Underlying mechanism by which adipose-derived mesenchymal stem cell transplantation alleviates renal ischemia-reperfusion injury in rats

doi:10.3969/j.issn.2095-4344.1550

Abstract

Abstract:

BACKGROUND: Acute renal injury induced by ischemia and reperfusion is a common clinical problem with high mortality, and there is no effective treatment. Mesenchymal stem cell transplantation has been used to improve the condition of the disease, but the possible mechanism is still unclear.
OBJECTIVE: To investigate the protective effect of adipose-derived mesenchymal stem cell transplantation on renal ischemia-reperfusion injury in rats and its possible mechanism.
METHODS: Adipose-derived mesenchymal stem cells were isolated from the inguinal adipose tissue of Sprague-Dawley rats (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. in China), cultured and identified. Thirty rat models of renal ischemia-reperfusion were established by 45 minutes of clipping the left renal artery and randomly divided into transplantation control group and stem cell transplantation group, 15 rats in each group. Another 15 rats were selected as sham operation group. Adipose-derived mesenchymal stem cells were injected into the tail vein of the stem cell transplantation group and the normal saline was injected into the tail vein of the control group. The following tests were performed 5, 15 and 30 days after transplantation: frozen sections of rat kidney tissue were observed by fluorescence microscope for the colonization of adipose-derived mesenchymal stem cells; serum creatinine and urea nitrogen levels were detected by ELISA; malondialdehyde content in the renal tissue was detected by biochemical method; renal pathological structure was observed by hematoxylin-eosin staining in paraffin slices and renal tubule damage was scored; glutathione peroxidase 4 expression in renal tissue was detected by western blot.
RESULTS AND CONCLUSION: Adipose-derived mesenchymal stem cells from Sprague-Dawley rats expressed CD90 and CD44, and showed multidirectional differentiation. There was no green fluorescence in the renal tissue of transplantation control group, but green fluorescence was observed in the stem cell transplantation group at 5 days after transplantation. Compared with the transplantation control group, the serum creatinine and urea nitrogen levels, malondialdehyde content in renal tissue and renal tubular injury score were significantly lower in the stem cell transplantation group (P < 0.05), while the expression of glutathione peroxidase 4 was significantly increased (P < 0.05). The renal tissue morphology of the stem cell transplantation group improved significantly compared to the transplantation control group. These results suggest that adipose-derived mesenchymal stem cell transplantation can alleviate renal ischemia-reperfusion injury in rats, and may play an anti-lipid peroxidation role by promoting the expression of glutathione peroxidase 4 in the renal tissue.

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: Tissue Engineering, Stem Cells, Mesenchymal Stem Cells

CLC Number:

R456
R364

Lei Yu, Liu Rongan, Zeng Fan . Underlying mechanism by which adipose-derived mesenchymal stem cell transplantation alleviates renal ischemia-reperfusion injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 749-755.

Figures/Tables 3

2.1 实验大鼠数量分析实验的50只SD大鼠中体外实验5只。体内实验45只，其中15只用于假手术组，其中1只术后死亡，30只用于造模，1只移植术后死亡，3只造模不成功；最后，假手术组14只大鼠、移植对照组13只大鼠、干细胞移植组13只大鼠。最终共40只大鼠的实验数据计入结果分析。 2.2 大鼠脂肪间充质干细胞的鉴定如图1A所示，流式细胞术检测间充质干细胞表面标志物的结果显示，所收集的大鼠脂肪间充质干细胞中黏附分子CD44和CD90的阳性率分别为97.9%和99.5%；成骨诱导21 d后，茜素红S染色后，光学显微镜下可见散在分布的深红色钙结节，见图1B，提示成骨分化阳性；成软骨诱导28 d后，阿利新蓝染色后，光镜下可见致密的块状组织被染成灰蓝色，说明组织内含有酸性黏多糖，见图1C，提示成软骨分化阳性；成脂诱导10 d后，油红O染色后，光学显微镜下可见胞质内有大小不一的黄色脂滴，见图1D，提示成脂分化阳性。以上结果证实，分离培养的是纯度较好的脂肪间充质干细胞。 2.3 脂肪间充质干细胞在大鼠肾脏中定植的观察如图2A，B所示，实验将用于标记干细胞的荧光染料注射至对照组大鼠体内，注射5 d后，荧光显微镜下肾脏血管球内可见绿色荧光，注射15 d后血管内绿色荧光消失；如图2C所示，注射5 d后，干细胞移植组大鼠肾组织中有沿肾小管上皮腔面分布的绿色荧光，提示大鼠肾脏内有脂肪间充质干细胞定植在肾小管；移植15和30 d，干细胞移植组大鼠的肾组织中未见绿色荧光，见图2D，E，说明随着移植时间的延长，脂肪间充质干细胞可能发生分化减弱了荧光强度。 2.4 脂肪间充质干细胞对大鼠肾功能的影响实验取移植后5，15和30 d检测大鼠肌酐和尿素氮水平。如图3所示，与假手术组比较，移植后5，15和30 d，移植对照组和干细胞移植组大鼠肌酐和尿素氮水平升高(P < 0.01)；与移植对照组比较，移植后5，15和30 d干细胞移植组大鼠肌酐和尿素氮水平降低(P < 0.01)；与移植后5 d比较，干细胞移植组移植15和30 d后大鼠肌酐和尿素氮水平降低(P < 0.01)。说明移植脂肪间充质干细胞能改善肾功能。"

2.6 脂肪间充质干细胞对大鼠肾组织病理学变化的影响光学显微镜下观察肾组织苏木精-伊红染色切片，如图5A-G所示，与假手术组比较，移植对照组和干细胞移植组大鼠肾组织存在不同程度的病理变化，主要表现为肾皮质内的肾小管上皮细胞水肿、变性及坏死，肾小管管腔扩张，肾间质水肿和炎细胞浸润；与移植对照组比较，干细胞移植组随着移植时间的延长，肾组织损伤程度减轻明显。为了进一步评价肾小管的损伤程度，实验进行了肾小管损伤评分，如图5H所示，与假手术组比较，移植对照组和干细胞移植组在移植后5，15和30 d肾小管损伤评分均升高(P < 0.05)；与移植对照组比较，干细胞移植组在移植后5，15和30 d肾小管评分降低(P < 0.05)；与移植后5 d比较，干细胞移植组在移植后15和30 d肾小管评分降低(P < 0.05)；与移植后15 d比较，干细胞移植组在移植后30 d肾小管评分降低(P < 0.05)提示移植脂肪间充质干细胞能减轻肾小管损伤。 2.7 脂肪间充质干细胞对大鼠肾组织谷胱甘肽过氧化酶4表达的影响为了进一步探讨脂肪间充质干细胞抗脂质过氧化作用的机制，实验应用Western blot检测3组大鼠肾组织中谷胱甘肽过氧化酶4蛋白的表达情况，如图6所示，与假手术组比较，移植后5和15 d，移植对照组和干细胞移植组大鼠肾组织谷胱甘肽过氧化酶4表达降低(P < 0.05)；与移植对照组比较，移植后5和15 d干细胞移植组大鼠肾组织谷胱甘肽过氧化酶4表达升高(P < 0.05)；与移植后5 d比较，干细胞移植组在移植后15 d大鼠肾组织谷胱甘肽过氧化酶4表达升高(P < 0.05)；与移植后15 d比较，干细胞移植组在移植后30 d大鼠肾组织谷胱甘肽过氧化酶4表达降低(P < 0.05)。提示移植脂肪间充质干细胞能促进谷胱甘肽过氧化酶4的表达，但随着时间延长作用减弱。"

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