significantly increased and malondialdehyde (MDA) decreased in groups 2 and 4 by the administration of TXL.
Conclusions  Our study demonstrates the following: (1) immediate intramyocardial injection of MSCs after AMI and reperfusion resulted in limited survival and differentiation potential of implanted cells in vivo, thus being incapable of beneficially affecting post-hearts; (2) TXL-facilitation resulted in a significant survival and differentiation potential of implanted cells in vivo via inhibition of apoptosis and oxidative stress, accompanied by significant benefits in cardiac function.

     

骨髓间充质干细胞治疗内毒素血症小鼠的实验研究

目的 明确骨髓间充质干细胞对内毒素血症小鼠的治疗作用。方法 实验分为四组,对照组（Control group）,内毒素血症组（LPS group）,间充质干细胞治疗组（LPS+MSCs group）,间充质干细胞组（MSCs group）。分别在LPS注射24h和7天后观察小鼠心功能的变化,ELISA方法检测血清细胞因子的水平,组织学方法观察对心肌、肝脏、肺脏和肾脏形态学改变的影响。结果 与对照组小鼠相比,内毒素血症组经LPS刺激后血清IL-1 和TNF-含量增高,间充质干细胞治疗组血清IL-1 和TNF-含量明显降低;内毒素血症组小鼠心功能明显下降,间充质干细胞治疗组心功能明显恢复;内毒素血症小鼠心肌细胞和肝细胞凋亡增加,肺间质和肺泡水肿,间充质干细胞治疗组上述组织损伤明显改善。结论 MSCs移植抑制了内毒素血症小鼠的炎症反应,改善了心功能,减轻了对心脏、肝脏和肺脏的损害。     

静脉移植骨髓干细胞治疗大鼠急性心肌梗死

目的　探讨急性心肌梗死 (AMI)早期经静脉途径移植骨髓基质干细胞对心功能的影响。方法　大鼠急性心肌梗死模型通过结扎冠状动脉前降支制作。同种异体大鼠骨髓基质干细胞体外培养、扩增、标记 ,并通过尾静脉于心肌梗死后 1d移植入AMI大鼠体内。分别进行心脏超声检查 ,以及组织学和免疫化学检测。结果　心肌梗死3周后 ,免疫组化分析表明部分干细胞迁移至梗死心肌周围并存活下来 ;干细胞移植组大鼠心功能较移植前及对照组明显改善 (P <0 0 5 )。结论　心肌梗死后 1d经静脉移植的骨髓基质干细胞可以归巢至梗死心肌处 ,并且可以改善损伤的心功能。     

谷氨酰胺联合脐血间充质干细胞移植在大鼠肠缺血再灌注损伤中的作用

目的探讨谷氨酰胺联合脐血间充质干细胞(MSCs)移植在大鼠肠缺血再灌注损伤中作用。方法体外复苏并培养脐血间充质干细胞移植前备用,观察CM-Di I荧光标记后脐血间充质干细胞的去向。80只SD大鼠随机分为正常对照组,缺血再灌注损伤组,谷氨酰胺组,MSCs移植组及联合组每组各15只。对照组采用生理盐水灌肠,损伤组采用TNB(S乙醇稀释)灌肠,在TNBS建模后1 h,谷氨酰胺组于尾静脉输入谷氨酰胺0.45 g/kg、MSCs移植组于尾静脉输入1×10~(10)/L脐血间充质干细胞悬液,联合组尾静脉输入谷氨酰胺0.45 g/kg+脐血间充质干细胞悬液1×10~(10)/L。通过ELISA法检测各组大鼠血清中肠脂肪酸结合蛋白(IFABP)、白介素-6(IL-6)、超氧化物歧化酶(SOD)的含量;各组于再灌注1 h、3 h后检测肠组织含水率;通过RT-PCR、Western blot观察大鼠肠黏膜上皮细胞caspase-3、NF-k B、Bcl-2在谷氨酰胺联合MSCs移植后的mRNA和蛋白的表达情况。结果通过荧光示踪法观察到移植的MSCs细胞分布于肠粘膜淋巴组织内和腺上皮细胞间,表明MSCs可能参与了肠缺血再灌注损伤的修复过程。各组大鼠血清中SOD、IFABP、IL-6的含量变化比较,损伤组血清中IFABP、IL-6的含量较对照组显著增加,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著减少,联合组减少更为明显,损伤组血清中SOD的含量较对照组显著减少,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著增高,联合组增高更为明显(P0.05)。再灌注1 h和3 h,损伤组肠组织含水率均明显高于对照组;与损伤组相比,谷氨酰胺组、MSCs移植组及联合组肠组织含水率值均显著降低,联合组降低更为明显,而谷氨酰胺组、MSCs移植组差异无统计学意义(P0.05)。与对照组比较,损伤组肠黏膜上皮细胞caspase-3、NF-k B的mRNA和蛋白表达明显上调,Bcl-2的mRNA和蛋白表达明显下调(P0.05),而谷氨酰胺组、MSCs移植组及联合组与之比较,caspase-3、NF-k B的mRNA和蛋白表达明显下调,Bcl-2的mRNA和蛋白表达明显上调(P0.05),谷氨酰胺组及MSCs移植组之间无统计学差异(P0.05),但两组与联合组比较,差异明显(P0.05)。结论谷氨酰胺组及MSCs移植后,明显减轻了大鼠肠缺血再灌注损伤程度,其可能通过抑制caspase-3、NF-k B表达和促进Bcl-2表达减轻肠黏膜缺血再灌注损伤。     

