人羊水间充质干细胞对膜性肾病大鼠的治疗作用

doi:10.3877/cma.j.issn.2095-3216.2023.04.001

中华肾病研究电子杂志 ›› 2023, Vol. 12 ›› Issue (04) : 181 -186. doi: 10.3877/cma.j.issn.2095-3216.2023.04.001

论著

人羊水间充质干细胞对膜性肾病大鼠的治疗作用

杨蕴钊, 周诚, 石美涵, 赵静, 白雪源^†(

)

100853　北京，解放军医学院；100853　北京，解放军总医院第一医学中心肾脏病医学部、解放军肾脏病研究所、肾脏疾病国家重点实验室、国家慢性肾病临床医学研究中心、肾脏疾病研究北京市重点实验室
100853　北京，解放军总医院第一医学中心肾脏病医学部、解放军肾脏病研究所、肾脏疾病国家重点实验室、国家慢性肾病临床医学研究中心、肾脏疾病研究北京市重点实验室

收稿日期:2022-12-03 出版日期:2023-08-28
通信作者: 白雪源
基金资助:
国家重点研发计划项目(2020YFA0113004); 国家自然科学基金重点项目(81830060)

Therapeutic effect of human amniotic fluid-derived mesenchymal stem cells on membranous nephropathy in rats

Yunzhao Yang, Cheng Zhou, Meihan Shi, Jing Zhao, Xueyuan Bai^†()

Chinese PLA Medical School; Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Diseases; Beijing 100853, China
Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Diseases; Beijing 100853, China

Received:2022-12-03 Published:2023-08-28
Corresponding author: Xueyuan Bai

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引用本文：

杨蕴钊, 周诚, 石美涵, 赵静, 白雪源. 人羊水间充质干细胞对膜性肾病大鼠的治疗作用[J]. 中华肾病研究电子杂志, 2023, 12(04): 181-186.

Yunzhao Yang, Cheng Zhou, Meihan Shi, Jing Zhao, Xueyuan Bai. Therapeutic effect of human amniotic fluid-derived mesenchymal stem cells on membranous nephropathy in rats[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2023, 12(04): 181-186.

目的

研究人羊水间充质干细胞(AF-MSC)对膜性肾病大鼠的治疗作用。

方法

24只健康雄性SD大鼠随机分为正常对照组(Control组)、模型组(Model组)和AF-MSC治疗组(AF-MSC组)，每组8只。在模型组和AF-MSC组注射羊抗大鼠Fx1A抗体构建膜性肾病大鼠模型。造模成功后连续4周尾静脉注射AF-MSC；每周检测大鼠尿蛋白/肌酐比值(UPCR)；治疗结束后，腹主动脉取血检测血清肌酐(Scr)、血尿素氮(BUN)、白蛋白(Alb)、甘油三酯(TG)、总胆固醇(TC)；肾脏组织切片经马松(Masson)和过碘酸-六胺银(PASM)染色，光镜下观察病理变化；透射电镜观察足细胞和基底膜结构变化；免疫荧光测定各组肾脏组织中IgG、膜攻击复合物C5b-9表达。免疫组织化学观测足细胞nephrin、podocin表达。

结果

与模型组相比，AF-MSC治疗组UPCR在第4周时显著下降(P<0.01)。血清白蛋白升高和胆固醇下降(均P<0.05)；病理结果显示肾小球基底膜免疫复合物沉积减少，基底膜增厚程度减少，足细胞形态相对完好；免疫荧光结果显示IgG和C5b-9沉积减弱；免疫组化结果显示AF-MSC组足细胞中nephrin及podocin蛋白表达增强(P<0.01)。

结论

AF-MSC可以明显改善膜性肾病大鼠的肾脏损伤。

关键词: 人羊水间充质干细胞, 膜性肾病, 大鼠

Objective

To investigate the therapeutic effect of human amniotic fluid-derived mesenchymal stem cells (AF-MSC) on membranous nephropathy in rats.

