Gegen Qin Lian Tang combined with human amniotic mesenchymal stem cell transplantation for early diabetic retinopathy

doi:10.3969/j.issn.2095-4344.2016.19.013

Abstract

Abstract:

BACKGROUND: Human amniotic mesenchymal stem cells have the potential to differentiate into insulin-secreting cells and have immunomodulatory effects. Additionally, it has been clinically proved that Gegen Qin Lian Tang can lower blood sugar.
OBJECTIVE: To investigate the effects of Gegen Qin Lian Tang combined with human amniotic mesenchymal stem cell transplantation on rat early diabetic retinopathy, by detecting pathological changes, levels of blood indicators and expressions of vascular endothelial growth factor in retinal tissues.
METHODS: By intraperitoneal injection of streptozotocin, 42 Wistar rat models with diabetes mellitus were prepared and then randomly divided into model group, human amniotic mesenchymal stem cell transplantation group (cell transplantation group) and combination of human amniotic mesenchymal stem cells and Gegen Qin Lian Tang group (combination group). At 4 weeks after transplantation, levels of blood sugar and serum insulin in diabetic rats were detected; pathological changes of the retina in diabetic rats were observed by hematoxylin-eosin staining. Besides, expressions of CD45 and vascular endothelial growth factor mRNA were detected using immunohistochemistry staining and RT-PCR technology, respectively.
RESULTS AND CONCLUSION: Compared with the model group, the levels of blood glucose and serum insulin in both cell transplantation and combination groups were significantly decreased and increased, respectively, and these changes were even more significant in the combination group (P < 0.05). In the model group, the retinal edema could be found, accompanied by structural disorder and irregular cell arrangement, while these retinal lesions were relatively milder in the cell transplantation group and significantly improved in the combination group. In addition, the CD45 expression and in the retina was highest in the model group subsequently followed by the cell transplantation group (P < 0.05) and lowest in the combination group (P < 0.05). Furthermore, compared with the model group, the expression of vascular endothelial growth factor mRNA was also significantly lower in the other two groups, especially in the combination group (P < 0.05). These data show that Gegen Qin Lian Tang combined with human amniotic mesenchymal stem cell transplantation improves the severity of diabetic retinopathy by inhibiting expressions of CD45 and vascular endothelial growth factor in the retinal tissues.

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: Pueraria, Amnion, Mesenchymal Stem Cell Transplantation, Diabetic Retinopathy, Tissue Engineering

CLC Number:

R394.2

Liu Liang, Wang Jin-liang. Gegen Qin Lian Tang combined with human amniotic mesenchymal stem cell transplantation for early diabetic retinopathy[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2824-2830.

Figures/Tables 2

2.1 人羊膜间充质干细胞的形态原代培养24 h后出现贴壁生长，形态多样，无明显扩增。两三天后贴壁细胞开始增多，细胞开始转变为梭形、多边形等(图1A)。7 d后细胞增殖加快，细胞呈漩涡状密布生长，胞体变得细长，成纤维样细胞进一步增多。细胞传代后生长速度增快，多呈单层放射状或漩涡状生长，3-7 d即可长满瓶底。传第3代时细胞生长融合度可达90%，呈漩涡状紧密排列，细胞形态无明显变化(图1B)。 2.2 各组糖尿病大鼠血糖、血清胰岛素的变化移植后各组糖尿病大鼠空腹血糖、血清胰岛素检测结果显示，与模型组比较，人羊膜间充质干细胞移植组和联合组大鼠空腹血糖明显降低，而联合组血糖降低较人羊膜间充质干细胞移植组更为明显；与模型组比较，人羊膜间充质干细胞移植组和联合组大鼠血清胰岛素含量增加，联合组血清胰岛素含量增加大于人羊膜间充质干细胞移植组，差异有显著性意义(P < 0.05)，见表1，2。 2.3 糖尿病大鼠视网膜形态学变化正常大鼠视网膜各层结构清晰，细胞排列紧密整齐，层次分明，神经节细胞单层排列；内核层较薄、外核层较厚，视网膜的内界膜完整，未见毛细血管内皮细胞突破内界膜。苏木精-伊红染色后光镜下观察到各组大鼠视网膜形态学变化：模型组大鼠视网膜水肿、结构紊乱、部分细胞突出于内界膜，细胞排列不规则；与模型组比较，人羊膜间充质干细胞移植组大鼠视网膜病变相对轻微，联合组可见视网膜病变明显好转，见图2。 2.4 免疫组织化学检测视网膜CD45的表达 CD45在糖尿病大鼠视网膜中呈高强度表达，表现为黄色或棕黄色。与模型组比较，人羊膜间充质干细胞移植组CD45在视网膜中的表达明显减弱，与人羊膜间充质干细胞移植组比较，联合组进一步减弱，见图3。 2.5 RT-PCR法检测视网膜血管内皮生长因子mRNA的表达与模型组比较，人羊膜间充质干细胞移植组和联合组血管内皮生长因子mRNA明显降低，联合组降低范围明显大于人羊膜间充质干细胞移植组，各组间比较差异有显著性意义(P < 0.05)，见图4。"

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