同种异体骨髓单个核细胞移植对急性心肌梗死后大鼠心肌细胞凋亡及相关基因表达的影响

目的:探讨大鼠同种异体骨髓单个核细胞(bone marrow mononuclear cells,BM-MNCs)急性心肌梗死区内移植对大鼠心肌细胞凋亡及相关基因表达的影响.方法:健康雄性Wistar大鼠24只,用结扎冠状动脉左前降支的方法建立大鼠心肌梗死模型后,分别将制备的培养基(对照组)和BM-MNCs悬液(移植组)由心外膜下植入梗死心肌周围.移植术后4周,超声心动图评价左室形态及功能,采用TUNEL法检测心肌细胞凋亡,免疫组化法检测心肌细胞中Bcl-2、Fas、FasL蛋白的表达水平,同时观察梗死区心肌内移植BM-MNCs及其周边区组织形态学特点.结果:移植术后4周,移植组左室收缩末期内径和左室舒张末期内径明显低于对照组(P＜0.01),左室射血分数、左室短轴缩短率明显高于对照组(P＜0.01).TUNEL结果显示:移植组心肌细胞凋亡指数(AI)明显低于对照组(P＜0.05).免疫组化结果显示:与对照组相比,移植组心肌细胞中Bcl-2蛋白平均光密度值显著升高(P＜0.05),Fas、FasL蛋白平均光密度值显著降低(P＜0.05),同时观察到移植组心肌梗死区内有BrdU标记阳性的BM-MNCs移植细胞存活.结论:同种异体BM-MNCs移植可以抑制心肌梗死后心肌细胞凋亡的发生,改善急性心肌梗死后的心脏功能,其机制可能与其调节Bcl-2、Fas、FasL的表达有关.     

Effects of heme oxygenase-1 gene modulated mesenchymal stem cells on vasculogenesis in ischemic swine hearts

Background  Mesenchymal stem cells (MSCs) transplantation may partially restore heart function in the treatment of acute myocardial infarction (AMI). The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 (HO-1) on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells.

Methods  In vitro, MSCs were divided into four groups: (1) non-transfected MSCs (MSCs group), (2) MSCs transfected with the pcDNA3.1-Lacz plasmid (Lacz-MSCs group), (3) MSCs transfected with pcDNA3.1-hHO-1 (HO-1-MSCs group), and (4) MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin (SnPP) (HO-1-MSCs+SnPP group). Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at <1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor (TGF-β) and fibroblast growth factor (FGF-2) in the supernatant. In vivo, 28 Chinese mini-pigs were randomly allocated to the following treatment groups: (1) control group (saline), (2) Lacz-MSCs group, (3) HO-1-MSCs group, and (4) HO-1-MSCs + SnPP group. About 1×10⁷ of autologous stem cells or an identical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging (MRI) assay and postmortem analysis were assessed four weeks after stem cell transplantation.