Methods

Twenty-four healthy male Sprague-Dawley (SD) rats were randomly divided into three groups with 8 rats each. The rat model of membranous nephropathy was established by injection of sheep anti-rat Fx1A antibody in both the model group and AF-MSC group. AF-MSC was injected by tail vein (4×10⁶/rat/week) for 4 weeks after model establishment. The urinary protein/creatinine ratio (UPCR) was measured weekly. After the treatment, serum creatinine (Scr), blood urea nitrogen (BUN), albumin (Alb), triglyceride (TG) and total cholesterol (TC) were detected with blood samples from abdominal aorta of rats. The kidney tissues were stained with the Masson and periodic acid-silver methenamine (PASM) staining methods, and the pathological changes were observed under light microscope. The structural changes of podocytes and basement membrane were observed by transmission electron microscope. The expression of IgG and membrane attack complex C5b-9 in renal tissues in each group were determined by immunofluorescence. The expressions of nephrin and podocin in podocytes were observed by immunohistochemistry.

Results

Compared with the model group, the AF-MSC group showed significantly lower level of UPCR at week 4 (P<0.01), as well as higher level of serum Alb (P<0.05) and lower level of TC (P<0.05). The pathological results showed that the immune complex deposition on glomerular basement membrane was reduced, and that basement membrane thickening was inhibited, with the morphology of podocytes being relatively intact. Immunofluorescence results showed less deposition of IgG and C5b-9 immune complex. Immunohistochemical results showed that the expressions of nephrin and podocin in podocytes were increased (P<0.01).

Conclusion

The human AF-MSC could effectively alleviate the kidney injury in the membranous nephropathy of the rats.

Key words: Human amniotic fluid-derived mesenchymal stem cells, Membranous nephropathy, Rat

图1 各组大鼠尿蛋白/肌酐比值的比较注：与Control组比较，^aP<0.01；与Model组比较，^bP<0.01

表1 各组大鼠血清白蛋白、胆固醇、甘油三酯、肌酐和尿素氮水平比较(±s，n＝8)

组别	ALB(g/L)	TC(mmol/L)	TG(mmol/L)	Scr(μmol/L)	BUN(μmol/L)
Control	41.53±0.87	1.77±0.17	1.60±0.39	56.24±9.28	7.94±0.85
Model	37.35±1.78^a	4.90±0.86^a	5.35±2.60^a	75.86±6.17^a	7.78±0.42
AF-MSC	39.33±1.39^b	3.60±1.08^b	3.74±1.175	63.4±17.23	7.59±0.99

表1 各组大鼠血清白蛋白、胆固醇、甘油三酯、肌酐和尿素氮水平比较(±s，n＝8)

组别	ALB(g/L)	TC(mmol/L)	TG(mmol/L)	Scr(μmol/L)	BUN(μmol/L)
Control	41.53±0.87	1.77±0.17	1.60±0.39	56.24±9.28	7.94±0.85
Model	37.35±1.78^a	4.90±0.86^a	5.35±2.60^a	75.86±6.17^a	7.78±0.42
AF-MSC	39.33±1.39^b	3.60±1.08^b	3.74±1.175	63.4±17.23	7.59±0.99

图2 各组大鼠肾脏病理注：A：Masson染色结果(400×)；B：PASM染色结果(400×)；C：扫描电镜结果(5 000×)；扫描电镜病理图片中红色箭头指示肾小球基底膜，可见Control组足细胞基底膜形态完好，Model组基底膜包绕着沉积物、足突广泛消失，AF-MSC组沉积物与基底膜反应不明显、仅见足突部分消失或变钝

图3 各组膜性肾病大鼠中IgG和C5b-9在肾脏组织中的沉积(免疫荧光染色×400)

图4 各组大鼠Nephrin和Podocin免疫组化结果及比较注：A：各组nephrin免疫组化染色(×400)；B：各组podocin免疫组化染色(×400)；C：各组nephrin平均光密度比较；D：各组podocin平均光密度比较；与Control组相比，^aP<0.01；与Model组相比，^bP<0.01

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