Effects of bone marrow mesenchymal stem cell transplantation on colon anastomotic healing

doi:10.3969/j.issn.2095-4344.2014.50.014

Abstract

Abstract:

BACKGROUND: With the advantage of strong proliferation ability, multi-directional differentiation potential, fully repair of damaged tissue, low immunogenicity and immune regulation, bone marrow mesenchymal stem cells can be used to treat refractory diseases which are difficult to cure by the traditional methods. Its immeasurable clinical application value makes it the new hot spots of cell transplantation therapy.
OBJECTIVE: To observe the colonization of bone marrow mesenchymal stem cells in intestinal submucosa and healing effects after local injecting bone marrow mesenchymal stem cells into rat models of ischemic colon anastomosis.
METHODS: Forty female Sprague-Dawley rats were randomly divided into four groups: group A (postoperative 4 days, control group), group B (postoperative 7 days, control group), group C (postoperative 4 days, bone marrow mesenchymal stem cells group), group C (postoperative 7 days, bone marrow mesenchymal stem cells group). In group A and group B, 0.5 mL fetal bovine serum was injected into the line of the left colon ischemic bowel mucosa after anastomosis. Similarly, about 1×107 cells suspended in 0.5 mL sterile physical serum were submucosally injected into the left colon ischemic bowel mucosa in group C and group D. Rat anastomotic bursting pressure and hydroxyproline content were detected respectively. The migration and engraftment of donor derived bone marrow mesenchymal stem cells was detected by Y chromosome in situ hybridization in colon tissue. Histopathological observation was scored though hematoxylin-eosin and Masson staining.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells transplantation augmented bursting pressure and hydroxyproline content at the site of ischemic colonic anastomosis. The histopathological features, except for the epithelization and inflammation, were significantly more favorable in the bone marrow mesenchymal stem cell groups, and the microvascular density was significantly higher than that in the control groups. These findings indicate that allogeneic bone marrow mesenchymal stem cells can be colonized in the receptor of ischemic colon anastomosis model, and significantly accelerate the recovery of ischemic colonic anastomosis. It contributes to the healing of ischemic colonic anastomosis through the paracrine activities of bone marrow mesenchymal stem cells, rather than direct differentiation.

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

全文链接：

Key words: bone marrow, mesenchymal stem cell transplantation, colon, anastomosis, surgical

CLC Number:

R394.2

Feng Tao, Zhao Xin, Xu Xing-wei, Zheng Peng, Kao Xiao-ming, Ji Wu. Effects of bone marrow mesenchymal stem cell transplantation on colon anastomotic healing[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(50): 8113-8121.

Figures/Tables 2

2.1 大鼠骨髓间充质干细胞的形态学特征刚接种的细胞呈圆形，大小不一，细胞膜清晰，细胞体透明，悬浮于培养液中，见图1A。培养6-8 h后部分细胞贴附在塑料培养皿底，开始亦呈圆形，部分细胞有伪足伸出，呈椭圆形、多角形、纺锤形或梭形，见图1B。24 h后大部分细胞贴壁，48 h后细胞基本完成贴壁，进行首次半量换液，后每3 d全量换液，见图1C。红细胞等杂细胞随换液逐渐被清除，随后贴壁细胞开始分裂增殖，四五天后部分区域开始形成集落，细胞呈成纤维细胞样外观，其后集落逐渐增大铺展，10 d左右细胞基本汇合长满瓶底，即为第1代，见图1D。传代后，细胞贴壁迅速，形态以梭形为主，不再以集落的方式生长，细胞呈放射状或平行排列，形态趋于一致，见图1E，7 d即可传代1次。 2.2 骨髓间充质干细胞的活力以及增殖能力评估锥虫蓝拒染实验显示骨髓间充质干细胞活力良好，细胞存活率为(95.69±2.17)%。CCK-8 法检测细胞增殖能力示显示骨髓间充质干细胞生长状态良好，增殖能力较强，培养第1，2天为相对潜伏期，接种第3天始细胞增殖活跃，步入对数增长期，第5-7天达到高峰，逐渐进入平台期，见图2。 2.3 骨髓间充质干细胞细胞表型鉴定体外培养骨髓间充质干细胞常用的标志物有CD29、CD44、CD90、CD106等，本实验选取CD29-PE，CD90-PE作为骨髓间充质干细胞阳性对照。CD45在淋巴造血干细胞表面高度表达，选取CD45-PE作为骨髓间充质干细胞的阴性对照，同型对照分别为 IgG1, κ Isotype Control-PE，IgG2，λ1 Isotype Control-PE，结果显示骨髓间充质干细胞CD29、CD90阳性表达率为97.58%，96.85%，而CD45阳性表达率仅为6.11%，见图3。 2.4 细胞培养上清中肝细胞生长因子、血管内皮细胞生长因子、转化生长因子β水平第3代骨髓间充质干细胞培养上清液肝细胞生长因子、血管内皮细胞生长因子、转化生长因子β水平分别为(498.42±39.78)，(249.90±34.57)，(172.28±39.57) ng/L，见图4。 2.5 骨髓间充质干细胞在肠黏膜归巢的检测 Y染色体示踪实验显示，在肠道黏膜固有层、肠黏膜上皮细胞层和肠黏膜下层均可见归巢的骨髓间充质干细胞，其中肠黏膜固有层居多。骨髓间充质干细胞移植4 d组和骨髓间充质干细胞移植7 d组细胞归巢率分别为(9.62±2.57)%、(11.69± 3.09)%，见图5。 2.6 吻合口爆破压的测定术后第4天，骨髓间充质干细胞移植组吻合口压力较对照组明显增高[(15.4±2.77) kPa，(8.25±2.54) kPa，P < 0.01]，同样，术后第7天，骨髓间充质干细胞移植组吻合口压力较对照组明显增高[(26.6± 2.94) kPa，(16.3±4.34) kPa，P < 0.01]，见图6。 2.7 羟脯氨酸含量的测定术后第4天，骨髓间充质干细胞移植组羟脯氨酸含量较对照组明显增高[(0.66±0.11) mg/g，(0.36±0.12) mg/g，P < 0.05]，同样，术后第7天，骨髓间充质干细胞移植组羟脯氨酸含量较对照组明显增高[(0.71±0.13) mg/g，(0.47±0. 09) mg/g，P < 0.05]，见图7。 2.8 组织病理学特征骨髓间充质干细胞移植组黏膜坏死、新生血管形成、胶原蛋白沉积量及成纤维细胞活性评分与对照组有明显区别，差异有显著性意义(P < 0.05)，骨髓间充质干细胞移植组上皮形成、炎症反应评分和对照组比较差异无显著性意义(P > 0.05)，见图8，表1。"

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