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

全文 ( ) 下载PDF ( ) 导出引用

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/OL]. 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/OL]. 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

[1]	Ronco P, Beck L, Debiec H, et al. Membranous nephropathy [J]. Nat Rev Dis primers, 2021, 7(1): 69.
[2]	Chen Y, Yu Q, Hu Y, et al. Current research and use of mesenchymal stem cells in the therapy of autoimmune diseases [J]. Curr Stem Cell Res Ther, 2019, 14(7): 579-582.
[3]	Rota C, Morigi M, Imberti B. Stem cell therapies in kidney diseases: progress and challenges [J]. Int J Mol Sci, 2019, 20(11): 2790.
[4]	Mareschi K, Castiglia S, Sanavio F, et al. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta [J]. Exp Hematol, 2016, 44(2): 138-150.
[5]	Perin L, Giuliani S, Jin D, et al. Renal differentiation of amniotic fluid stem cells [J]. Cell Prolif, 2007, 40(6): 936-948.
[6]	Da SS, Perin L, Sedrakyan S. Amniotic fluid cells: current progress and emerging challenges in renal regeneration [J]. Pediatr Nephrol, 2018, 33(6): 935-945.
[7]	Sedrakyan S, Da SS, Milanesi A, et al. Injection of amniotic fluid stem cells delays progression of renal fibrosis [J]. J Am Soc Nephrol, 2012, 23(4): 661-673.
[8]	Cybulsky AV, Quigg RJ, Salant DJ. Experimental membranous nephropathy redux [J]. Am J Physiol Renal Physiol, 2005, 289(4): F660-F671.
[9]	Fei X, Jiang S, Zhang S, et al. Isolation, culture, and identification of amniotic fluid-derived mesenchymal stem cells [J]. Cell Biochem Biophys, 2013, 67(2): 689-694.
[10]	张峻铭，张英杰，白雪源，等. 人羊水间充质干细胞的分离鉴定及其对抗Thy-1肾炎的治疗作用[J/CD]. 中华肾病研究电子杂志，2020, 9(4): 145-151.
[11]	Nakatsue T, Koike H, Han GD, et al. Nephrin and podocin dissociate at the onset of proteinuria in experimental membranous nephropathy [J]. Kidney Int, 2005, 67(6): 2239-2253.
[12]	Loukogeorgakis SP, De Coppi P. Stem cells from amniotic fluid--potential for regenerative medicine [J]. Best Pract Res Clin Obstet Gynaecol, 2016, 31: 45-57.
[13]	Ornellas FM, Ramalho RJ, Fanelli C, et al. Mesenchymal stromal cells induce podocyte protection in the puromycin injury model [J]. Sci Rep, 2019, 9(1): 19604.
[14]	Loh JT, Zhang B, Teo J, et al. Mechanism for the attenuation of neutrophil and complement hyperactivity by MSC exosomes [J]. Cytotherapy, 2022, 24(7): 711-719.
[15]	Prodromidi EI, Poulsom R, Jeffery R, et al. Bone marrow-derived cells contribute to podocyte regeneration and amelioration of renal disease in a mouse model of Alport syndrome [J]. Stem Cells, 2006, 24(11): 2448-2455.

[1]	刘思锐, 赵辰阳, 张睿, 张一休, 杨萌. 多普勒超声对孕鼠子宫动脉不同节段血流动力学参数的评估[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(09): 877-883.
[2]	李朋, 苗立帅, 朱智奇. 四氢嘧啶对大鼠关节软骨细胞的保护作用[J/OL]. 中华关节外科杂志(电子版), 2024, 18(01): 69-77.
[3]	林琳, 田思萌, 于永华, 徐飞飞, 黄明莉. 干细胞及其外泌体治疗宫腔黏连的研究现状[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(03): 271-275.
[4]	邓健, 王少华, 陈尊, 邹振庄. Keap1/Nrf2信号通路在脂多糖诱导宫内感染致新生鼠支气管肺发育不良的作用机制[J/OL]. 中华妇幼临床医学杂志(电子版), 2023, 19(06): 665-674.
[5]	张晓燕, 肖东琼, 高沪, 陈琳, 唐发娟, 李熙鸿. 转录因子12过表达对脓毒症相关性脑病大鼠大脑皮质的保护作用及其机制[J/OL]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[6]	杨文飞, 郝嘉文, 鲁梦远, 赵学刚, 李聪颖, 盖晨阳, 张晶, 张庆富. 高压电烧伤对大鼠心肌氧化应激的影响及N-乙酰半胱氨酸的干预作用[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(02): 106-112.
[7]	张礼刚, 邹志辉, 许顺, 蔡可可, 胡永涛, 梁朝朝. 酒精对慢性非细菌性前列腺炎中T淋巴细胞变化的影响研究[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(01): 74-81.
[8]	赛甫丁·艾比布拉, 买买提·依斯热依力, 李义亮, 王永康, 王志, 克力木·阿不都热依木. 不同材质补片修补对腹壁疝大鼠腹横筋膜组织转化生长因子-β1及Collagen合成代谢的作用[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(02): 161-167.
[9]	刘锦程, 王斌, 张雯, 张明周, 刘禹, 叶东樊, 黄赞胜, 邱凌霄, 卿斌, 王创业, 王南博, 王苹, 郭宇航, 周培花, 程秋霞, 徐智. 肺泡灌洗液RASSF1A及SHOX2甲基化联合径向超声特征对肺结节性质鉴别诊断的意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(04): 505-511.
[10]	施麟宵, 洪兰兰, 阳柳, 芶碧珍, 刘畅, 吴欣. 钠-葡萄糖协同转运蛋白2 抑制剂治疗原发性膜性肾病的疗效及其对Th1/Th2 的影响[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 261-267.
[11]	张勤灵, 王盼飞, 赵倩文. 尿β2-微球蛋白在原发性膜性肾病预后评估中的作用[J/OL]. 中华肾病研究电子杂志, 2024, 13(04): 195-200.
[12]	王琳娜, 郭存霞, 乔东鸽, 赵旭, 阎磊, 邵凤民, 陈香美. 无高血压和糖尿病的特发性膜性肾病患者肾内动脉病变特点及其预后影响分析[J/OL]. 中华肾病研究电子杂志, 2024, 13(01): 39-45.
[13]	代叶梅, 苏晓乐, 王利华. 膜性肾病靶抗原的研究进展[J/OL]. 中华肾病研究电子杂志, 2023, 12(05): 282-286.
[14]	萨仁高娃, 张英霞, 邓伟, 闫诺, 樊宁. 超声引导下鼠肝消融术后组织病理特征的变化规律及影响[J/OL]. 中华消化病与影像杂志(电子版), 2023, 13(06): 394-398.
[15]	王小红, 钱晶, 翁文俊, 周国雄, 朱顺星, 祁小鸣, 刘春, 王萍, 沈伟, 程睿智, 秦璟灏. 巯基丙酮酸硫基转移酶调控核因子κB信号介导自噬对重症急性胰腺炎大鼠的影响及机制[J/OL]. 中华消化病与影像杂志(电子版), 2023, 13(06): 422-426.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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