Development of the therapy for hepatic congenital metabolic disease by In-Utero-Manipulation.

大鼠胎肝处于发育和增殖期，通过子宫膜可见，是一种较好的细胞移植模型。我们这次选择的羊膜上皮细胞作为供体细胞，被认为是可能进入神经和肝脏组织的基因载体。这种细胞的优点是：1)伦理问题少，2)有神经和肝脏干细胞样细胞，3)细胞可以冷冻保存，4)细胞可以相对长期存活。然而，经门静脉大量移植羊膜细胞可能会导致血栓，并且细胞在体外只能分裂两到三次。因此，我们进行了羊水细胞胎肝移植。正如人类羊膜上皮细胞所报道的那样，在培养期间一定数量的羊膜细胞表达白蛋白(J Hum Genet 45 171 2000)。这些细胞可以被冷冻保存，而不会对细胞的活力造成明显损害。胚胎早期(第14天)细胞平均分裂倍数为2.7倍，端粒酶活性较高，提示端粒酶基因可能促进细胞增殖。胎肝细胞移植(LacZ基因转染法，1~8×10~6/10~25μ~1)后(18.5~20.5天)，存活率为24~85%。移植的细胞在出生后至少14天就可以在肝脏中检测到。与同时期胎肝内直接注射Ad-LacZ载体相比，羊膜细胞移植后LacZ阳性细胞存活时间更长。因此，目前的宫内操作将是细胞介导的先天性肝病基因治疗的有用策略。

Rat fetal liver is a good model for choice to develop cell transplantation because the liver is under developmental and proliferative stage and visible through uteric memebrane. Amniotic epithelial cells, which we chose as donor cell this time, are proposed to be possible gene carrier into neural and hepatic tissue. The advantages of the cells are that 1) ethical problem are few, 2) there are neural and hepatic stem-like cells, 3) cells can be stored frozen, and 4) cells survive for relatively long-term. However, mass transplantation of amniotic cells via portal vein may cause embolism and the cells intrinsically proliferate only by two or three division in vitro. Therefore, we performed transplantation of amniotic cells into fetal liver. The certain population of the amniotic cells expressed albumin during culture period as was reported by human amniotic epithelial cells(J Hum Genet 45 171 2000). The cells can be stored frozen without marked impairment of viability. The cell division was 2.7 times at average and telomerase activity was detectable in the cells from fetuses at early stage(day 14), suggesting that telomerase transfection may accelerate cell proliferation. After cell transplantation(lacZ-transfected, 1〜8x10^6/10〜25μ1)to the liver of fetus(day18.5-20.5), 24-85% of the rats survived to be born. The transplanted cells were detectable in the liver at least 14 days after birth. The lacZ-positive cell remained longer with amniotic cell transplantation than direct injection of Ad-lacZ vector into fetal liver at the same stage. Therefore, present In-Utero-Manipulation will be useful strategy for cell mediated gene therapy for congenital liver disease.