心脏瓣膜组织工程中理想种植细胞的来源比较

Ideal seeding cells for tissue engineering heart valves

背景:现有的人工瓣膜,无论是机械瓣膜还是生物瓣膜,都存在与取材有关、无法从工艺制作方式改进解决的固有缺陷。组织工程心脏瓣膜概念的提出为解决该难题提供了新的方向。目的:通过比较成熟分化血管内皮细胞和骨髓间充质干细胞骨的获取手段、体外增殖能力以及在脱细胞基质上的生长情况,分析何者更适合作为制备组织工程人工心脏瓣膜的种植细胞。设计、时间及地点:单一样本观察,于2008-06/09在上海市胸科医院动物实验基地完成。材料:用胰蛋白酶(胰酶)-EDTA、TritonX-100、DNase、RNase消化猪主动脉瓣膜获得脱细胞基质。从1只12kg2岁雄性杂交犬大隐静脉、股外侧动脉及肋骨骨髓获取种植细胞。方法:用胰酶消化并收集内皮细胞,收集骨髓细胞,分别进行培养。通过贴壁法获得内皮细胞及骨髓间充质干细胞。利用内皮细胞生长因子诱导骨髓间充质干细胞向内皮细胞分化,并绘制细胞生长曲线。以抗Ⅷ因子抗体以及抗血管内皮生长因子抗体荧光染色,荧光显微镜观察。将细胞种植于脱细胞瓣膜上培养3d后进行扫描电镜观察。主要观察指标:①试验犬伤口愈合情况。②细胞培养、增殖情况。③骨髓来源骨髓间充质干细胞表型分析以及形态学观察。结果:成熟血管内皮细胞体外增殖能力低下,骨髓间充质干细胞则增殖旺盛。生长曲线显示骨髓间充质干细胞的倍增时间约30h,而成熟内皮细胞生长曲线平直。对骨髓间充质干细胞进行抗Ⅷ因子抗体以及抗VEGF抗体荧光染色,可见荧光染色阳性。扫描电镜见种植骨髓间充质干细胞的脱细胞瓣膜上散在星状细胞贴附。结论:骨髓间充质干细胞体外增殖能力强,在内皮细胞生长因子诱导下可向内皮细胞转化,并可以生长贴附于脱细胞瓣膜,是作为组织工程人工心脏瓣膜制备的理想细胞来源。

BACKGROUND： Every existing prosthetic valve, either mechanical valve prosthesis or biovalve, has its inherent defects that are determined by its material itself and can not be avoided no matter what manufacturing technique is used. Fortunately, the concept of tissue engineering heart valve （TEHV） provides a new direction for the solution of the above-mentioned dilemma. OBJECTIVE： To analyze which kind of cells is the more suitable seeding cells for tissue engineering heart valves, well-differentiated vascular endothelial cells or bone marrow mesenchymal stem cells （MSCs）, by comparing the obtaining methods of the two, as well as their in vitro proliferation abilities and their growth potentials on acellular matrixes. DESIGN, TIME AND SETTING： A single sample observation was completed in the Animal Experiment Base of Shanghai Chest Hospital from June to September in 2008. MATERIALS： Acellular matrixes were obtained by digesting aortic valves of pigs with Trypsin-EDTA, Triton X-100, DNase and RNase. Seeding cells were obtained from the great saphenous veins, the lateral femoral arteries and the ribs marrows of a male hybrid dog of two years old, weighing 12 kg. METHODS： Trypsin digestion was adopted to collect endothelial cells which, together with the bone marrow cells, were cultured separately. The endothelial cells and MSCs were harvested with adherence method. Then, the MSCs were induced into endothelial cells using endothelial cell growth factors, and the cell growth curve was drawn simultaneously. After that, MSCs were stained with the anti-Ⅶ factor antibody and the anti-vascular endothelial growth factor （anti-VEGF） antibody, and then were observed under the fluorescence microscope. After 3 days of culture on acellular valves, seeding cells were observed under the scanning electron microscope. MAIN OUTCOME MEASURES： ①The wound healing of the experiment dog; ②The culture and proliferation of cells; ③The phenotype analyses and morphology observation of MSCs. RESULTS： Compared with well-differentiated vascular endothelial cells, MSCs showed a much greater expanding ability in vitro. The doubling time of MSCs was 30 hours according to the cell growth curve, which is a well contrast to the flat one of the mature endothelial cells. Moreover, MSCs showed positive fluorescence staining after being stained with fluorescent anti-factor Ⅷ antibody and fluorescent anti-VEGF antibody. Under scanning electron microscope, adherent stellate cells could be seen scattering on the culturing acellular valves of MSCs. CONCLUSION： MSCs not only have a strong ability of in vitro proliferation, but also can be induced into endothelial ceils by endothelial cell growth factors. What＇s more, they can grow on and adhere to acellular valves. All of these show that MSCs are the ideal seeding cells for TEHV preparation.