人羊膜上皮细胞移植治疗肝纤维化免疫大鼠

TRANSPLANTATION OF HUMAN AMNIOTIC EPITHELIAL CELLS IN TREATMENT OF HEPATIC FIBROSIS IN IMMUNE RATS

目的 观察人羊膜上皮细胞（human amniotic epithelial cells，hAECs）在正常免疫大鼠体内存活、迁徙及对大鼠肝纤维化程度的影响，为 hAECs 应用于临床提供实验依据。方法 10 周龄雄性 SD 大鼠 64 只，体重 220 ～ 280 g，随机分为 4 组，每组 16 只。A 组为肝纤维化 ＋ 细胞移植（移植细胞量 4×10^6个）组，B 组为肝纤维化 ＋ 生理盐水组，C 组为肝纤维化组，D 组为空白对照组。移植后 2 周，取 A 组大鼠心、肝、脾、肾、肺、脑组织采用 DNA-PCR 法检测人 Alu 基因重复序列，免疫组织化学染色观察人白细胞抗原 G（human leucocyte antigen G，HLA-G）表达，并比较各脏器阳性表达百分比（脾脏为细胞注入器官不纳入统计）；取各组肝组织，行 HE 染色观察并采用肝纤维化组织学半定量计分系统（SSS）行肝纤维化半定量分析，行 TGF-β1免疫组织化学染色观察并行免疫组织化学评分（immunohistochemical score，ISH）；取各组血清行谷草转氨酶（aspartate transaminase，AST）、谷丙转氨酶（alanine aminotransferase，ALT）、白蛋白（albumin，ALB）浓度检测，定量分析大鼠肝纤维化程度。 结果 A 组各脏器中人 Alu 基因重复序列及 HLA-G 均表达阳性，肝组织中阳性表达百分比显著高于其余组织，比较差异有统计学意义（P〈0.05）。A、B、C组肝纤维化组织学半定量计分分别为（10.47±3.20）（、13.84±3.46）（、13.85±3.16）分，A 组显著低于 B、C 组（P〈0.05）；B、C 组间差异无统计学意义（P〉0.05）。A、B、C、D 组 TGF-β1的 ISH 评分分别为（3.60±1.50）、（5.38±2.60）、（5.50±2.40）、（1.87±1.36）分，A、B、C 组显著高于 D 组，A 组显著低于 B、C 组（P〈0.05）；B、C 组间差异无统计学意义（P〉0.05）。A、B、C 组血清 AST、ALT 浓度显著高于 D 组，A 组显著低于 B、C 组（P〈0.05），B、C 组间差异无统计学意义（P〉0.05）；ALB 浓度显著低于 D 组，A 组显著高于 B、C 组（P〈0.05）；B、C 组间差异无统计学意义（P〉0.05）。结论 hAECs 可在正常免疫大鼠体内存活，可缓解肝纤维化程度并改善血清肝功能指标。

Objective To observe the survival, migration, and effect of human amniotic epithelial cells （hAECs） on hepatic fibrosis in immune rats so as to provide the experimental theory for the clinical treatment with hAECs. Methods Sixty-four 10-week-old male Sprague Dawley rats （weighing, 220-280 g） were randomly divided into 4 groups, sixteen rats in each group. Rat hepatic fibrosis model was induced in groups A, B, and C; hepatic fibrosis rats were injected with 4×10^6 hAECs in group A, and with normal saline in group B, and no treatment was given in group C; group D served as control group. After 2 weeks of transplantation, the expression of human Alu gene repeat sequence was detected by DNA-PCR method and human leucocyte antigen G （HLA-G） by immunohistochemical staining in heart, liver, spleen, kidney, lung, and brain in group A, and then the percentage of positive expression was compared between organs except spleen. Semi-quantitative analysis was done for liver fibrosis with HE staining according to Chevallier semi-quantitative histological liver fibrosis scoring system, and immunohistochemical staining for TGF-β, was used to record immunohistochemical score （ISH）, the concentrations of aspartate transaminase （AST）, alanine aminotransferase （ALT）, and albumin （ALB） were determined to analyze hepatic fibrosis. Results Alu gene repeat sequence and HLA-G could be detected in liver, heart, brain, lung, and kidney in group A, the percentage of positive expression in the liver was significantly higher than that in the other organs （P〈0.05）. The histological semi-quantitative score of group A （10.47±3.20） was significantly lower than that of groups B and C [（13.84±3.46） and （13.85±3.16）]（P〈0.05）, but no significant differencewas found between groups B and C （P〉0.05）. The ISH scores in groups A, B, C, and D were 3.60±1.50, 5.38±2.60, 5.50±2.40, and 1.87±1.36, respectively; groups A, B, and C were significantly higher than group D, and group A was significantly lower than groups B and C （P〈0.05）, but there was no significant difference between groups B and C （P〉0.05）. The concentrations of ALT and AST in groups A, B, and C were significantly higher than those in group D, and group A was significantly lower than groups B and C （P〈0.05）, but there was no significant difference between groups B and C （P〉0.05）. The concentration of ALB in groups A, B, and C was significantly lower than that in group D, and group A was significantly higher than groups B and C （P〈0.05）, but there was no significant difference between groups B and C （P〉0.05）. Conclusion hAECs can survive in immune rats by intrasplenic transplantation and migrate to liver, heart, brain, lung, and kidney, and the liver shows the largest migration. The transplantation of hAECs in immune rat with cirrhosis can alleviate hepatic fibrosis and improve the serum indexes of liver function.