重组人粒细胞集落刺激因子动员后自体外周血干细胞在大鼠纤维化肝脏内的发育

Development of autologous peripheral blood stem cells in the fibrous liver of rats after recombinant human granulocyte colony stimulating factor mobilization

目的:观察重组人粒细胞集落刺激因子(rhG-CSF)动员后的自体外周血干细胞在大鼠纤维化肝脏内的发育情况及自体外周血干细胞移植的治疗作用。方法:实验于2005-04/2006-04在大连医科大学附属第一医院中心实验室、大连医科大学中日临床病理中心和大连医科大学中心实验室完成。①实验动物:采用四氯化碳皮下注射建立大鼠肝纤维化模型,将肝纤维化造模成功的大鼠32只随机分为4组,每组8只(雌雄各半):rhG-CSF单纯动员组、rhG-CSF动员后自体移植组、自然恢复组以及对照组。②实验方法:rhG-CSF单纯动员组仅皮下注射rhG-CSF。rhG-CSF动员后自体移植组皮下注射rhG-CSF,通过密度梯度离心分离外周血单个核细胞(内含外周血干细胞),对所分离的外周血单个核细胞进行PKH26-GL标记后自体移植入肝内。自然恢复组不经任何处理。于6周后采集rhG-CSF动员组、rhG-CSF动员后自体移植组、自然恢复组血清和肝组织。对照组为肝纤维化模型成功后即时处死组,留取血清和肝组织以备进行检测。③实验评估:观察各组血生化指标(丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、白蛋白和总胆红素)和肝脏病理变化情况。通过免疫荧光检测自体移植的外周血干细胞在肝脏内的发育情况。结果:32只大鼠均进入结果分析。①rhG-CSF单纯动员组、rhG-CSF动员后自体移植组总胆红素水平低于自然恢复组(P<0.05),白蛋白水平高于自然恢复组(P<0.05)。rhG-CSF单纯动员组与rhG-CSF动员后自体移植组的丙氨酸氨基转移酶、天冬氨酸氨基转移酶、白蛋白和总胆红素水平差异无显著性(P>0.05)。②rhG-CSF单纯动员组、rhG-CSF动员后自体移植组肝纤维化程度较自然恢复组有明显改善(P<0.05)。rhG-CSF单纯动员组与rhG-CSF动员后自体移植组之间肝纤维化程度无明显差异。③PKH26-GL标记的外周血干细胞在纤维化肝脏内少量表达白蛋白。结论:经rhG-CSF动员后的大鼠自体外周血干细胞可以在肝纤维化环境中定植并可以转化为有白蛋白表达的细胞。rhG-CSF动员可以减轻肝纤维化程度。

AIM： To study the development of autologous peripheral blood stem cells in the fibrous liver after mobilizing recombinant human granulocyte colony stimulating factor and the therapeutical effect of autologous peripheral blood stem cell transplantation. METHODS：Experiments were performed at the Central Laboratory of First Affiliated Hospital, China-Japan Clinical Pathological Center and Central Laboratory of Dalian Medical University from April 2005 to April 2006. （1）Rat models of hepatic fibrosis were made by injecting carbon tetrachloride subcutaneously. Thirty-two successfully established rat models of hepatic fibrosis were divided into group A, group B, group C and group D with 8 in each group, male and female （half and half）. （2）Rat models of group A were only administered with recombinant human granulocyte colony stimulating factor subcutaneously. Rat models of group B were administered with recombinant human granulocyte colony stimulating factor subcutaneously. Peripheral blood mononuclear cells containing peripheral blood stem cells were collected by density gradient centrifugation. These peripheral blood mononuclear cells were grafted in liver after labeling with PKH26-GL. Rat models of group C did not undergo any management. Serum and hepatic tissues were collected from rat models of group A, group B and group C six weeks later. Successful rat models of liver fibrosis of group D were sacrificed instantly after the last time of carbon tetrachloride administration. Serum and hepatic tissues were collected for measurement. （3）The alanine aminotransferase, aspartate aminotransferase, albumin and total bilirubin levels and hepatic pathological changes of rat models of all the four groups were measured. Immunofluorescent method was employed to observe the development of autologous peripheral blood stem cells in the liver. RESULTS： A total of 32 rats were involved in the result analysis. （1）Total bilirubin levels were lower in group A and group B than that of group C （P 〈 0.05）, while albumin level was higher （P 〈 0.05）. No significant difference was found in alanine aminotransferase, aspartate aminotransferase, albumin and total bilirubin levels of rat models of group A and group B （P 〉 0.05）. （2）Hepatic fibrosis was significantly improved in the group A and group B as compared with the group C （P 〈 0.05）. No significant difference was found in hepatic fibrosis between the group A and group B. （3）Autologous peripheral blood stem cells labeled with PKH26-GL expressed albumin in fibrous liver of rats. CONCLUSION： In the environment of liver fibrosis, mobilized autologous peripheral blood stem cells can differentiate into cells that can express albumin. Recombinant human granulocyte colony stimulating factor can accelerate the recovery of the rat liver with hepatic fibrosis.