粒细胞集落刺激因子动员自体骨髓干细胞促进大鼠部分肝移植物的肝再生

Granulocyte colony-stimulating factor-mobilized autologous bone marrow stem cells promote the liver regeneration of partial liver transplant: an experiment with rats

目的探讨粒细胞集落刺激因子(G-CSF)动员自体骨髓干细胞是否会促进部分肝移植物的再生,减轻肝损伤。方法建立性别交叉的大鼠(雌性→雄性)50%部分肝移植模型(PLTx),移植前动员组:移植前受体皮下注射G-CSF5d;移植后动员组:移植后3h,注射G-CSF5d;未动员组:移植后3h,皮下注射生理盐水5d。分别与1、3、5、7和14d取肝脏。检测移植物内CD34干细胞标志,确定干细胞迁移入肝脏的情况,观察肝脏的有丝分裂相和溴脱氧核苷尿嘧啶(BrdU)掺入检测肝再生情况,并检测sry+细胞确认细胞来源。结果用G-CSF动员后发现,移植后动员组14d生存率(90%)较移植前动员组(60%)和未动员组(50%)高,差异有统计学意义。与未动员组相比,移植后第3天,肝坏死灶少,有丝分裂指数高(30%±5%vs24%±6%,P<0·05),BrdU掺入高(42%±6%vs34%±4%,P<0·05),差异有统计学意义。移植后第3至14天,移植后动员组汇管区可见较多的CD34+的成堆或散在分布的淋巴细胞样细胞;移植后第3天,移植前动员组见汇管区较多的CD34+细胞。移植后第5天各组在肝窦和汇管区偶见sry阳性细胞,第14天,G-CSF动员两组汇管区周围sry阳性细胞增多,未动员组较少。结论移植后用G-CSF动员自体骨髓干细胞,受体生存率高,移植肝再生明显,肝损伤轻。

Objective To investigate the effects of granulocyte colony-stimulating factor （G-CSF）- mobilized autologous bone marrow stem cells on the liver regeneration of partial liver transplant. Methods A 50% partial liver transplantation model as established by transplanting parts of the liver of female rats to the male rats with the equal weights that had had parts of their livers resected. Then the transplanted male rats were randomly divided into 3 equal groups ： G-CSF ＋ PLTx group that was injected hypodermically with recombinant human G-CSF （rhG-CSF） daily for 5 days and then underwent liver resection and 50% partial liver transplantation （PLTx）; PLTx ＋ G-CSF group that underwent PLTx and 3 hours after the transplantation received injection of rhG-CSF daily for 5 days, and PLTx control group that underwent PLTx and 3 hours later received injection of normal saline daily for 5 days. One, 3, 5, 7, and 14 days after the PLTx blood samples and livers were collected from 6 rats from each group, and one hour before the liver was taken bromodeoxyuridine （BrdU） was injected intraperitoneally to be integrated into the synthesis of DNA in the liver cells. Immunohistochemistry was used to detect the CD34 and BrdU-positive cells. In situ hybridization was used to detect the sry （ sex-determining region） gene so as to determine the origin of the proliferating cells in the transplanted liver. Results The 14-day survival rate of the G-CSF ＋ PLTx group was 90%, significantly higher than those of the G-CSF ＋ PLTx （ 60% ） and PLTx group （ 50% ） （ both P 〈 0.05 ） with a significant difference between the latter 2 groups too （ P 〈 0.05 ）. The mitosis index of liver cells 3 days after the transplantation of the PLTx ＋ G-CSF group was 30%±5% , significantly higher than those of the G-CSF ＋ PLTx group （24%± 7% ） and PLTx group （ 24%± 6% ） （ both P 〈 0.05 ） without a significant difference between the latter 2 groups. The rate of BrdU-positive cells of the PLTx ＋ G-CSF group was 42%±6%, significantly higher than those of the G-CSF ＋ PLTx group （38% ±4% ） and PLTx group （34% ± 8% ） （ both P 〈 0. 05 ）. The transplantation the mitosis index and rate of BrdU-positive cells decreased since the 7 th days after transplantation in all 3 groups, however, with the same relationship among them. The numbers of CD34^＋ cell around the portal area of the 2 G-CSF groups increased since the 3 rd day after transplantation in comparison with the PLTx group, was the highest on the 3 rd day for the GCSF ＋ PLTx group, and continued to increase in the PLTx ＋ G-CSF group. Since the 3rd day after transplantation, sry-positive cells were seen in the hepatic sinousoid and portal area in the 2 G-CSF groups, and were rarely seen in the PLTx group. Conclusion G-CSF treatment after 50% PLTx significantly promotes liver regeneration and ameliorates the liver damage, thus raising the survival rate.