骨髓间充质干细胞对大鼠梗死心肌胶原重构的双重调节作用

目的 探讨骨髓间充质干细胞(MSC)对梗死心肌胶原重构的调节作用。方法 采用结扎冠状动脉前降支的方法复制大鼠心肌梗死(MI)模型,随机分为假手术组（仅穿线不结扎冠状动脉,n=8）、MI+ PBS组（结扎冠状动脉+心肌注射PBS溶液,n=8）和MI+ MSC组（结扎冠状动脉+心肌注射MSC,n=8）。通过心脏超声检查、血液动力学检查和组织学染色方法分别检测左心室射血分数(LVEF)、短轴缩短率(FS)、左心室收缩末压力(LVSP)、左心室舒张末压力(LVEDP)、左心室压最大升降速率（±dp/dtmax）、心肌梗死面积和梗死扩张指数等指标,评价MSC对大鼠心功能及心室重构的影响。同时采用免疫组化、RT-PCR、Western blot等方法,测量胶原蛋白表达情况。结果 (1)MI大鼠心室重构和心脏功能指标的检测结果：MI+ MSC组大鼠心肌梗死面积显著小于MI+ PBS组[(38.27±2.70)％比(46.20±3.17)％,t=5.386,P＜0.001],FS显著高于MI+ PBS组[(29.98±4.50)％比(23.43 ±3.34)％,t=-3.305,P=0.005],LVSP显著高于MI+ PBS组[(113.63±10.81)mm Hg(1 mm Hg=0.133 kPa)比（99.25±16.76）mm Hg,P＜0.05],LVEDP显著低于MI+PBS组[(12.10±4.28) mm Hg比(20.08±4.26) mm Hg,P＜0.05],+dp/dtmax显著高于MI+ PBS组[（4616.63±363.34）mum Hg/s比(3912.75±248.79) mm Hg/s,P＜0.05],- dp/dtmax显著高于MI+ PBS组[(4254.63±324.34) mm Hg/s比（3530.88±309.71）mm Hg/s,P＜0.05]。(2)Ⅰ型和Ⅲ型胶原蛋白表达水平的检测结果：MI+ MSC组大鼠梗死区Ⅰ型和Ⅲ型胶原蛋白表达均显著高于MI+ PBS组,而非梗死区Ⅰ型和Ⅲ型胶原蛋白表达均显著低于MI+ PBS组（P均＜0.05）。结论 MSC通过促进MI大鼠梗死区胶原蛋白修复性合成,减少非梗死区胶原蛋白沉积,从而抑制心室重构,改善心脏功能。     

人羊膜间充质干细胞静脉移植后在心肌梗死大鼠体内的定植及分化

目的 观察大鼠心肌梗死(MI)后经静脉移植的人羊膜间充质干细胞(hAD-MSCs)在体内的定植、分化及对心功能的影响.方法 SD大鼠随机分为hAD-MSCs移植组,模型组和假手术组各12只.分离hAD-MSCs用流式细胞仪和免疫组化染色鉴定其表型特征.制模后1w经舌下静脉移植Brdu标记的hAD-MSCs,于移植后6 w将大鼠心脏取出并切片行组织病理学、免疫组织化学、双重免疫荧光染色等检测,以观察植入的细胞在MI区的定植、分化,在肝、脾中的分布情况.结果 分离的hAD-MSCs具有典型的间充质干细胞表型特征,高表达CD44和波形蛋白;静脉移植后6w,心脏MI区、肺脏和脾脏中均可见Brdu阳性细胞,但在肝脏中未见阳性细胞,移植的hAD-MSCs表达心肌特异蛋白连接蛋白43和α-辅肌动蛋白.移植后6 w,移植组大鼠心功能没有进一步恶化.结论 hAD-MSCs经静脉移植能够定植于大鼠MI区域,可分化为心肌细胞,并改善心功能.     

