Ang Ⅱ诱导大鼠BMSCs联合脱细胞牛心包体外构建工程化心肌组织的实验研究  

Experimental study of in vitro construction of engineering myocardial tissue based on rat BMSCs induced by Ang Ⅱ and accellular bovine pericardium

作　　者：石静宝[1] 邢万红[1] 

机构地区：[1]山西医科大学第二医院心胸外科,太原030001

出　　处：《国际生物医学工程杂志》2015年第3期133-137,I0001,I0002,共7页International Journal of Biomedical Engineering

基　　金：山西省科技攻关项目（20120313021-3）;山西省留学归国人员科研项目（2012091）

摘　　要：目的探讨血管紧张素Ⅱ（AngⅡ）诱导骨髓间充质干细胞（BMSCs）分化为心肌细胞的能力，及诱导后BMSCs体外联合脱细胞牛心包构建工程化组织心肌的可行性。方法分离培养大鼠BMSCs，用含0．1μmol／LAngⅡ的完全培养基诱导培养第3代BMSCs24h，然后换用完全培养基连续培养；对照组采用完全培养基连续培养。采用免疫荧光法检测诱导后的BMSCs表达心肌特异蛋白cTnT、β-MHC情况；透射电镜观察诱导后的细胞超微结构。用去污剂一酶消化法对新鲜牛心包行脱细胞处理，然后将诱导后4周的BMSCs接种于脱细胞牛心包生物支架上培养3d后，对其进行观察检测。结果AngⅡ诱导后的BMSCs向心肌细胞分化，可表达cTnT和β—MHC，对照组则不表达cTnT和β—MHC。电镜结果显示诱导后的细胞间可见类肌节组织及明显的桥粒连接；新鲜的牛心包经去污剂一酶联合四步法脱细胞处理及京尼平交联后，HE染色观察脱细胞的效率接近100％。扫描电镜观察发现脱细胞处理后的牛心包表面无细胞残留且表面排列杂乱，放大后可见有2μm小孔。观察体外构建的工程化心肌显示诱导后的BMSCs可良好地黏附于脱细胞牛心包生物支架表面并生长增殖，且少量可渗透到支架内部。结论AngⅡ诱导的大鼠BMSCs可向心肌细胞方向分化，表达心肌特异蛋白cTnT和β-MHC。将其种植于脱细胞牛心包生物支架上，表面黏附良好，且可渗透到支架内部及血管周围，有望用于构建具有血管网络化的组织工程化心肌。Objective To investigate the feasibility of inducing rat bone marrow mesenchymal stem cells （BMSCs） into myocardial cells by angiotensin Ⅱ（Ang Ⅱ） and the approach of applying the induced BMSCs to construct myocardial tissue in vitro. Methods BMSCs from rats were isolated and cultured. The third generation of BMSCs were incubated in complete culture medium, in which 0.1 μmol/L Ang Ⅱ was added, for 24 h. The BMSCs were switched to complete medium and cultured. The control group was cultured by complete medium without Ang Ⅱ. The protein cTnT and 13-MHC were tested by immunofluorescence staining and the structure of induced cells was observed by transmission electron microscopy. The bovine pericardiums were managed by the detergent-enzyme digestion method. The induced BMSCs were planted onto the acellular bovine pericardium and cultured for 3 d before being tested. Results The cells of induced group were induced into cardiomyocytes by Ang Ⅱ, which could express cTnT and β-MHC, while cells in the control group couldn＇t. The result of electron microscopy showed that desmosome could be observed in the induction group. The surface of acellular bovine pericardiums was detected by scanning electron microscopy and no cells remaining on the surface except 2 μm-aperture were observed in the acellular bovine pericardiums. The induced BMSCs adhered to acellular bovine pericardium and grew well. Conclusions BMSCs can be induced into cardiomyocyte in vitro and express cTnT and β-MHC. The ceils can adhere well on the surface and the inside of acellular bovine pericardium. BMSCs have the potential to form engineered cardiac muscle tissue with blood vessel.

关 键 词：骨髓间充质干细胞 心肌细胞 组织工程 血管紧张素Ⅱ 

分 类 号：R318[医药卫生—生物医学工程] Q813[医药卫生—基础医学]