骨髓间充质干细胞免疫调节作用对急性肝功能衰竭大鼠肝再生的影响

The immunoregulation effect of bone marrow mesenchymal stem cells on liver regeneration of rats with acute liver failure

目的动态分析和研究骨髓间充质干细胞（BMMSC）移植对急性肝功能衰竭（ALF）大鼠的免疫调节作用及其对肝再生的影响。方法D-GalN诱导建立ALF大鼠模型24h后，模型组尾静脉注射0．9％氯化钠溶液1mL，移植组尾静脉注射含1×10^6个BMMSC细胞悬液1mL，分别在1、2、3、7和14d取两组大鼠血标本和肝组织；另取6只健康大鼠作为正常对照组。全自动生化分析仪检测大鼠血清ALT、AST水平，ELISA法检测血清TNF-α、IL-6水平，HE染色观察肝组织病理学变化，免疫组织化学法检测肝组织中增殖细胞核抗原（PCNA）的表达，流式细胞检测PBMC中Treg比例变化。两样本均数比较采用Studentt检验，率的比较采用χ^2检验。结果移植组死亡大鼠的平均存活时间为（38．71±5．73）h，模型组为（29．90±6．17）h，两组差异有统计学意义（t=2．92，P〈0．05）。移植组ALT峰值较模型组显著下降，差异有统计学意义（t=2．76，P〈0．05），而AST水平在移植后1、2、3d均较模型组显著下降，差异均有统计学意义（t值分别为3．22、3．22和3．79，均P〈0．05）。移植组1、2、3、7dTNF-α（t值分别为3．81、6．44、4．13和5．38，均P〈0．05）、IL-6水平（t值分别为8．10、3．80、3．92和4．37，均P〈0．05）较模型组同一时间点显著下降，差异均有统计学意义。HE染色结果提示，移植组大鼠肝脏炎性反应较模型组明显好转。移植组PCNA表达量在移植后1、2、3、7、14d均显著高于模型组，差异均有统计学意义（t值分别为3．13、6．37、12．60、2．31和6．20，均P〈0．05）。移植组大鼠Treg占CD4^＋T淋巴细胞比例在1、2、3、7d均显著高于正常对照组，差异有统计学意义（t值分别为5．91、10．88、9．50和2．99，均P〈0．05），与模型组同一时间点比较，差异亦均有统计学意义（t值分别为3．29、4．13、3．34和3．89，均P〈0．05）。结论BMMSC移植可抑制ALF体内炎性反应，从而有利于改善肝功能，促进肝再生，Treg可能参与介导了BMMSC的负向免疫调节功能。

Objective To study the immunoregulation effect of bone marrow mesenchymal stem cells （BMMSC） transplantation on liver regeneration of rats with acute liver failure （ALF）. Methods The ALF model of rats was induced by D-galactosamine （D-GaIN）. Then the ALF rats were randomly divided into ALF model group and BMMSC transplantation group at 24 h after the injection of D-GaIN. Rats in ALF model group were injected with 0. 9% saline via tail vein, while those in BMMSC transplantation group were injected with 1 mL BMMSC cell suspension containing 1 × 10^6 cells. Blood and liver tissue specimens of ALF rats were collected at day 1, 2, 3, 7 and 14, respectively after transplantation. Another six healthy rats were enrolled as control group. The levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected by automatic biochemistry analyzer. Tumor necrosis factor （TNF）-α and interleukin （IL）-6 in serum were detected by enzyme-linked immunosorbent assay （ELISA）. The changes of liver pathology were observed with hematoxylin-eosin （HE） staining. The expression of proliferating cell nuclear antigen （PCNA） protein was detected by immunohistochemistry method. The ratio of regulatory T lymphocyte （Treg） cells to CD4^＋ T cells in PBMC was assayed by flow cytometry. Continuous variables were compared using Student t test, while categorical variables were compared using χ^2 test. Results The mean survival time of rats in BMMSC transplantation group was （38. 7 1±5.73） h, which was significantly longer than that in ALF model group （[29. 90±6. 17] h, t= 2. 92, P〈0. 05）. Compared with ALF model group, the peak ALT level in transplantation group decreased significantly （t = 2.76, P〈0.05）, and the levels of AST on 1, 2, 3 d after transplantation in transplantation group were all significantly lower than model group （t = 3. 22, 3.22, and 3. 79; all P〈0. 05）. The levels of TNF-α in transplantation group were decreased more obviously than those in model group （t=3.81, 6.44, 4.13, and 5.38i all P〈0.05）. The changes of IL- 6 were as similar as TNF-α （t=8.10, 3.80, 3.92, and 4.37; all P〈0.05）. Moreover, the improvement of liver pathology was also pronounced than that in model group. At the same time, the expressions of PCNA protein on 1, 2, 3, 7, 14 d after transplantation in transplantation group were all increased significantly compared to those in model group （t=3.13,6.37, 12.60, 2.31 and 6.20; all P〈0.05）. The ratio of Treg cells to CD4＋ T cells increased significantly in ALF model rats. But the increases were more pronounced in transplantation group on 1, 2, 3, 7 d after transplantation （t=3.30, 4.13, 3.34, and 3.89; all P〈0.05）. Conclusions BMMSC transplantation in ALF rats is effective in inhibiting inflammation, which is favorable to improve liver function and regeneration. The negative immunoregulation role of BMMSC may be mediated, at least in part, through the regulation of Treg cells.