The effects of mesenchymal stem cells transduced with Akt in a porcine myocardial infarction model

OBJECTIVE: This study was designed to examine whether mesenchymal stem cells (MSCs) transduced with Akt enhance cardiac repair after transplantation into the ischemic porcine heart. METHODS: MSCs isolated from porcine bone marrow and transduced with myr-Akt were transplanted into porcine hearts after experimental myocardial infarction (MI) using intracoronary injection [Group I, vehicle; Group II, MSCs; Group III, Akt-MSCs]. Myocardial single photon emission tomography (M-SPECT) was performed to assess myocardial function and the infarcted area. Pigs were also sacrificed for immunohistochemical characterization and histologic analysis. In addition, in vitro assays were performed to examine the resistance of Akt-MSCs to H(2)O(2) stimulation. RESULTS: Transplantation of MSCs into the ischemic porcine myocardium (Group II) increased the left ventricular ejection fraction (DeltaLV EF; -6.3 +/- 15.1% versus 0.5 +/- 6.4%, P < 0.001) and decreased the Deltaarea of MI (6.8+/-5.6% versus -5.0+/-5.3%, P < 0.001) compared with the vehicle control (Group I). Transplantation of MSCs transduced with myr-Akt (Group III) resulted in further improvement in DeltaLV EF (-6.3 +/- 15.1% versus 5.8 +/- 11.3%, P < 0.001) and in Deltaarea of MI (6.8 +/- 5.6% versus -17.0 +/- 7.6%, P < 0.001). Akt-MSCs were more resistant to apoptosis, and the levels of extracellular signal-regulated protein kinase (ERK) activation and vascular endothelial growth factor (VEGF) were higher in H(2)O(2)-stimulated Akt-MSCs. CONCLUSION: Cellular transplantation of Akt-MSCs further enhances the repair of injured myocardium compared to MSC transplantation alone by increasing the number of viable MSCs after cellular transplantation.     

Transplantation of mesenchymal stem cells attenuates myocardial injury and dysfunction in a rat model of acute myocarditis

Acute myocarditis is a non-ischemic inflammatory disease of the myocardium for which there is currently no specific treatment. We have previously shown that mesenchymal stem cells (MSC) can ameliorate heart injury during acute ischemia and in dilated cardiomyopathy; however, the therapeutic potential in acute myocarditis is unclear. In this study, we investigated the ability of MSC to attenuate myocardial injury and dysfunction during the acute phase of experimental myocarditis. Ten-week-old male Lewis rats were injected with porcine myosin to induce myocarditis. Cultured MSC (3x10(6) cells/rat) were injected intravenously 7 days after myosin injection. At 3 weeks, myosin injection resulted in severe inflammation and significant deterioration of cardiac function. MSC transplantation attenuated increases in CD68-positive inflammatory cells and monocyte chemoattractant protein-1 (MCP-1) expression in myocardium, and improved cardiac function in this model. Furthermore, myocardial capillary density was higher in myocarditis tissue, and was further increased by MSC transplantation. In vitro, cultured adult rat cardiomyocytes were injured in response to MCP-1, whereas this effect was attenuated by MSC-derived conditioned medium, suggesting cardioprotective effects of MSC acting in a paracrine manner. MSC transplantation attenuated myocardial injury and dysfunction in a rat model of acute myocarditis, at least in part through paracrine effects of MSC.     

Differentiation of rat embryonic stem cells into functional cardiomyocytes

Rats(Rattus norvegicus) have many advantages over mice in scientific studies,for example, they are more relevant to human in physiological and pharmacological responses.Therefore,rats are broadly used in experimental studies.The recent breakthrough in the generation of rat embryonic stem cells(rESCs) opens the door to application of gene targeting to create models for the study of human diseases.In addition,the in vitro differentiation of rESCs into derivatives of three germ lines will serve as a powerful tool and resource for the investigation of mammalian development,cell function, tissue repair,and drug discovery.However, the distinct culture condition and signal inhibitor-depended maintenance of rESCs stand as a considerable challenge for its in vitro differentiation.To address it,we investigated whether rESCs are capable of forming terminal differentiated cardiomyocytes. We found that the embryoid bodies(EBs)-based method used in mouse ESC(mESC) differentiation failed to work in the cultivation of rESCs.We then modified the differentiation protocol and successfully developed an in vitro differentiation system to differentiate rESCs into three embryonic germ layers.By using this method,the rESCs form spontaneous beating cardiomyocytes with the properties similar to those derived from fetal rat hearts and mESCs.This unique cellular system will provide a new approach to study the early development and cardiac function as well as to perform pharmacological test and cell therapy study(Grants:the State Major Research Program of China(2009ZX09503-024,2010CB945603) and CAS(XDA01030000).     