Results  Post hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs ((874.88±68.23) pg/ml) compared with Lacz-MSCs ((687.81±57.64) pg/ml, P <0.001). FGF-2 was also significantly increased in the HO-1-MSCs ((1106.48±107.06) pg/ml) compared with the Lacz-MSCs ((853.85±74.44) pg/ml, P <0.001). In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area compared with the Lacz-MSCs group (14.24±1.66/HPFs vs. 11.51±1.34/HPFs, P <0.001). Arteriolar density was also significantly higher in HO-1-MSCs group than in the Lacz-MSCs group (7.86±2.00/HPFs vs. 6.45±1.74/HPFs, P=0.001). At the same time, the cardiac function was significantly improved in the HO-1-MSCs group compared with the Lacz-MSCs group ((53.17±3.55)% vs. (48.82±2.98)%, P <0.05). However, all these effects were significantly abrogated by SnPP.

Conclusion  MSCs provided a beneficial effect on cardiac function after ischemia/reperfusion by the induction of therapeutic angiogenesis, and this effect was amplified by HO-1 overexpression.

     

Transplantation of magnetically labeled mesenchymal stem cells improves cardiac function in a swine myocardial infarction model

Background Mesenchymal stem cells （MSCs） transplantation provides a new approach for myocardial repair. However, many important fundamental questions about MSCs transplantation remain unanswered. There is an urgent need to identify MSCs from the beating heart and analyze the efficacy of this new approach. This study aimed to localize the magnetically labeled MSCs （MR-MSCs） and monitor the restorative effects of MR-MSCs with magnetic resonance （MR） imaging. Methods Acute myocardial infarction （AMI） was created in swine by a balloon occlusion of the left anterior descending coronary artery. Cells were delivered via intracoronary infusion after myocardial infarction. Infarct size change and cardiac function were assessed with 3.0T MR scanner. The results were then confirmed by histological and western blot analysis. All statistical procedures were performed with Systat （SPSS version 12.01）. Results A total of 26 swine were divided into four groups （sham-operated group, n=6; AMI group with PBS transplantation, n=6; labeled MSCs group, n=7; unlabeled MSCs group, n=7）. MSCs, MR-MSCs （10~cells） or PBS were delivered by intracoronary injection after MI and serial cardiac MR imaging studies were performed at 0, 4 and 8 weeks after transplantation. MR imaging demonstrated MI size decreased after MSCs transplantation in labeled and unlabeled groups, however, increases were seen in the AMI group at 8 weeks after MI. The left ventricular ejection fraction （LVEF） was slightly increased in the AMI group （（41.87~2.45）% vs （39.04~2.80）%, P 〉0.05）, but significantly improved in the MR-MSCs group （（56.85~1.29）% vs （40.67~2.00）%, P 〈0.05） and unlabeled group （（55.38~1.07）% vs （41.78~2.08）%, P 〈0.05） at 8 weeks after treatment. MR-MSCs were further confirmed by Prussian blue and immunofluorescent staining. Western blot analysis demonstrated that there was an increased expression of cardiomyocyte markers such as myosin heavy chain and troponin T in the MSCs trea     

水飞蓟素通过抑制心肌细胞凋亡减轻心肌梗死

目的：观察水飞蓟素对心肌梗死小鼠的血流动力学、梗死面积及梗死边缘区凋亡蛋白表达情况。方法：将60只小鼠随机分为心肌梗死组、假手术组、心肌梗死+水飞蓟素组和心肌梗死溶剂组。建模成功4周后检测小鼠血流动力学变化,进行心脏超声检查,评价梗死面积、细胞凋亡指数以及凋亡蛋白Bcl-2、Bax、Cleaved-Caspase3的表达。结果：与心肌梗死组小鼠相比,水飞蓟素可显著减轻心肌梗死,改善心梗小鼠心功能,降低心肌细胞凋亡指数,增强Bcl-2蛋白表达和减弱Bax和Cleaved-Caspase3蛋白表达。结论：水飞蓟素能够减轻心肌梗死,改善心梗小鼠心室收缩功能,保护心肌,减少心肌细胞的凋亡,其机制与升高Bcl-2蛋白、降低Bax和Cleaved-Caspase3蛋白表达水平有关。     

