脂肪间充质干细胞与胰岛联合移植对术后免疫状态及移植效果的影响

目的研究同系脂肪间充质干细胞(AMSC)与同种异体胰岛联合移植对胰岛移植术后免疫状态及移植效果的影响。 方法选择Lewis大鼠建立链脲佐菌素诱导的糖尿病大鼠模型作为胰岛移植受体，AMSC＋胰岛移植组经左肾包膜下联合移植Lewis大鼠AMSC及Wistar大鼠胰岛，单纯胰岛移植组经左肾包膜下单独移植Wistar大鼠胰岛，单纯AMSC移植组经左肾包膜下单独移植Lewis大鼠AMSC，阳性对照组为未进行移植的Lewis糖尿病大鼠，阴性对照组为正常Lewis大鼠。ELISA法检测血清细胞因子IFN-γ、IL-2、IL-4和IL-10浓度，流式细胞技术检测外周血CD4^＋、CD8^＋ T细胞比例，HE染色观察移植胰岛淋巴细胞浸润情况，观察血糖、胰岛素水平及胰岛移植物存活时间。采用单因素方差分析比较5组大鼠外周血淋巴细胞亚群比例和血清细胞因子水平，采用LSD法进行组间两两比较；血糖及血清胰岛素变化采用重复测量资料方差分析；采用独立样本t检验比较AMSC＋胰岛移植组与单纯胰岛移植组胰岛移植物淋巴细胞计数。采用Kaplan-Meier法绘制AMSC＋胰岛移植组与单纯胰岛移植组胰岛移植物生存曲线，并采用log-rank检验进行比较。P<0.05为差异有统计学意义。 结果AMSC＋胰岛移植组胰岛移植物平均生存时间为(26.8±3.0) d，长于单纯胰岛移植组的(19.0±1.3) d，两组大鼠胰岛移植物生存曲线差异有统计学意义(χ²＝4.494，P<0.05)。胰岛移植后各时间点，AMSC＋胰岛移植组大鼠移植后血糖和胰岛素水平均优于单纯胰岛移植组(P均<0.05)。胰岛移植前，5组大鼠外周血CD4^＋、CD8^＋ T细胞比例以及细胞因子IFN-γ、IL-2、IL-4和IL-10差异均无统计学意义(F＝0.425、0.476、0.256、0.418、0.281和0.313，P均>0.05)。胰岛移植后第7、14、28天，AMSC＋胰岛移植组大鼠外周血CD4^＋、CD8^＋T细胞比例均低于单纯胰岛移植组(P均<0.05)，血清IFN-γ和IL-2浓度均低于单纯胰岛移植组(P均<0.05)，IL-4和IL-10浓度均高于单纯胰岛移植组(P均<0.05)。胰岛移植第7天，AMSC＋胰岛移植组胰岛移植物平均淋巴细胞计数为(142±21)个/mm²，低于单纯胰岛移植组的(311±36)个/mm²(t＝8.245，P<0.05)。 结论与胰岛联合移植的AMSC能够提高胰岛移植的效果，可调节糖尿病大鼠体内IFN-γ、IL-2、IL-4和IL-10表达及抑制同种异体胰岛刺激的T细胞增殖，减轻胰岛移植物的免疫损伤。

The influence of adipose mesenchymal stem cell on immune status and transplantation effect when co-transplanted with allogenic islet

ObjectiveTo investigate the influence of adipose mesenchymal stem cell (AMSC) on immune status and transplantation effect when co-transplanted with allogenic islets. MethodsThe streptozotocin induced diabetic Lewis rats were selected as islet transplantation recipients. The diabetic rats which were co-transplanted with islets from Wistar rats and AMSC from Lewis rats were set as the AMSC+ islet transplantation group. The rats in islet transplantation group were transplanted with islets from Wistar rats. The rats in AMSC transplantation group were transplanted with AMSC from Lewis rats. All the islets and AMSC were transplanted through subrenal capsular of left kidney. The rats in positive control group were diabetic Lewis rats. The rats in negtive control group were normal Lewis rats. The concentrations of IFN-γ, IL-2, IL-4 and IL-10 in peripheraI blood serum was detected with ELISA, the average percentage of CD4⁺ T and CD8⁺ T cells in peripheral blood was tested with flow cytometry, the infiltration of lymphocytes in the islets grafts was observed by HE staining. Blood glucose and serum insulin levels of the rats and survival time of the islet grafts were observed. ResultsThe mean survival time of the islet grafts was longer in AMSC+ islet transplantation group (26.8±3.0) days] than in islet transplantation group (19.0±1.3) days], and statistical differences was also found for survival curve of islets grafts between the two groups (χ²=4.494, P<0.05). The blood glucose and serum insulin levels of the diabetic rats in the AMSC+ islet transplantation group were better than those in the islet transplantation group at different time points after transplantation (P all <0.05). Before islets transplantation, there were no statistical differences for the indexes including the average percentage of peripheral blood CD4⁺ T, CD8⁺ T cells and serum concentrations of IFN-γ, IL-2, IL-4 and IL-10 among the 5 groups (F=0.425, 0.476, 0.256, 0.418, 0.281, 0.313, P all >0.05). On the 7th, 14th and 28th days after transplantation, the serum concentrations of IFN-γ and IL-2 in the AMSC+ islet transplantation group were significantly lower than those in the islet transplantation group (P all <0.05), and the serum concentrations of IL-4 and IL-10 significantly higher than those in the islet transplantation group (P all <0.05), the average percentage of peripheral blood CD4⁺ T and CD8⁺ T cells less than those in the islet transplantation group (P all <0.05). On the 7th day after transplantation, the infiltrating lymphocyte counts of islet grafts in the AMSC+ islet transplantation group were significantly lower than those in the islet transplantation group (t=8.245, P<0.05). ConclusionsIn diabetic rats, AMSC could improve the effects of islets transplantation, regulate the expression of IFN-γ, IL-2, IL-4 and IL-10, suppress the T lymphocyte proliferation, and reduce islet grafts immune injury.