人脐带间充质干细胞向胰岛样分泌细胞的诱导分化及胰岛样分泌细胞治疗效果评价

Differentiation of Islet Like Cells from the Umbilical Cord Mesenchymal Stem Cells in vitro and Its Function Evaluation

目的对人脐带间充质干细胞(mesenchymal stem cells,MSCs)在体外诱导分化为胰岛样分泌细胞进行观察,并对获得的胰岛样分泌细胞治疗效果进行评价。方法选择雄性SD大鼠32只,随机分为正常对照组(8只)、糖尿病对照组(12只)和糖尿病实验组(12只)。糖尿病对照组和糖尿病实验组给予链脲佐菌素腹腔注射进行糖尿病大鼠造模,正常对照组予0.9%氯化钠注射液腹腔注射。分离、培养脐带Wharton's Jelly(WJ)中的MSCs,将培养至第3代MSCs与雄性SD大鼠胰岛细胞共同培养14 d,培养出的干细胞移植至糖尿病实验组肾包膜下,观察56 d结束实验。通过HE染色和免疫组织化学染色观察实验结束后3组肾脏形态和细胞变化,并比较基线时与实验结束后3组血糖和体重变化。结果实验结束后正常对照组和糖尿病对照组肾脏HE染色示肾被膜边缘完整,形态清楚;免疫组织化学染色示肾被膜下无胰岛素及外源性抗原溴脱氧尿嘧啶核苷(Brdu)染色阳性的细胞。实验结束后糖尿病实验组肾脏HE染色示移植细胞部位肾被膜和肾皮质之间有大量的细胞,部分肾被膜出现断裂现象;免疫组织化学染色示肾被膜和肾皮质之间有胰岛素和Brdu阳性的细胞。基线时糖尿病实验组和糖尿病对照组血糖明显高于正常对照组,体重低于正常对照组,差异均有统计学意义(P<0.01或P<0.05)。实验结束后糖尿病实验组血糖较基线时下降,体重较基线时有所增加,差异有统计学意义(P<0.05);3组血糖、体重比较差异均有统计学意义(P<0.05);糖尿病实验组血糖明显低于糖尿病对照组,差异有统计学意义(P<0.05);糖尿病实验组、糖尿病对照组体重均低于正常对照组,糖尿病实验组体重高于糖尿病对照组,差异均有统计学意义(P<0.05)。结论脐带MSCs在胰岛细胞微环境下可诱导分化为胰岛样分泌细胞,对糖尿病鼠具有降糖和恢复体重的作用。

Objective To induce and observe the differentiation of islet like cells from human umbilicalcord mesen-chymal stem cell （ hUC-MSCs） in vitro, and to make evaluation of the function and effect of islet like cells diabetes. Methods 32 male SD rats were randomly divided into normal control group （8 rats）, diabetic control group （12 rats） and diabetic group （12 rats）. Rats in the diabetic control group and the diabetic experimental group were intraperitoneally injected with Streptozocin （STZ） injection while rats in normal control group were intraperitoneally injected with 0. 9% sodium chloride in-jection of equal volume. The hUC-MSCs form Wharton＇s Jelly （WJ） was isolated and the 3rd generation of hUC-MSCs was cul-tured with the rat islet cells for 14 days, and the changes of hUC-MSCs was observed under inverted microscopy, and 56 d end text as well. The islet like cells was transplanted under the renal capsule of diabetic rats of the experimental group and the changes of renal morphology and cell were observed through HE staining and immunohistochemical staining as well as the changes of the weight and blood glucose of diabetic rats during the baseline and after the experiment. Results After the ex-periment, the HE dyeing showed that the verge of renal capsule was integrity and clear in normal control group and diabeticnbsp;control group. The immunohistochemistry dyeing showed that there were not positive cells for insulin or Brdu below the renal capsule in normal control group and diabetic control group. There were considerable cells between renal capsule and renal cor-tex in the diabetic experimental group. The renal capsule appeared to be broken. There were some positive cells for insulin whose endochylemas were brown as well as some positive cells for Brdu whose nucelus were brown between renal capsule and renal cortex in the diabetic experimental group. At the baseline, the blood glucose of the diabetic experimental group and the diabetic control group were significantly higher than those of the normal control group while the weight was lower with statisti-cally significant difference （ P 〈0. 01 or P 〈0. 05 ） . After the experiment, the blood glucose of the diabetic experimental group was decreased compared with baseline, but the weight increased, and the difference was statistically significant （ P〈0. 05）, difference of blood glucose and weight between the 3 groups were statistically significant （P〈0. 05）. The blood glu-cose of diabetic experimental group was lower than that of the diabetes control group, and the difference was statistically signif-icant （P〈0. 05）. The weight of diabetic experimental group and diabetic control group was lower than that of the normal con-trol group, while the weight of diabetic experimental group was higher than that of the diabetes control group, and the differ-ence was statistically significant （P〈0. 05）. Conclusion In the islet cell microenvironment, the umbilical cord MSCs can be induced and differentiated into islet like cells, which can reduce blood glucose level and rebalance the weight of diabetic patients.