Umbilical cord blood mesenchymal stem cell transplantation for acute intestinal ischemia-reperfusion injury

doi:10.3969/j.issn.2095-4344.2016.19.012

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells can repair intestinal ischemia-reperfusion injuny by interfering inflammatory reactions after intestinal ischemia-reperfusion to protect intestinal barrier functions. In recent years, umbilical cord blood mesenchymal stem cells are gradually used as a substitute source of bone marrow mesenchymal stem cells.
OBJECTIVE: To investigate the effects of umbilical cord blood mesenchymal stem cells on acute intestinal ischemia-reperfusion injury.
METHODS: Umbilical cord blood mesenchymal stem cells were induced, isolated in vitro and tracked by CM-DiI fluorescent labeling. Sixty-three Sprague-Dawley rats were equivalently randomized into three groups: control group received normal saline enema, intestinal ischemia-reperfusion injury group with ethanol diluted trinitro-benzene-sulfonic acid and transplantation group administrated with 1×10¹⁰/L umbilical cord blood mesenchymal stem cell suspension via the tail vein at 1 hour after trinitro-benzene-sulfonic acid modeling. At 3 days after transplantation, colon tissues were removed in each group to observe pathological changes of the intestinal tract by hematoxylin-eosin staining. Besides, expression of leptin mRNA in the colon tissues and cyclooxygenase-2 in the mucosa were detected by RT-PCR and immunohistochemistry method, respectively.
RESULTS AND CONCLUSION: Transplanted umbilical cord blood mesenchymal stem cells distributed in the intestinal lymphoid tissue and among glandular epithelial cells, suggesting that these stem cells might be involved in the process of intestinal ischemia-reperfusion injury repair. Compared with the control group, intestinal injury in the injury group was significantly aggravated, and most intestinal epithelial cells shed; and the transplantation group appeared to have significantly reduced intestinal damage and significantly less cell shedding. Expression of leptin mRNA was significantly higher in the injury group than the transplantation group followed by the control group, and there were significant differences among the three groups (P < 0.05). Additionally, expression of cyclooxygenase-2 in the injury group was significantly higher than that in the control group (P < 0.05); compared with the injury group, expression of cyclooxygenase-2 was significantly lower in the transplantation group (P < 0.05). To conclude, leptin and cyclooxygenase-2 may be involved in acute intestinal ischemia-reperfusion injury, and umbilical cord blood mesenchymal stem cell transplantation significantly lessens intestinal ischemia-reperfusion injury, which provides an experimental basis for human treating acute intestinal ischemic injury.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Cord blood Cell Transplantation, Mesenchymal Stem Cells, Reperfusion Injury, Tissue Engineering

CLC Number:

R394.2

Wang Bing-jie, Hu Yan-wei, Zhao Ye-fang, Zhang Shi-dong. Umbilical cord blood mesenchymal stem cell transplantation for acute intestinal ischemia-reperfusion injury[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2817-2823.

Figures/Tables 1

2.1 实验动物数量分析选择63只SD大鼠，按随机数字表法分为3组，单独放于3个笼子里。其中造模过程中死亡1只，做脐血间充质干细胞培养时处死2只，将剩余的动物分为3组，每组20只。 2.2 脐血间充质干细胞的形态观察脐血间充质干细胞在培养5 d左右时，贴壁细胞明显增多，形态以长梭形或多角形为主，部分细胞呈集落状生长，可见有丝分裂。培养八至九天时，细胞铺满培养瓶底面，成漩涡状生长，细胞集落不断增多。10-14 d细胞集落进一步扩大，互相融合，可达80%融合率，传代脐血间充质干细胞很快贴壁，呈梭形均匀分布，且增殖迅速。经多次传代后，脐血间充质干细胞呈漩涡状排列，见图1。流式细胞仪检测结果显示CD29、CD34、CD45、CD49、CD90及CD105阳性，脐血间充质干细胞均一性好，纯度达95%以上。 2.3 CM-Dil标记的脐血间充质干细胞形态观察通过荧光示踪法观察到CM-Dil标记的脐血间充质干细胞在荧光显微镜下呈红色荧光，FCM检测细胞标记率达100%，见图2A。进一步观察可见CM-Dil标记的红色脐血间充质干细胞分布于肠黏膜淋巴组织内和腺上皮细胞间，表明脐血间充质干细胞可能参与了肠缺血再灌注损伤的修复过程，见图2B。 2.4 苏木精-伊红染色观察肠道病理变化苏木精-伊红染色观察到与对照组相比，缺血/再灌注损伤组肠道损伤明显加重，肠黏膜可见较大面积的溃疡、糜烂、充血及水肿，肠黏膜上皮细胞脱落较多。脐血间充质干细胞移植组肠道损伤明显减轻，溃疡面积显著减轻，细胞充血、脱落现象明显好转。各组大鼠于移植后3 d肠道病理变化比较见图3。 2.5 RT-PCR法检测结肠组织中leptin mRNA的表达 RT-PCR法检测结果显示，leptin mRNA在缺血/再灌注损伤组呈现高表达，其表达量明显大于对照组及脐血间充质干细胞移植组，脐血间充质干细胞移植组又明显大于对照组，对照组的leptin mRNA呈弱表达，各组间比较差异有显著性意义(P < 0.05)，见表1及图4。 2.6 免疫组化法检测肠黏膜中环氧化酶2表达免疫组化法检测到，对照组肠黏膜环氧化酶2呈阴性表达，损伤组肠黏膜环氧化酶2的阳性表达率为100%，明显高于对照组(P < 0.05)，脐血间充质干细胞移植组环氧化酶2表达明显低于损伤组，但较对照组高(P < 0.05)。各组大鼠肠黏膜环氧化酶2表达变化见表2。"

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