大鼠肝卵圆细胞的分离、鉴定及体内移植  

Isolation Identification and Transplantation of Rat Hepatic Oval Cells

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,300170

出　　处：《天津医药》2008年第2期122-125,共4页Tianjin Medical Journal

基　　金：国家863课题(项目编号:2001AA216051)

摘　　要：目的:探讨大鼠肝卵圆细胞分离、纯化方法,并对暴发性肝功能衰竭大鼠模型进行体内移植。方法:以雄性Wistar大鼠肝卵圆细胞增殖模型为供体,Seglen胶原酶原位灌注结合Percoll密度梯度离心分离纯化大鼠卵圆细胞,并进行鉴定。D-氨基半乳糖(1200mg/kg)诱导雌性大鼠暴发性肝功能衰竭模型,肝脏注射移植细胞0.4mL,移植前及移植后取血测定血清谷丙转氨酶(ALT)、总胆红素(TBiL),PCR方法测定受体肝脏组织性别决定因子。结果:平均细胞产量为(5.10±0.40)×106,细胞活性率(94.30±1.89)%。光镜、电镜及免疫组化染色符合卵圆细胞特点,体外培养可形成克隆样结构。移植组暴发性肝功能衰竭大鼠中位生存时间延长(P<0.05),血清ALT、TBiL水平下降,各时间点组间比较均低于对照组(P<0.01)。肝脏病理损伤减轻,受体肝脏组织中性别决定因子呈阳性表达,且随时间延长而表达增强。结论:本方法可成功诱导、分离、纯化大鼠肝卵圆细胞。体内移植延长暴发性肝功能衰竭大鼠中位生存时间,改善肝功能及病理指标。Objective： To study the isolation,identification of hepatic oval cell （HOC）, and its transplantation in treatment of fulminant hepatic failure （FHF） rat. Methods： The HOC proliferation models were induced in male rats with administeration of 2-AAF combined with 2/3 partial hepatectomy,and then HOC were isolated by collagenase perfusion method and purified by Percoll gradient centrifugation. The obtained cells were indentified by microscope,electron microscopic examination and SABC immunohistochemical staining. 0.4 mL cell suspension was injected directly into the liver in female rat with FHF induced by D-galactosamine （ 1 200 mg/kg）. The serum ALT and TBiL levels were measured pre and post transplantation at different time points. Sex determining region of the Y （Sty）was examined by PCR technique after transplantation. Results： The stem cell yield/rat was（5.10 ± 0.40）×10^6,the viability was（94.30 ± 1.89）%. The obtained cells met the characteristic of oval cell under phase-contrast microscope, electron microscopic examination expressed both phenotypic makers of hepatocytes and that of cholangiocytes. The isolated cell could clonally proliferate and form colonies in vitro culture. The survival time of HSC transplantation group was longer than that of control group （P 〈 0.05 ）.Biochemical parameters were declined after HSC transplantation as the time goes by, and lower than that of the control group at each time points （P 〈 0.01 ）.The hepatocytes necrosis in rat liver sections were improved after HOC transplantion. Sry was detected in female recipient rat liver after transplantation. Conclusion： HOC could be isolated and purified from this preliferation model successfully by introduced technique. Stem cell transplantation could prolong the suvival time of recipients, correct their biochemical disorders and liver pathological injury of rats with FHF.

关 键 词：干细胞 大鼠 WISTAR 卵母细胞 细胞分离 细胞移植 肝功能衰竭 急性 

分 类 号：R575[医药卫生—消化系统] R285.5[医药卫生—内科学]