骨髓间充质干细胞对氧诱导心肌细胞凋亡的保护作用

目的 探讨骨髓间充质干细胞（MSC）对缺氧诱导心肌细胞凋亡的保护作用及可能机制。方法 分离培养成年大鼠MSC和乳鼠心肌细胞,将培养的乳鼠心肌细胞进行缺氧处理后,加入MSC或其条件培养液继续在无氧（95％N2＋5％CO2,持续缺氧组）或正常氧（缺氧／复氧组）条件下共培养72h,采用Hoechst细胞核染色,计算凋亡细胞百分率;采用蛋白质免疫印迹法检测凋亡相关蛋白Bcl-2和Bax的变化。结果持续缺氧可以诱导心肌细胞凋亡,凋亡率为51．6％±2．4％,与MSC或其条件培养液共培养,心肌细胞凋亡率显著减低,分别为15．1％±5．4％和24．0％±4．2％（均P〈0．01）;缺氧／复氧损伤可以引起心肌细胞凋亡,凋亡率为20．9％±2．7％,心肌细胞与MSC共培养,凋亡率显著减低（11．5％±3．7％,P〈0．05）;心肌细胞与MSC条件培养液共培养,凋亡率略有减低（20．1％±4．2％,P〉0．05）。蛋白质免疫印迹显示凋亡时心肌表达Bax水平较高,与MSC或其条件培养液共培养时,Bax表达水平呈不同程度降低,与心肌细胞凋亡发生率减低一致,Bcl-2无明显变化。结论 MSC对体外缺氧诱导的心肌细胞的凋亡有保护作用,其作用机制可能是通过细胞间直接接触和旁分泌细胞因子,影响了Bcl-2家族部分蛋白分子在心肌细胞的表达。     

同种异体骨髓间充质干细胞在大鼠心脏的迁移及分化

目的探讨同种异体骨髓间充质干细胞(MSC)在大鼠心脏定居存活情况、同种异体移植是否可行。正常心脏微环境与梗死心脏微环境对骨髓间充质干细胞(MSC)迁移、分化的影响。方法雌性Wistar大鼠35只,①正常心脏MSC移植组(10只);②急性心肌梗死对照组(AMI,10只);③心肌梗死MSC移植组(10只);④单个核细胞治疗组(5只)。结扎左冠状动脉前降支造成心肌梗死模型。分离纯化来自雄性Wistar大鼠的骨髓MSC,于冠脉结扎后1～3h植入到雌性正常大鼠和心肌梗死大鼠心脏组织,模拟同种异体细胞移植治疗。于细胞移植后10周取心脏检测各种指标。结果(1)经纯化的大鼠MSC在同种异体大鼠心脏可定居、生存,而未经纯化的单个核细胞移植在同种异体大鼠心脏未见存活;(2)在正常心脏微环境中,带蓝色荧光的供体MSC细胞核呈小岛屿状分布,与宿主心肌纤维排列无关;而在心肌梗死模型大鼠心脏微环境中,蓝色细胞核分布广泛,呈椭圆形,似心肌细胞核,其排列方向与宿主心肌纤维排列方向一致,免疫组化检测胞浆心肌特异蛋白染色阳性。结论纯化的MSC具有免疫耐受特性,无需加用免疫抑制剂,可进行同种异体移植;同种异体MSC在正常心脏微环境中不发生迁移、分化;而在急性梗死大鼠心脏中具有向缺血梗死区迁移并分化为心肌样细胞的特点。     