SD大鼠羊水间充质干细胞向神经样细胞体外诱导分化

目的:探讨体外诱导羊水间充质干细胞(AF-MSCs)分化成神经干细胞.方法:10只SD怀孕大鼠,180-240 g,胎龄16d,过量腹腔麻醉致死,开腹全部切除子宫,在无菌的条件下抽取全羊水,采用贴壁法分离AF-MSCs,体外培养扩增,观察细胞生长特性,流式细胞仪检测AF-MSCs表面抗原表达及细胞周期.取第3代AF-MSCs,加入预诱导液(DMEM/F12、100 mL/L FBS、1 mmol/Lβ-MEM)诱导24 h,然后加入诱导液(DMEM/F12、100 mL/L FBS、5 mmol/L β-MEM)诱导24 h,最后在分化维持培养液(DMEM/F12、20μg/L EGF、20μg/L bFGF)中培养,行免疫荧光染色,检测NSE和GFAP的表达.结果:成功分离、培养和传代SD大鼠AF-MSCs,流式细胞术检测提示大鼠AF-MSCs的G0/G1期细胞比例约为87.5%、S+G2+M期比例为12.5%,表达CD44为98.3%、GD29为70%、和CD105为33.2%、不表达CD45为0.5%,CD34为1%,DLA-DR(MHC class Ⅱ)为0.1%.诱导后,AF-MSCs能表达神经细胞标示物NSE和GFAP,且可在体外继续存活2 wk左右.结论:羊水中也存在MSCs,与其他来源的MSCs特点相符:羊水可以作为一种新的胎儿MSCs来源,而且能在体外诱导分化为神经细胞.     

Effect of mesenchymal stem cell transplantation on the engraftment of human hematopoietic stem cells and leukemic cells in mice model

We investigated the effect of human bone marrow-derived mesenchymal stem cells on engraftment of human umbilical cord blood CD34⁺ cells and acute myelogenous leukemia cells and also assessed the homing capability of MSCs. Forty-two NOD/SCID mice were administered sublethal irradiation followed by various cell doses of intravenous UCB CD34⁺ cells with or without MSCs. Another 12 NOD/SCID mice were also sublethally irradiated followed by intravenous injection of AML cells with or without MSCs. In ten of these mice, MSCs were genetically modified with an adenoviral vector encoding eGFP gene for tracking purpose. Cotransplantation of UCB CD34⁺ cells and MSCs resulted in a significant increase in bone marrow engraftment after 6 weeks, and the engraftment promoting effect of MSCs was proportional to the dose of MSCs and obvious when low doses of UCB CD34⁺ cells were given. There was no effect of MSCs on AML cells engraftment. All of the ten mice transplanted with eGFP-transfected MSCs showed positive for eGFP in their major organs. These data demonstrate that MSCs promote engraftment of UCB CD34⁺cells in bone marrow, but exert no effect on engraftment of AML cells, and are capable of homing to the major organs including bone marrow following intravenous infusion. This investigation was supported by a CMB Yuhan Research Grant of Yonsei University College of Medicine for 2003.     

Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells

AIM: To explore the possibility of marrow mesenchymal stem cells (MSC) in vitro differentiating into functional isletlike cells and to test the diabetes therapeutic potency of Islet-like cells. METHODS: Rat MSCs were isolated from Wistar rats and cultured. Passaged MSCs were induced to differentiate into islet-like cells under following conditions: pre-induction with L-DMEM including 10 mmol/L nicotinamide+l mmol/L β-mercaptoethanol+200 mL/L fetal calf serum (FSC) for 24 h, followed by induction with serum free H-DMEM solution including 10 mmol/L nicotinamide+1 mmol/L,β-mercaptoethanol for 10 h. Differentiated cells were observed under inverse microscopy, insulin and nestin expressed in differentiated cells were detected with immunocytochemistry. Insulin excreted from differentiated cells was tested with radioimmunoassay. Rat diabetic models were made to test in vivo function of differentiated MSCs. RESULTS: Typical islet-like clustered cells were observed. Insulin mRNA and protein expressions were positive in differentiated cells, and nestin could be detected in predifferentiated cells. Insulin excreted from differentiated MSCs (446.93&#177;102.28 IU/L) was much higher than that from pre-differentiated MSCs (2.45+0.81 IU/L (P&lt;0.01). Injected differentiated MSCs cells could down-regulate glucose level in diabetic rats. CONCLUSION: Islet-like functional cells can be differentiated from marrow mesenchymal stem cells, which may be a new procedure for clinical diabetes stem -cell therapy, these cells can control blood glucose level in diabetic rats. MSCs may play an important role in diabetes therapy by islet differentiation and transplantation.     

自体骨髓间充质干细胞在大鼠移植肝中的多向分化

我们将自体全骨髓细胞及骨髓间充质干细胞(MSCs)输入移植肝中,观察移植物存活时间、肝功能、病理变化及自体骨髓细胞的分化情况,探索自体骨髓细胞减少排斥反应、诱导移植物长期存活的可能机制.     

Focused ultrasound-induced stimulation of microbubbles augments site-targeted engraftment of mesenchymal stem cells after acute myocardial infarction

Intravascular transplantation of bone marrow-derived mesenchymal stem cells (MSCs) is a promising therapeutic approach after acute myocardial infarction. Efficacy and targeting of myocardial cell engraftment are crucial variables determining the therapeutic value of MSC transplantation. Highly focused ultrasound-mediated stimulation of microbubbles (hf-UMS) allows locoregional pre-treatment of target tissue. In a “proof of concept” study, we investigated augmentation of site-targeted MSC engraftment with hf-UMS. We further evaluated the ability of transplanted MSCs to transmigrate across the endothelial barrier into non-ischemic and post-ischemic myocardium in vivo. After acute myocardial ischemia and reperfusion, rats received hf-UMS focused on the anterior left-ventricular wall followed by intravascular transplantation of MSCs. Global and regional myocardial engraftment of MSCs was quantified by means of confocal laser-scanning microscopy; endothelial adhesion, transendothelial migration and invasion of basement membrane were distinguished. Targeted myocardium exhibited higher amount of transplanted MSCs vs. non-targeted tissue. The rate of transendothelial migration was lowest in non-ischemic (41.2 ± 2%) compared to post-ischemic myocardium (53 ± 5.7%, p < 0.01). Hf-UMS significantly increased the transmigration rate to 50 ±6.1% (p < 0.05) and 64 ± 8.9% (p < 0.05), respectively. Additionally, myocardial segments exposed to hf-UMS revealed an onset of protease activity. Signs of undesired biological effects, such as induction of apoptosis and/or myocardial necrosis were not observed. This study provides the first evidence of the migration of MSCs across the myocardial endothelium in vivo. Hf-UMS not only improves myocardial engraftment of MSCs but also allows locoregional targeting of post-ischemic myocardium.     

心肌细胞裂解液诱导骨髓间充质干细胞向心肌细胞的分化作用

目的:用心肌细胞裂解液体外诱导分化骨髓间充质干细胞(SMCs),观察SMCs能否表达脑钠肽(BNP)及β1受体mRNA,是否具有受体后信号转导通路.方法:分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年小鼠长骨骨髓中分离MSCs,并用含有心肌细胞裂解液的培养基培养1周.用逆转录聚合酶链反应方法定性检测MSCs中BNP及β1受体mRNA水平,用放射免疫方法测定MSCs中环磷酸腺苷(cAMP)的含量.结果:诱导组SMCs能表达BNP及β1受体mRNA,对照组不表达BNP和β1受体mRNA.诱导的SMCs经10-8,10-7,10-6,10-5mol/L异丙肾上腺素(ISO)作用2 h后,均能增加细胞内cAMP含量,且具有ISO浓度依赖性(P＜0.05或P＜0.01).诱导的SMCs经不同浓度的美托洛尔(MET)10-6,10-5 mmol/L作用10 min后,和1×10-7mol/L ISO再一起作用110 min后,测定细胞内cAMP含量与空白组比较,差异无统计学意义;与10-7mol/L ISO作用结果比较,差异有统计学意义(P＜0.01),表明MET能完全阻断ISO升高细胞内cAMP作用.结论:心肌细胞裂解液能体外模拟心肌微环境诱导分化SMCs表达β1受体及BNP mRNA,并具有受体后活性的信号转导通路.     

