人胎儿肝干细胞分离鉴定及体内移植  

Isolation and identification of human fetal hepatic stem cell and its transplantation in treatment of acute liver injury SCID mice

作　　者：张金卷[1] 杜智[1] 李涛[1] 朱争艳[1] 高英堂[1] 王毅军[1] 聂福华[1] 宋继昌[1] 

机构地区：[1]天津市第三中心医院肝胆外科,卫生部人工细胞工程技术研究中心,天津300170

出　　处：《生物医学工程与临床》2006年第4期214-217,共4页Biomedical Engineering and Clinical Medicine

基　　金：国家863课题(编号:2001AA216051)

摘　　要：目的分离鉴定人胎儿肝干细胞,并观察其对急性肝损伤严重联合免疫缺陷(SCID)小鼠进行异种移植的治疗效果。方法以改进Seglen胶原酶(Ⅳ型)原位灌注结合Percoll密度梯度离心法分离纯化要求终止中期妊娠而流产的男性胎儿肝干细胞,经光镜、电镜下观察细胞特点,进行肝干细胞细胞标志SABC免疫组化染色。经肝脏直接注射移植人胎儿肝干细胞(106细胞)以治疗D-氨基半乳糖(800mg/kg)诱发的急性肝损伤雌性SCID小鼠,于移植前及移植后24、48、72h、1周取血测定ALT、TBiL,于移植后1、2、4周取受体肝脏组织,PCR方法检测受体肝组织性别决定因子。结果人胎儿肝干细胞平均细胞产量(2.10±0.50)×107,细胞活性率为(92.30±1.23)%;光镜下呈游离状态、大小不等的单个细胞,约为正常肝细胞1/5～1/3大小;电镜下细胞表面可见少量短而小的微绒毛状突起,卵圆形细胞核,核/浆比例较大等特点;甲胎蛋白、细胞角蛋白8、19及α-1-抗胰蛋白酶免疫组化染色呈阳性,白蛋白及白细胞共同抗原染色呈阴性表现,培养3周可形成克隆样结构。人胎儿肝干细胞移植组急性肝损伤SCID小鼠移植24h后血清ALT、TBiL水平降低,各时间点均低于对照组(P<0.05),且肝脏病理损伤逐渐减轻;存活2周小鼠肝脏组织中PCR测定性别决定因子呈阳性表达,随时间延长表达增强。结论该方法能成功分离高产量、高活性人胎儿肝干细胞,在急性肝损伤SCID小鼠体内移植存活,并改善其肝功能、肝脏病理指标。Objective To isolate and identify human fetal hepatic stem cell （HSC）, and to study the feasibility and effectiveness of its heterotransplantation to acute liver injury （ALI） in mice with severe combined immunodeficiency disease （SCID）. Methods Human fetal HSC were obtained from liver in abortion fetus in the second trimester of pregnancy. Liver cells were isolated by using collagenase （type Ⅳ） digestion method, and purified by Pereoll gradient centrifugation. The Albumin, cytokeratin（CK）18,CK19,α-feto protein （AFP） and leucocyte common antigen （LCA） were identified by immunohistochemical staining. The purified cells （10^6） were transplanted into the hver of female SCID mice with ALl induced by D-galactosamine （800 mg/kg）;The serum ALT and TBil levels were measured pre and post transplantation at 24,48,72 hours and 1 week after transplantation. Sex determining region of the Y（SRY） of hepatic cells in female mice adjacent to the hetero graft were identified by PCR technique. Results The purified human fetal HSC yield： （2.10 ± 0.50） ×10^7, viability rate： （92.30 ± 1.23） %.The diameter of cells was less than 1/3 of the normal hepatoeytes under phase contrast microscopic. The electron microscopic examination showed there were protruding microvilli on surface, oval shaped nuclei and high nuclear/cytoplasm ratio; there were CKS, CK19, AFP and AAT positive in stem cell, their Albumin and LCA were not stained. The isolated cell could clonally proliferate and form colonies in primary culture within 3 weeks. The biochemical parameters including serum ALT and TBiL levels were decreasing after hepatic stem cell transplantation （P 〈 0.05） in FHF model, and lower than that in control group at each time points （P 〈 0.05）. The pathological injury of liver was improved; SRY was detected in the livers of female recipient mice at 2 weeks after transplantation. Conclusion Liver stem cells could be easily isolated from human fetus by collagenase perfusion technique

关 键 词：人胎儿 肝干细胞 细胞分离 细胞鉴定 细胞移植 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]