氧糖剥夺条件下p75神经营养素受体在人神经母细胞瘤细胞中的表达和作用

目的 探讨氧糖剥夺（OGD）条件下p75神经营养素受体（p75NTR）在人神经母细胞瘤细胞（SH-SY5Y）中的表达变化和作用。方法 SH-SY5Y细胞OGD模型的建立采用三气培养箱无糖无血清培养方法。模型成功建立后设立3组：无血清常规培养组（对照组）、OGD组和OGD+p75NTR竞争性阻断剂LM11A-31处理组（OGD+LM11A-31组）。在细胞培养12 h时用利用四甲基偶氮唑盐（MTT）法检测3组细胞存活率,乳酸脱氢酶（LDH）活力检测试剂盒测定LDH释放活力,流式细胞术检测凋亡细胞比例,蛋白质印迹法检测p75NTR的蛋白表达。结果 成功构建SH-SY5Y细胞OGD模型。细胞培养12 h时,对照组、OGD组和OGD+LM11A-31组细胞存活率分别为（94.80±4.06）%、（50.34±5.55）%、（64.68±4.59）%,LDH释放活力分别为（46.93±5.49）U/L、（353.09±30.67）U/L和（282.20±25.60）U/L,凋亡细胞比例分别为（1.82±0.45）%、（14.98±2.59）%和（7.36±1.98）%,p75NTR蛋白相对表达量分别为0.06±0.01、0.41±0.02和0.19±0.03,差异均有统计学意义（F=67.94、142.10、36.28、221.20,P均<0.05）。多重比较显示,OGD组细胞存活率低于对照组,LDH释放活力高于对照组,凋亡细胞比例高于对照组,p75NTR蛋白表达高于对照组,差异均有统计学意义（Bonferroni法,P''均<0.05）;而LM11A-31处理后OGD+LM11A-31组细胞存活率高于OGD组,LDH释放活力低于OGD组,凋亡细胞比例低于OGD组,p75NTR蛋白表达低于OGD组,差异均有统计学意义（Bonferroni法,P''均<0.05）。结论 OGD条件下p75NTR在人神经母细胞瘤细胞SH-SY5Y中表达增加,可能促进了神经损伤和凋亡。     

经冠状动脉自体骨髓间充质干细胞移植治疗小型猪急性心肌梗死

目的评价经冠状动脉移植体外分离培养自体骨髓间充质干细胞(MSCs)治疗急性心肌梗死(AMI)的可行性及疗效.方法通过结扎冠状动脉左前降支90 min后恢复血流的方法建立AMI模型.采用密度梯度离心法分离骨髓单个核细胞、传代培养后,在AMI造模10～14 d后将培养扩增的自体MSCs经冠状动脉植入梗死区,应用超声心动图观察移植前后心功能变化情况,冰冻切片荧光显微镜示踪4',6-二脒-2-苯基吲哚(DAPI)标记的MSCs,Ⅷ因子免疫荧光染色测定新生血管数目.结果MSCs生长呈纺锤样,生长旺盛呈旋涡样;DAPI标记率为l00%.AMI造模10～14 d后MSCs移植数量平均为(4～6)×107个,移植术后3个月,MSCs治疗组与对照组比较,左心室射血分数(LVEF)显著升高[(54.65±3.39)%vs(43.98±4.21)%(P＜0.01)],可见DAPI标记胞核为蓝色荧光的移植细胞,MSCs治疗组与对照组比较,新生血管数目显著升高[(13.28±4.39)vs(4.27 2.28)个,(P＜0.01)].结论通过体外培养扩增在短期内可以获得相当数量的生物性状稳定的MSCs,在AMI造模10～14 d后进行经冠状动脉MSCs自体移植,促进左室功能的恢复.MSCs移植与目前广泛运用的介入治疗相结合,方法简便、经济、有效,可望改善AMI患者的预后,具有良好的临床应用前景.     

Influence of Transplantation of Allogenic Bone Marrow Mononuclear Cells on the Left Ventricular Remodeling of Rat after Acute Myocardial Infarction

Ventricular remodeling after acute myocardial in-farction (AMI) not only significantly increases the complications and deaths during the acute period but also results in the progressive impairment of ventricular func-tion progress, finally leading to congestive heart failure. Therefore, restricting or reducing the ventricular remod-eling after AMI has become one of important topic in cardiovascular studies. In recent years, the study on stem cells transplantation has been making steady progr…     

经冠状动脉病变血管植入体外培养扩增的自体骨髓间充质干细胞对急性心肌梗死区的疗效研究

目的 探讨经冠状动脉病变血管植入体外培养扩增的自体骨髓间充质干细胞(MSCs)对急性心肌梗死区的影响.方法 体外扩增培养巴马香猪MSCs.用球囊扩张封堵冠状动脉前降支制备巴马香猪急性心肌梗死模型,成模1个月后,经冠状动脉前降支植入体外扩增培养的自体MSCs至急性心肌梗死区,观察其对心肌梗死区的影响.结果 梯度离心法分离...     