大鼠脂肪间充质干细胞在部分肝切除模型中向肝细胞分化的研究

目的探讨脂肪间充质干细胞（ADMSCs）在部分肝切除模型中向肝细胞的分化。方法从大鼠脂肪组织中分离出干细胞，并进行体外扩增、传代，取第2代ADMSCs用PKH26标记，制作部分肝切除模型，将标记细胞经门静脉自体植入体内。2周后切下肝脏制成冰冻切片，荧光显微镜下观察标记细胞在肝脏的定位，进行免疫荧光染色检测标记细胞白蛋白的表达。结果从脂肪组织中分离出的ADMSCs能在体外大量扩增，PKH26标记后细胞在荧光显微镜下发红色荧光，细胞标记率约95％；荧光显微镜下可见肝脏冰冻切片中散在分布红色标记细胞，免疫荧光染色显示大多数标记细胞白蛋白染色阳性。结论 大鼠脂肪间充质干细胞在肝再生环境中能向肝细胞分化，有可能在肝部分切除后参与肝再生。     

心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究

目的通过向骨髓间充质干细胞(MSCs)培养体系中添加心肌细胞裂解液的方法,体外模拟心肌微环境,观察MSCs向心肌细胞分化的诱导作用,并与诱导分化剂5-氮杂胞苷(5-aza)比较。方法分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年大鼠骨髓中分离MSCs,用含有心肌细胞裂解液的培养基(A组)、含有5-aza的培养基(B组)、含有5-aza和心肌细胞裂解液的培养基(c组)以及普通培养基(对照组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达α-肌动蛋白、心脏特异性肌钙蛋白T(cTnT)、连接蛋白43及CD31的情况。结果A、B组的MSCs在培养1周后均形成肌细胞形态,并且均表达α-肌动蛋白和cTnT;A组MSCs分化的肌样细胞所含的肌纤维较B组更丰实,细胞生长趋势也优于B组,并且可以表达CD31;B组MSCs分化的肌样细胞不表达CD31;对照组细胞仅表达α-肌动蛋白。结论心肌细胞裂解液是体外诱导MSCs分化为心肌样细胞的理想条件,优于传统的5-aza,在心肌细胞移植技术中可以用于体外模拟心肌细胞微环境。     

同种异体移植骨髓间充质干细胞治疗大鼠心肌梗死

目的　探讨同种异体骨髓间充质干细胞 (MSCs)在梗死大鼠心脏局部存活、迁移、分化及对心功能的影响 ;明确同种异体细胞移植治疗心肌梗死 (MI)的可行性及效果。方法　雌性Wistar大鼠 3 5只 ,随机分为正常对照组、急性心肌梗死 (AMI)组及MI MSCs治疗组。分离纯化雄性Wistar大鼠骨髓MSCs ,于左冠状动脉前降支结扎后 1～ 3h植入到雌性大鼠心脏组织 ,移植后 10周检测心功能并取心脏检测各种相关指标。结果　异体大鼠MSCs经纯化后可在梗死心脏组织定居、生存 ,并与宿主心肌纤维排列方向一致 ,免疫组化检测胞质心肌特异蛋白染色阳性 ,与MI组比较 ,异体细胞移植组左室收缩压升高 (P <0 0 5) ,舒张末压明显降低 (P <0 0 1)、左心室内压最大上升和下降速率显著增快 (P <0 0 5) ,梗死边缘区心肌面毛细血管数目明显增加 (P <0 0 5) ,多功能真彩色病理图像分析系统显示MI面积缩小 (P <0 0 5)。结论　同种异体MSCs移植治疗MI可行、有效