摘要：
Under physiological conditions,thrombomodulin (TM) can combined with thrombin into a compound,which can prevent blood coagulation and promote fibrinolysis by activating protein C;and can be absorbed by endothelial cells,where the thrombin is decomposed by intracellular lysosomes.In addition,TM can be combined with coagulation factor Xa specificity,inhibit the activation of prothrombin,so that makes the generation of thrombin decreased significantly.When pig (Sus scrofa) blood vessels expose to the human (Homo sapiens) blood after xenotransplantation,the pig TM can't combine with human thrombin,which is the reason of coagulopathy.Wuzhishan Miniature pigs are best candidate for organ xenotransplantation donor with characteristics of highly similar anatomy and physiology to human.There are three aims of this research:produce transgenetic Wuzhishan Minipigs expressing human thrombomodulin (hTM),research hTM expression patten in endothelial cells of the transgenetic pigs,and provide practical basis for solve disorders in blood coagulation after xenotransplantation.Taking the Large White Pig's TM bacterial artificial chromosome (BAC) as a template,constructed a PBR322-catch vector that had homologous short arm.A 7 kb promoter of porcine TM gene was sub-cloned from BAC of Large White Pig by gap-repair method mediated by red homologous recombination system in E.Coli.,positive vector pBR322-catch-pProwas identified by enzyme.hTM amplified from human cDNA and bovine Growth Hormone PolyA (bGHpA) amplified by PCR,were linked to the pBlueScript Ⅱ (SK-)vector.Vector pBR322-catch-pProwas was digested by enzyme to obtain the Large White Pig's TM promoter,then connected it with vector pBlueScript Ⅱ (SK-)to construct the specific expression pBS-hTM vector containing puromycin.The Xhol Ⅰ linearized pBS-hTM was integrated into the fetal fibroblasts cell lines (WPF167 and WPF169) by electroblot.Screening cells using culture solution contained 1.0 μg/mL puromycin for 10～15 days.Two cell clones with better growth characters were used as donor cells to conduct nuclear transfer to acquire reconstructed embryo.972 reconstructed embryos were harvested,and then transferred into 5 Wuzhishan Miniature receptor pigs.New born piglets were identified by genomic DNA PCR,and umbilical cord tissue RT-PCR and Western blot.As results,positive catch vector pBR322-catch-pPro and expression vector pBS-hTM were successfully acquired.354 cell clones were gained after 10～15 days screening,in which 339 cell clones were positive.The reconstructed embryo had no significant differences with the normal reconstructed embryos (P＞0.05).Two receptor pigs showed pregnant by B-ultrasonic determination after 30 days and 5 piglets were born after 120 days.4 of new born piglets were identified positive and the expression of hTM specifically on the surface of endothelial cells.As a conclusion,transgenic cloned Wuzhishan Miniature pigs were successfully achieved,which specifically expressed hTM in vascular endothelial cells.This study could provide practical basis for further in-depth study in coagulation disorders after xenotransplantation.%生理条件下,血栓调节蛋白(thrombomodulin,TM)可与凝血酶结合成复合物,该复合物既可通过激活蛋白C达到抗凝和促纤溶作用;又可经内皮细胞胞吞,通过细胞内溶酶体降解、清除体内凝血酶.另外,TM可与凝血因子Xa特异性结合,抑制凝血酶原的活化,进而使凝血酶的生成速率明显降低.移植后,猪(Sus scrofa)的血管暴露在人(Homo sapiens)血液中,猪TM无法与人的凝血酶发生结合从而使凝血功能紊乱,因此,在猪体内表达人TM是目前解决凝血功能异常的一个新的思路.本研究制备了血管内皮特异性表达人血栓调节蛋白(human thrombomodulin,hTM)的转基因克隆猪,研究了hTM在五指山小型猪血管内皮细胞表达情况.利用Red重组系统从细菌人工染色体(bacterial artificial chromosome,BAC)上抓捕获得猪的内源TM启动子.PCR扩增获得hTM的cDNA及牛生长素PolyA(bGHpA)序列,依次酶切连入pBlueScriptⅡSK(-),构建血管内皮特异性表达hTM的载体.获得的载体pBS-h TM-puro经Xho Ⅰ线性化,电转染五指山小型猪胎儿成纤维细胞,经1.0 μg/mL嘌呤霉素筛选10～15 d得到339个转hTM基因的阳性克隆,取两个生长较好的克隆用于核移植,随后获得重构胚972枚.胚胎移植后产仔猪5头;其中4头经基因组DNA、组织RT-PCR检验为阳性,Western blot结果显示hTM为血管内皮细胞特异性表达.成功获得可特异性表达hTM转基因细胞系及转基因克隆猪,为解决异种器官移植后出现的凝血紊乱问题提供了资料基础.
