利用人骨髓基质干细胞构建组织工程心脏瓣膜的体外实验

背景：目前临床应用的人工瓣膜,无论机械瓣还是生物瓣都存在着诸如易感染、出血和血栓形成并发症,因瓣口狭窄需再手术等自身难以克服的缺陷。理论上具有生物活性的组织工程心脏瓣膜可克服上述缺点,但种子细胞和瓣膜支架的选取尚存在争议。目的：探讨应用人骨髓基质干细胞及脱细胞猪主动脉瓣支架体外构建组织工程瓣膜的可行性。方法：采用去垢剂一核酸酶消化法处理,去除猪主动脉瓣叶细胞成分,获取施行简单先心病修补患者胸骨来源的骨髓基质干细胞,将其种植于脱细胞猪主动脉瓣支架共培养5d。结果与结论：流式细胞技术证实接种的种子细胞的表面抗原符合人骨髓基质干细胞的特征;光镜及电镜检查证实,猪主动脉瓣膜中的细胞成分可完全去除,获得完整无细胞的纤维网状支架;瓣叶去细胞前后的生物力学性能无明显变化;种植的人骨髓基质干细胞可在脱细胞猪主动脉瓣支架表面形成一层连续的细胞层;接种的人骨髓基质干细胞有向成纤维细胞分化的趋势。以上结果提示种植人骨髓基质干细胞于脱细胞猪主动脉瓣支架上,可构建出组织工程心脏瓣膜。     

大鼠室壁瘤左心室重建模型的建立

目的探讨建立大鼠室壁瘤左心室重建(LVR)动物模型的可行性。方法对35只成年雄性Sprague-Dawley(SD)大鼠实施左冠状动脉前降支结扎以制作心肌梗死后室壁瘤(LVA)模型,4周后,对符合入选标准的16只LVA模型实施LVR手术,作为实验组;另10只模型行开胸手术作为对照组,在术后3 d、2周、4周分别以超声心动图评价两组大鼠心功能情况,以直视摄片、Masson’s Trichrome染色评价室壁瘤切除的彻底程度。结果 LVA模型及LVR模型的死亡率分别为11.4%(4/35)及18.8%(3/16),建模成功率分别为74.3%(26/35)和81.3%(13/16)。直视摄片及Masson’s Trichrome染色提示补片完全替代心肌梗死瘢痕。超声心动图提示:行LVR手术的实验组动物的左心室收缩期末内径(LVESD)及左心室短轴缩短率(FS)在术后3 d即较对照组有所改善[术后3 d:LVESD(5.00±0.87)mm vs.(5.90±0.92)mm,P<0.05;FS(34.20%±6.80%)vs.(26.60%±6.12%),P<0.01],随着时间的推移,施行LVR手术的实验组大鼠比对照组更好地维持了左心形态及收缩功能[术后2周:左心室舒张期末内径(LVEDD)(7.60±0.56)mm vs.(8.50±1.08)mm,P<0.01;LVESD(5.10±0.65)mm vs.(6.69±0.89)mm,P<0.001;FS(31.90%±6.90%)vs.(21.10%±6.17%),P<0.001;术后4周:LVEDD(7.70±0.50)mm vs(.9.10±0.89)mm,P<0.001;LVESD(5.20±0.39)mm vs.(7.20±0.95)mm,P<0.001;FS(31.80%±2.40%)vs.(20.20%±4.17%),P<0.001]。结论大鼠LVR模型可作为组织工程心肌补片(EHT)研究中一种稳定、可靠、经济的初选模型。     

利用人骨髓基质干细胞构建组织工程心脏瓣膜的体外实验

BACKGROUND:Nowadays, mechanical or biological valve recipients used in the clinic are still at the risk of infection, hemorrhage, thrombosis and reoperation owing to valve stenosis. Tissue-engineered heart valve with biological activity can overcome the disadvantages above. While, the optimal choice of scaffolds and seeding cells remains disputable. OBJECTIVE:To explore the feasibility to construct tissue-engineered heart valve with acellularized porcine aortic valve scaffold and human bone marrow stromal stem cells in vitro. METHODS:The porcine aortic valves were decellularized with the detergent and enzymatic extraction process to remove the cellular components. Human bone marrow stromal stem cells were aspirated from sternum of the patients with simple congenital heart malformation, and then the cells were seeded on the acellularized porcine aortic valve scaffold and cultured for 5 days. RESULTS AND CONCLUSION:Flow cytometry identified that the characteristics of surface antigen of the inoculated seed cells were in line with those of human bone marrow stromal stem cells. Light microscopy and electron microscopy confirmed that the cellular components in the porcine aortic valves could be removed to obtain the complete acellular fiber mesh stent. There was no significant difference in biomechanical property between before and after acellularization. The human bone marrow stromal stem cells implanted on the acellularized porcine aortic valve scaffold could form a continuous cell layer on the surfaces of the scaffold. The inoculated bone marrow stromal stem cells could be differentiated into fibroblasts. The implantation of human bone marrow stromal stem cells on the acellularized porcine aortic valve scaffold can construct the tissue-engineered heart valve.     

共价结合生长因子的胶原补片修补大鼠左心室室壁瘤的实验研究

目的 比较采用碳化二亚胺法(EDC法)处理共价结合不同生长因子的胶原补片修补大鼠左心室室壁瘤后的再血管化情况,及其对大鼠左心功能的影响.方法 直径5 mm的圆形胶原补片经EDC法处理作对照组,再分别结合血管内皮细胞生长因子(VEGF)或VEGF+碱性成纤维细胞生长因子(bFGF)后作实验组.成年雄性SD大鼠行左冠状动脉前降支结扎制作透壁心梗模型.4周后,心脏彩超筛选心梗面积占左心室前壁25％～ 35％者入选.随机分成3组,以不同补片行室璧瘤修补,对照组(8只),VEGF组(10只),VEGF+ bFGF组(10只).术后1周、2周、4周分别行心脏彩超监测左心功能,至实验终点取材,免疫荧光法检测补片边缘毛细血管(vWFⅧ染色)及成熟血管(SMA染色)的生成情况.结果 全组死亡比例15％(6/40只).修补1周后,3组动物心功能均明显改善;4周后,结合生长因子的两组心功能较对照组明显改善(对照组对VEGF组,P＜0.05;对照组对VEGF+ bFGF组,P＜0.01).组织学检查显示,两组结合有生长因子的毛细血管生成情况均较对照组明显改善(P ＜0.05);VEGF+bFGF组的再血管化较其他两组明显改善(P＜0.01).相关性分析显示,大鼠心功能参数(FS)与再血管化呈正相关(P=0.0297,r2=0.998).结论 EDC法可有效改善胶原补片的机械性能,共价结合生长因子后可显著增加补片内血管生成,再血管化有助于左心功能的维持.