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构建人源性肝硬化组织细胞外基质生物支架的新方法
New method for constructing extracellular matrix bioscaffold derived from human cirrhotic liver tissues
- 第三军医大学学报 2019年41卷第18期 页码：1758-1762
- 作者机构：
400014 重庆，重庆医科大学附属儿童医院肝胆外科，儿童发育疾病研究教育部重点实验，儿童发育重大疾病国家国际科技合作基地
- 作者简介：
[ 张明满，E-mail:zhangmingman-a@163.com ZHANG Mingman, E-mail:zhangmingman-a@163.com
- 基金信息：国家自然科学基金青年科学基金(81501736);重庆市科卫联合医学科研项目(2018ZDXM041);重庆市卫生健康委员会第四批中青年医学高端后备人才项目(2018年)
DOI：10.16016/j.1000-5404.201903204
中图分类号： R318.08;R322.47;R329-33收稿：2019-03-24，
修回：2019-06-02，
纸质出版：2019-09-30
- 稿件说明：
移动端阅览
熊强, 邓玉华, 张祯祯, 等. 构建人源性肝硬化组织细胞外基质生物支架的新方法[J]. 第三军医大学学报, 2019,41(18):1758-1762.
Qiang XIONG, Yuhua DENG, Zhenzhen ZHANG, et al. New method for constructing extracellular matrix bioscaffold derived from human cirrhotic liver tissues[J]. Journal of Third Military Medical University, 2019, 41(18): 1758-1762.
熊强, 邓玉华, 张祯祯, 等. 构建人源性肝硬化组织细胞外基质生物支架的新方法[J]. 第三军医大学学报, 2019,41(18):1758-1762. DOI： 10.16016/j.1000-5404.201903204.
Qiang XIONG, Yuhua DENG, Zhenzhen ZHANG, et al. New method for constructing extracellular matrix bioscaffold derived from human cirrhotic liver tissues[J]. Journal of Third Military Medical University, 2019, 41(18): 1758-1762. DOI： 10.16016/j.1000-5404.201903204.
摘要
目的
2
通过理化方法制备人源性肝硬化组织细胞外基质(extracelluar matrix，ECM)生物支架，并利用肝硬化组织ECM对人原代肝细胞(primary human hepatocytes，PHH)进行立体培养，建立以肝硬化组织ECM为基础的细胞立体培养平台。
方法
2
收集肝移植患者移除的硬化肝脏，肝素生理盐水灌洗去除血细胞，将肝脏切割成125 mm
3
组织块。依次将组织块置于超纯水、去污剂中反复振荡，破坏细胞膜、DNA及免疫源性物质，制备出肝硬化组织ECM。采用悬滴法将PHH植入ECM进行细胞培养，连续培养9 d后，组织学检测PHH植入生物支架情况。qPCR比较离体培养及平
面培养模型下肝细胞的特异性基因表达差异。
结果
2
去除细胞的肝硬化组织呈无色透明，HE染色及扫描电镜未发现残留细胞核，残余DNA鉴定显示DNA残留约为10 ng/mg，提示该法成功去除肝硬化组织原有细胞及基因组DNA。将人PHH接种入人源性肝硬化组织ECM，连续培养9 d后，HE染色发现PHH能够植入人源性肝硬化组织ECM中并生长良好，立体培养条件下，PHH特异性基因表达水平明显高于传统平面培养。
结论
2
本研究利用人源性肝硬化组织成功制备无细胞ECM生物支架，并证实其在细胞立体培养的可行性。
Abstract
Objective
2
To develop a novel decellularized extracellular matrix (ECM) bioscaffold derived from human cirrhotic liver tissues and construct a 3-dimensional cell culture system using this bioscaffold material.
Methods
2
Clinical samples of cirrhotic liver tissues were collected from patients undergoing liver transplantation for hepatic cirrhosis. The cirrhotic liver tissues were cleansed
cut into small tissue blocks (5 mm×5 mm×5 mm)
and agitated in deionized water and detergents to obtain decellularized cirrhotic liver scaffold. Human primary human hepatocytes (PHHs) were seeded into the prepared ECM scaffold and cultured continuously for 9 d. Histological studies of the seeded cells were performed to assess the cell engraftment
and qPCR was used to detect the expression of the liver-specific genes in the cultured cells.
Results
2
The cirrhotic liver tissue became transparent after decellularization
and HE staining and scanning electron microscopy showed no residual nuclei in the bioscaffold. Residual DNA identification showed that the scaffold material had a residue DNA level less than 10 ng/mg of the tissue. After 9 d of continuous culture in the ECM scaffold
the PHHs showed good engraftment as shown by HE staining; the results of qPCR showed that the cells cultured in this 3D culture system expressed higher levels of liver-specific genes as compared with the cells in conventional 2D culture.
Conclusion
2
We successfully construct decellularized ECM bioscaffold derived from human cirrhotic liver tissues and confirmed the feasibility of 3D cell culture using this new bioscaffold material.
关键词
Keywords
references
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