课题背景：课题受国家自然科学基金（39870651）及湖南省自然科学基金（04JJ6048）资助，自1993年始采用脐血输注治疗慢性肝炎，1995年始采用脐血输注治疗重型肝炎肝衰竭，共治疗近千例患者，取得了较好的临床效果，并同时进行了相应的基础实验研究，先后发表相关论文十余篇，其中SCI收录2篇，CSCD收录5篇，分别于1997年及2003年获湖南省医药卫生科技成果奖及湖南省科技进步奖。

应用要点：①直接利用人流胚胎作为干细胞来源，而不是利用细胞株，故试验所用细胞再生及分化能力强。②将胚胎原始生殖细胞直接移植，利用肝衰竭的体内环境促使其定向分化为肝细胞，从而治疗肝衰竭。

偏倚或不足：①如能以同位素标记原始生殖细胞后动态观察其在大鼠体内的分布，更具有说服力，可直接说明原始生殖细胞能在大鼠肝脏内定植。②如能在鼠肝中对已分化的人肝细胞特异基因片段进行检测，则能够直接说明原始生殖细胞可在大鼠肝脏内分化为肝细胞。

摘要
目的：胚胎原始生殖细胞能以原始胚胎状态无限增殖，并可通过异体移植或适宜的体外环境自然分化为胚胎生殖层。实验拟进一步观察体外培养的胚胎原始生殖细胞对急性肝损伤大鼠的治疗效果，及其能否在大鼠体内分化为肝细胞。
方法：实验于2003-09/2005-05在中南大学肝病研究所完成。①细胞及动物：经中南大学湘雅二医院医学伦理委员会批准，取孕4~12周流产人胚胎，其双亲3代内无遗传性疾病，孕妇对流产胚胎用于实验均签署知情同意书。清洁级健康成年SD大鼠50只，随机数字表法分为5组：培养基对照组、单纯人胚原始生殖细胞组、人胚原始生殖细胞+环磷酰胺组、单纯鼠胚原始生殖细胞组、鼠胚原始生殖细胞+环磷酰胺组，10只/组。另选取健康成年SD孕鼠4只用于分离鼠胚原始生殖细胞。动物均由中南大学湘雅二医院动物实验中心提供，实验过程中对动物的处置符合动物伦理学标准。②实验方法：从人胚胎中分离出原始生殖嵴组织，悬浮培养获取人胚原始生殖细胞，用含15%胎牛血清的高糖DMEM基础培养基调节细胞浓度至1×108 L-1。取SD孕鼠胚胎分离生殖嵴，消化离心培养鼠胚原始生殖细胞，浓度1×108 L-1。各组大鼠均于腹腔内注射D-氨基半乳糖建立急性肝损伤模型，造模后48 h，培养基对照组自尾静脉输注基础培养基，其余4组给予对应细胞悬液及免疫抑制剂环磷酰胺。③实验评估：从细胞形态、表面标记及体外分化等方面检测人胚原始生殖细胞的生物学特性；比较各组大鼠肝功能、增殖细胞核抗原阳性率、糖原染色阳性率、肝脏病理改变；检测各组大鼠肝脏病理切片中人白蛋白、甲胎蛋白的表达。
结果：①人胚原始生殖细胞的生物学特性：培养1 d后单个原始生殖细胞形成细胞集落，以后集落逐渐增大并隆起，形成鸟巢状，周边界限清晰，内部细胞排列紧密。传代后原始生殖细胞集落呈自发分化趋势，周边出现成纤维样细胞，最后分化为梭形细胞、多边形细胞等。原代至第3代未分化的人胚原始生殖细胞集落碱性磷酸酶染色均呈阳性，分化的细胞及成纤维细胞呈阴性。②肝功能检测：细胞输注前各组丙氨酸转氨酶、天冬氨酸转氨酶、总胆红素水平均基本相似(t=0.361~1.183，P均 > 0.05)。输注后7 d与培养基对照组比较，单纯鼠胚原始生殖细胞组、鼠胚原始生殖细胞+环磷酰胺组上述3项指标水平均明显降低(t=2.532~8.361，P均 < 0.05)；后两组间比较差异无显著性意义(t=0.340~1.712，P均 > 0.05)。③增殖细胞核抗原阳性率：细胞输注后7d与培养基对照组比较，单纯鼠胚原始生殖细胞组、鼠胚原始生殖细胞+环磷酰胺组的增殖细胞核抗原阳性率均明显升高（t=9.020~10.747，P均 < 0.001）；后两组间比较差异无显著性意义(t=0.752，P=0.462)。④糖原染色：细胞输注后7 d，培养基对照组着色相对浅淡，呈细颗粒状，；单纯鼠胚原始生殖细胞组、鼠胚原始生殖细胞+环磷酰胺组着色相对深粗，呈斑块状聚集。⑤病理检测：细胞输注后7 d，培养基对照组肝索、肝窦结构排列欠规则，坏死区可见少量肝细胞再生，汇管区有大量炎症细胞浸润；单纯鼠胚原始生殖细胞组、鼠胚原始生殖细胞+环磷酰胺组各项病理情况均显著改善。⑥大鼠肝组织人白蛋白、甲胎蛋白的表达：细胞输注后14，21 d，单纯人胚原始生殖细胞组、人胚原始生殖细胞+环磷酰胺组表达人白蛋白及甲胎蛋白阳性细胞，培养基对照组未见表达。
结论：①胚胎原始生殖细胞移植有利于激发急性肝损伤大鼠肝细胞的增殖，改善肝功能，加速受损肝细胞的病理修复。②胚胎原始生殖细胞可在受体肝脏组织中生存，并分化为具有合成白蛋白及甲胎蛋白功能的肝细胞样细胞。
关键词：原始生殖细胞；胚胎干细胞；急性肝损伤；细胞移植

