猪兔鼠肾生物支架制备及体外细胞共培养探究

doi:10.13418/j.issn.1001-165x.2018.02.011

中国临床解剖学杂志 ›› 2018, Vol. 36 ›› Issue (2): 174-181.doi: 10.13418/j.issn.1001-165x.2018.02.011

猪兔鼠肾生物支架制备及体外细胞共培养探究

王新旺^1,4，周乐斌²，梅劲²，　张德明³，　刘丹¹，　杜奥玲¹，　陈胜华¹

1.南华大学医学院应用解剖与生殖医学研究所，湖南衡阳 421001； 2. 温州医科大学解剖学教研室，浙江温州 325035；
3. 浙江大学机械工程学院，杭州 310058； 4. 中国人民解放军69250部队，乌鲁木齐 830000

收稿日期:2018-01-08 出版日期:2018-03-25 发布日期:2018-05-04
通讯作者: 陈胜华，副教授，硕士生导师，E- mail:chenshenghuaa@163.com
作者简介:王新旺(1989-)，在读硕士, 主要从事临床应用解剖学方面的研究，E-mail: wxw0841@126.com
基金资助:
国家自然科学基金项目（80215086）

Production of decellularized porcine, leporine and murine renal biological scaffold and co-culture exploration of renal scaffolds slices with cells in vitro

WANG Xin-wang^1,4， ZHOU Le-bin ²， MEI Jin²， ZHANG De-ming ³， LIU Dan ¹， DU Ao-ling ¹，CHEN Sheng-hua ¹

1. Clinical Anatomy & Reproductive Medicine Application Institute, Medical college, University of South China, Hengyang 421001, Hunan Province, China; 2. Department of Anatomy, Wenzhou Medical University, Wenzhou 325035, Zhejiang Province, China; 3.College of Mechanical Engineering, Zhejiang University, Hangzhou 310058, China; 4. Troops 69250 of the PLA, Urumqi 830000, China

Received:2018-01-08 Online:2018-03-25 Published:2018-05-04

摘要/Abstract

摘要：

目的　灌注制备家猪、新西兰白兔、SD大鼠肾去细胞生物支架，探究三种肾支架对共培养种子细胞HEK的影响。方法　取健康成年家猪10头、新西兰白兔28只、SD大鼠28只，分别将取出的肾脏随机均等分为正常组和支架组，支架组由肾动脉依次灌入肝素、1% Triton X-100和1% SDS溶液完成去细胞化。两组肾分别作组织形态学鉴定并检测机械力学性质。去细胞支架作组织爬片与人胚肾上皮细胞共培养，观察细胞在支架爬片上的生长状况，免疫荧光检测人胚肾上皮细胞PCNA及DAPI表达量作灰度分析。结果　家猪、新西兰白兔、SD大鼠肾脏经灌注去细胞后HE核染色阴性， Masson染色显示胶原蛋白阳性，Collagen I和Collagen IV荧光染色阳性，电镜扫描可见去细胞支架内蜂窝状孔洞结构，并可见典型的肾小球龛样结构；支架组弹性模量与正常组肾弹性模量差异无显著性，支架组PCNA/DAPI值均高于空白对照组，而三种支架组之间PCNA/DAPI值无显著性差异。结论　本研究灌注去细胞方法可去除家猪、新西兰白兔、SD大鼠肾内的细胞及细胞核，保留细胞外基质，维持细胞外基质的三维空间结构和机械力学强度，是一种可靠有效的制备三者肾去细胞生物支架方法，灌注制备的去细胞支架均可提高异种共培养人胚肾上皮细胞HEK的增殖活性，且这种提高作用在家猪、新西兰白兔和SD大鼠之间并无物种差异性。

关键词: 去细胞, 　生物支架, 　肾再生, 　共培养, 　组织工程

Abstract:

Objective　To produce the decellularized biological renal scaffold of the pig, New Zealand white rabbit and SD rat, and then to explore the influence of porcine, leporine and murine renal biological scaffolds on the co-cultured seed cells HEK.　Methods　Adult healthy kidneys from 10 pigs, 28 New Zealand white rabbits, and 28 SD rats were harvested and divided into normal groups and scaffold groups randomly and equally. The scaffold group kidneys were perfused for decellularization with heparinized PBS, 1% Triton X-100 and 1% SDS through the renal artery successively. The kidneys in each group were characterized in histomorphology and detected for mechanical properties. And the co-culture experiments were conducted using the slices of each group and HEK cells, and the expression of PCNA in HEK cells was analyzed by immunofluorescence assay using the value of the gray level.　Results　In each scaffold group after decellularization, the HE staining of cell nucleus in the slices was negative, Masson's trichrome staining of collagen was positive, and immunofluorescence staining of Collagen I and Collagen IV was also positive. And with the scanning electron microscopy, the faveolate orifice structure and the typical glomerulus-like structure were observed. Characterized by the mechanical properties, the Young's modulus of each scaffold group is similar with that of the corresponding normal group. After the co-culture, the PCNA/DAPI value of every renal scaffold group is higher than that of the blank control. Furthermore, there was no significant difference on the PCNA/DAPI of HEK between the pig, New Zealand white rabbit and SD rat renal scaffolds. Conclusions　The decellularization method of our study can eliminate the cells in the porcine, leporine and murine kidney, retain the extracellular matrices and preserve the spatial structure and similar mechanical strength. And it is an effective way to produce the decellularized pig, New Zealand white rabbit and SD rat renal biological scaffolds. Importantly, the produced decellularized porcine, leporine and murine kidney scaffold slices can improve the proliferation activity of heterogeneous HEK cells when co-cultured in vitro, and there is no species difference on the improvement between the pig, New Zealand white rabbit and SD rat.

Key words: , Decellularization, 　Biological scaffold, 　Kidney regeneration, 　Co-cuture, 　Tissue engineering

王新旺,周乐斌,梅劲,张德明,刘丹,杜奥玲,陈胜华. 猪兔鼠肾生物支架制备及体外细胞共培养探究[J]. 中国临床解剖学杂志, 2018, 36(2): 174-181.

WANG Xin-wang,ZHOU Le-bin,MEI Jin,ZHANG De-ming,LIU Dan,DU Ao-ling,CHEN Sheng-hua. Production of decellularized porcine, leporine and murine renal biological scaffold and co-culture exploration of renal scaffolds slices with cells in vitro[J]. Chinese Journal Of Clinical Anatomy, 2018, 36(2): 174-181.

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猪兔鼠肾生物支架制备及体外细胞共培养探究

Production of decellularized porcine, leporine and murine renal biological scaffold and co-culture exploration of renal scaffolds slices with cells in vitro

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