Dynamic Expression of Tumor Necrosis Factor-α and Vascular Endothelial Growth Factor in Rat Model of Pulmonary Emphysema Induced by Smoke Exposure

In order to explore the roles of tumor necrosis factor-α(TNF-α) and vascular endothelial growth factor(VEGF) in the pathogenesis of pulmonary emphysema,male Wistar rats were randomized into group A1,group A2.5 and group A4,each with smoke exposure for 1 month,2.5 months or 4 months,respectively.Group B1,group B2.5 and group B4 were used as non smoking controls at corresponding time points.TNF-α in bronchoalveolar lavage fluid(BALF) and expression of VEGF in lung tissue was determined by ELISA or by SABC immunohistochemistry assay either.Lung slices were stained with hematoxylin and eosin(HE).Results showed that in animal with smoke exposure the mean linear interceptor(Lm),an index of pulmonary emphysema and the content of TNF-α in BALF increased gradually,on contrary,the expression of VEGF in lung tissue decreased(P<0.05).This phenomenon was not obvious in animals without smoke exposure.Lm was negatively correlated to the VEGF expression(γ=-0.81,P<0.01) and positively correlated to TNF-α concentration(γ = 0.52,P<0.004),which implies that smoke exposure decreased the expression of VEGF and increased the expression of TNF-α.It is plausible to speculate that the imbalance of TNF-α and VEGF may play an important role in the pathogenesis of smoke-induced pulmonary emphysema.     

Fth1基因标记对骨髓间充质干细胞生物学特性及体外MRI表现的影响

摘要 目的: 观察铁蛋白报告基因Fth1标记对骨髓间充质干细胞(MSCs)生物学特性的影响及体外MRI表现。方法：原代分离培养大鼠MSCs,构建携带铁蛋白重链基因Fth1的慢病毒载体,取第4代MSCs进行转染,并在培养基内加入柠檬酸铁进行培养。普鲁士蓝染色检测转染MSCs的摄铁能力,台盼蓝染色活细胞计数检测转染细胞的活力,CCK-8测定转染MSCs的增殖活性。应用MRI的FSE T2WI和SWAN序列观察转染MSCs和普通MSCs MRI信号的差异。结果：Fth1基因可成功转染MSCs,转染MSCs的普鲁士蓝染色标记效率为87%;未加含铁培养液的转染MSCs,活细胞比率为92.17±1.91,OD值为1.094±0.0682,与普通MSCs对照组比较差异均无统计学意义（P>0.05）,加入含铁培养液培养3天后的转染MSCs活细胞比率为77.47±4.10,OD值为0.4929 ±0.0239,与未加含铁培养液的标记MSCs及对照组比较差异均有统计学意义（P<0.05）;MRI扫描FSE T2WI和SWAN序列显示转染MSCs信号强度为847.1±10.54,与未加含铁培养液的转染MSCs及对照组比较差异均有统计学意义(P<0.05),而未加含铁培养液的转染MSCs与对照组比较差异无统计学意义（P>0.05）。结论：Fth1报告基因可成功转染MSCs、并高效表达与摄取铁,不影响细胞活力及增殖活性,但在铁浓度1mol/L含铁培养液中细胞活性受到一定影响;经含铁培养液培养5天后,MRI可体外检测标记MSCs,其FSE T2WI和SWAN序列呈低信号改变。     

老年骨髓基质细胞移植治疗急性心肌梗死实验研究

目的 :观察老年大鼠骨髓基质细胞 (MSC)能否在急性心肌梗死环境中存活、增殖和向心肌样细胞分化。方法 :选用老年近交系Wistar大鼠 ,取供体鼠MSC ,体外扩增 ,DAPI荧光标记。受体鼠经左冠状动脉结扎建立急性心肌梗死模型。 1周后将标记的MSC用直接注射方式移植到梗死灶中 ,移植后 3、7、14d取材 ,观察梗死灶中MSC分布、扩散及心肌特异性蛋白T(TnT)的表达。结果 :在不同移植时间梗死灶中都发现DAPI阳性的细胞 ,且细胞计数随移植时间延长而增多。部分细胞表达TnT ,其中少数细胞出现心肌样横纹。结论 :老年大鼠MSC可在急性心肌梗死环境中存活、增殖 ,有向心肌样细胞分化的能力。