唐晓鹏，陈丽敏，唐元媛，陈丁，张旻.人胚胎原始生殖细胞移植治疗大鼠急性肝损伤[J].中国组织工程研究与临床康复，2008，12(12):2285-2290 [www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2285(ps).pdf]

Human embryonic primordial germ cell transplantation for treating rats with acute liver injury

Abstract

AIM： Embryonic primordial germ cells can proliferate infinitely in the form of primary embryo and differentiate into embryonic germ-layer by heterogeneous cell transplantation or suitable extraorgan condition. This study is planed to further observe the therapeutic efficacy of cultured in vitro primordial germ cells (PGCs) on acute hepatic injury rats and whether PGCs can differentiate into hepatocytes in rats.

METHODS: Experiments were performed from September 2003 to May 2005 in the Institute of Hepatology of Central South University. ①After the agreement of Ethics Committee of Xiangya Second Hospital of Central South University and signature of informed consent by the pregnant woman for the use of aborted embryo in the study, the aborted 4-12 week gestated human embryos were collected. No hereditary disease was found in the pregnant women and their 3-generation parents. Fifty clean healthy adult SD rats were divided into 5 groups by means of random digits table and 10 rats in each group, medium control group, human PGCs group, human PGCs and cyclophosphamide group, rat PGCs group, rat PGCs and cyclophosphamide group. Meanwhile, rat PGCs were isolated from 4 healthy pregnant SD rats. All animals were supplied by the Experimental Animal Center of Xiangya Second Hospital of Central South University. The animals were treated in accordance with ethical standard for animals. ②Human primordial germ tissue was isolated from human embryo. Then human PGCs were collected by suspension culture and the concentration of PGCs was adjusted to 1×108 L-1 with high glucose DMEM medium containing 15% fetal bovine serum (FBS). Rat germ ridge was isolated from pregnant SD rats, and then the rat germ ridge was digested and centrifugated. Rat PGCs were cultured and the concentration of cells was adjusted to 1×108 L-1. An acute liver injury model was constructed by intraperitoneally injecting D-galactosamine into SD rat. After model was established 48 hours, the rats in medium control group were treated with DMEM medium by caudal vein injection. Animals in other groups were treated with suspension of corresponding human/rat PGCs and immunosuppressant cyclophosphamide respectively. ③The biological characteristics of human PGCs were detected in aspects of the appearance, surface marks and differentiation in vitro. The liver function, liver pathology, proliferating cell nuclear antigen (PCNA) and glycogen staining positive rate of rats in each group were compared. Meanwhile, the expression of human albumin (ALB) and alpha fetoprotein (AFP) in rat liver tissue were detected.

RESULTS: ①The biological characteristics of human PGCs: PGC colony was formed from single PGC after 1-day culture, and then increased in size and bird nest shape with clear boundary and the cells arrange tightly inside appeared. The PGCs colony had a tendency of spontaneous differentiation after passage and fibroblasts appeared and finally differentiated into fusiform cells and polygon cells. The colony of the first to third generation of undifferentiated human PGCs were alkaline phosphatase staining positive, but the colony of differentiated human PGCs and fibroblasts were alkaline phosphatase staining negative. ②Liver function determination: Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) were similar before PGCs infusion (t=0.361-1.183，P >0.05). The levels of ALT, AST and TBil were obviously decreased in rat PGCs group, rat PGCs and cyclophosphamide group when comparing to the medium control group after 7 days of PGCs infusion (t=2.532-8.361，P < 0.05). The difference between rat PGCs group and rat PGCs plus cyclophosphamide group was not significant (t=0.340-1.712，P > 0.05). ③Proliferating cell nuclear antigen (PCNA) staining positive rate: PCNA positive rate was much higher in the rat PGCs group, rat PGCs and cyclophosphamide group than in the medium control group after 7 days of PGCs infusion （t=9.020-10.747，P < 0.001）. The difference between the rat PGCs group and rat PGCs and cyclophosphamide group was not significant (t=0.752，P=0.462). ④Glycogen staining: The staining was light and fine grain in the medium control group after 7 days of PGCs infusion. However, the staining was dark and en plaque in the rat PGCs group, rat PGCs and cyclophosphamide group after 7 days of PGCs infusion. ⑤Pathology examination: The arrangement of hepatic cord and sinus hepaticus was not regular, and only a few of hepatocyte regeneration presented in zone of necrosis and much inflammatory cell infiltration can be found in the portal zone. The improvement of liver pathology was obvious in the rat PGCs group, rat PGCs and cyclophosphamide group. ⑥The expression of human ALB and human AFP in rat liver tissue: Human ALB and human AFP positive cells were present in the liver tissue of rats in the human PGCs group, human PGCs and cyclophosphamide group after 14, 21 days of human PGCs infusion, but no positive cell was found in the liver tissue of rats in the medium control group.

CONCLUSION: ①Transplantation of PGCs can induce hepatocyte proliferation and improve liver function in acute liver injury rats, and it is beneficial for the recovery of the damaged hepatocyte. ②PGCs can incorporate into the acute injury liver and differentiate into hepatocyte-like cells in the acute liver injury SD rats. These differentiated cells posses the ability to synthesize ALB and AFP.

Tang XP, Chen LM, Tang YY, Chen D, Zhang M.Human embryonic primordial germ cell transplantation for treating rats with acute liver injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2285-2290(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2285(ps).pdf]