人-猪异种生物瓣瓣膜材料的实验构建

目的　探讨用组织工程的方法实现异种生物瓣瓣膜材料的内皮化 ,制备新一代的生物瓣瓣膜材料。方法　取自肉联厂宰杀后 2小时内 4℃冰水保存的 8只猪心 ,在无菌条件下取出主动脉瓣分成 4组 :1组戊二醛 (GA) ,2组环氧氯丙烷 (EC) ,3组环氧氯丙烷 +左旋谷氨酸 (EC+L - GA) ,4组环氧氯丙烷 +左旋谷氨酸 +细胞提取液 (EC+L -GA +CE)。再将新生儿脐带 (离体后 6小时内 )用胰蛋白酶消化制备种子细胞 ,接种于加入 M199(10 %的胎牛血清 +2 0 %的混合血清 +黏附蛋白 +内皮细胞生长因子 )的培养液中进行种植 ,每日用倒置相差显微镜观察细胞生长情况 ,并进行内皮细胞 因子的定性检测、细胞生长覆盖密度的检测、光学和电子显微镜观察。　结果　倒置相差显微镜观察 :1组未见细胞生长 ,2组可见有细胞呈散在性生长覆盖 ,但较 3、4组的细胞数及覆盖率明显降低 (P<0 .0 1)。光镜和电镜观察 :1组未见有内皮细胞生长 ;2组可见有散在的内皮细胞生长 ;3组内皮细胞局部生长并可见细胞脱落 ;4组内皮细胞生长于原瓣叶的无细胞纤维支架上 ,并呈单层紧密排列 ,少见有细胞脱落或丢失 ,细胞沿纤维嵌合排列 ,细胞下组织呈间隙稀疏构成 ,有少量的基质成分和散在的胶原纤维 ,细胞沿纤维间隙排列、嵌合生长 ,形成空间三维排列生长的内皮

EXPERIMENTAL STUDY OF XENOGENEIC HEART VALVE MATERIAL

OBJECTIVE: To explore the possibility of improving the performance of tissue engineering valve by means of preendothelialization with cultured human umbilical vein endothelial cell(hUVEC) and to develop a new xenogenic bioprosthesis valve material. METHODS: The porcine aortic valves treated by use of glutaraldehyde(GA), epoxychloropropane(EC), L-glutamic acid(L-GA) and cellular extraction(CE) respectively were divided into four groups; group 1(GA), group 2(EC), group 3(EC + L-GA), and group 4(EC + L-GA + CE). The cultured hUVECs were seeded onto the treated porcine aortic valve, then that stuff were examined by means of EC VIII factor staining, living cells counting and microscopy. RESULTS: The cultured hUVEC could adhere to culturing bottle wall an hour later, and propagated to two passages after seven days. The cells increased with serial passage at a 7-day interval. But the hUVEC grew slowly when seeded onto the treated valve material except group 4. The cells in group 4 covered the surface of valve completely seven days later, which could also be seen in group 3 but not completely. There was no cell growing in group 1, and only fewer in group 2. The living cell in groups 3 and 4 were significantly more than in groups 1 and 2 on the 3rd, 7th and 14th days (P < 0.01), meanwhile, the number of cells in group 4 were also significantly more than that in group 3 (P < 0.05). The covering area of cultured cell on the valve material in groups 3 and 4 was significantly larger than that in groups 1 and 2. The covering area of cell in group 4 was over 95%, and higher than that in group 3(60%-70%). The hUVEC of group 4 arranged in pattern of three dimension. So it could resist rising of foreign power from the cardiac cavity of high pressure and flowing volume. There was no cell on the leaflet surface in group 1, and only a few pinch of cells could be seen in group 2. CONCLUSION: The porcine aortic valve can be used to be an ideal xenogeneic valve scaffold; the scaffold of porcine aortic valve should be treated by use of epoxy-chloropropane, L-glutamic acid and cellular extraction, so that a best growing environment to the hUVEC would be given; the cultured hUVECs used to be source of seed living cell had a boundless prospects; the growing velocity of cultured hUVEC was controllable, which facilitated clinical application; and the endothelial cells of xenogeneic valve material which grew compactly onto the scaffold can resist rising of foreign power from the cardiac cavity itself.