中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (18): 2847-2851.doi: 10.3969/j.issn.2095-4344.1682

• 细胞外基质材料 extracellular matrix materials • 上一篇    下一篇

肝脏细胞外基质水凝胶的制备及表征

曹玉伦，程  远，何国林，李  阳，彭  青，高  毅，潘明新
  

  1. 南方医科大学珠江医院肝胆外科，广东省广州市  510280
  • 收稿日期:2018-12-26 出版日期:2019-06-28 发布日期:2019-06-28
  • 通讯作者: 潘明新，教授，主任医师，博士生导师，南方医科大学珠江医院肝胆外科，广东省广州市 510280
  • 作者简介:曹玉伦，男，1992年生，河南省信阳市人，汉族，南方医科大学在读硕士，主要从事脱细胞支架及生物材料研究。
  • 基金资助:

    国家青年科学基金项目(81600489)，项目负责人：程远；广州市科技计划项目(201803010086)，项目负责人：彭青；广东省自然科学基金(2018A030313128)，项目负责人：彭青；广东省省科技计划项目(2015B020229002)，项目负责人：潘明新

Preparation and characterization of liver extracellular matrix hydrogel

Cao Yulun, Cheng Yuan, He Guolin, Li Yang, Peng Qing, Gao Yi, Pan Mingxin
  

  1. Pan Mingxin, Professor, Chief physician, Doctoral supervisor, Department of Hepatobiliary Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • Received:2018-12-26 Online:2019-06-28 Published:2019-06-28
  • Contact: Department of Hepatobiliary Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • About author:Cao Yulun, Master candidate, Department of Hepatobiliary Surgery, Zhujiang Hospital of Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation for the Youth of China, No. 81600489 (to CY); the Science and Technology Program of Guangzhou, No. 201803010086 (to PQ); the Natural Science Foundation of Guangdong Province, No. 2018A030313128 (to PQ); the Science and Technology Program of Guangdong Province, No. 2015B020229002 (to PMX)

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摘要：

文章快速阅读：

 

文题释义：
脱细胞技术：采用物理、化学、酶解等方法对组织或器官进行去除细胞成分，保留细胞外基质的结构和成分。通过去细胞技术得到的细胞外基质支架，可用于培养细胞。
肝脏细胞外基质水凝胶：采用脱细胞技术制备脱细胞肝脏基质，再通过研磨、消化等步骤及后续渗透压、pH值、温度的调节，多肽自组装为富含水分的三维网状立体结构水凝胶。肝脏基质水凝胶扩展了脱细胞肝脏支架的应用，是一种比较有应用前景的生物材料。
 
 
背景：研究表明，肝脏细胞外基质水凝胶可促进肝脏特异性细胞的行为更加接近体内的状态，可增强肝细胞的活性及功能，促进内皮细胞的血管化和胆管上皮细胞的胆管化，但对于肝脏基质胶本身性质的研究较少。
目的：采用温和去细胞技术制备肝脏细胞外基质水凝胶，并对水凝胶进行初步表征。
方法：取新鲜冰冻的猪肝组织切片，常温下放入去离子水中搅拌4次；移入预热到37 ℃的0.02%胰酶/ 0.05%EDTA溶液中，37 ℃搅拌1 h，去离子水清洗肝片，放入3%Triton X-100溶液中搅拌1.5 h；去离子水冲洗肝片，放入4%脱氧胆酸钠溶液中搅拌1.5 h；过量去离子水冲洗肝片，获得去细胞肝脏支架；将肝脏支架移入0.1%过氧乙酸溶液中搅拌2.0-3.0 h，置入1×PBS溶液中搅拌15 min、去离子水中搅拌浸泡2次、1×PBS冲洗15 min；再通过冻干、液氮研磨、消化成肝脏基质溶液，经过后续的配平，多肽分子自组装成肝脏细胞外基质水凝胶。检测肝脏细胞外基质水凝胶的脱细胞程度、DNA含量、组成成分、浊度动力学及微观结构。
结果与结论：①采用温和脱细胞技术得到了脱细胞肝脏支架，而且脱细胞较完全；②脱细胞肝脏支架的DNA含量较正常肝脏组织明显下降(P < 0.001)；③肝脏细胞外基质水凝胶前体溶液保留了许多猪肝细胞外基质成分，比如胶原蛋白、弹性蛋白、糖安聚糖及其前体等；④浊度动力学实验显示，随着肝脏细胞外基质凝胶质量浓度的增大，平台期的吸光度值升高；⑤扫描电镜显示，肝脏细胞外基质水凝胶呈现纤维网状多孔结构，纳米级细胞外基质纤维互相交错，并且随着肝脏细胞外基质水凝胶质量浓度的增加，纤维密度相对增加，直径无变化；⑥结果表明，采用温和去细胞技术制备的肝脏细胞外基质水凝胶，具有三维立体网络结构，为细胞的黏附、生长提供了结构基础。

关键词: 生物材料, 脱细胞支架, 水凝胶, 浊度动力学, 质谱, 微观结构, 细胞外基质, 肝脏细胞

Abstract:

BACKGROUND: Liver extracellular matrix hydrogel has been shown to promote the behavior of liver-specific cells closer to the body, enhance the activity and function of hepatocytes, and promote the vascularization of endothelial cells and the bile duct formation of bile duct epithelial cells, but the characteristics of liver matrigelis little known.
OBJECTIVE: To prepare the liver extracellular matrix hydrogel by mild decellularization technique and to investigate its characterization preliminarily.
METHODS: Fresh frozen pig liver tissue sections were taken and placed in deionized water at room temperature for 4 times, transferred to 0.02% trypsin/0.05% EDTA solution preheated at 37 °C, stirred at 37 °C for 1 hour, and washed with deionized water, put into 3% Triton X-100 solution and stirred for 1.5 hours. The liver piece was rinsed with deionized water, stirred in 4% sodium deoxycholate solution for 1.5 hours, and rinsed with excess deionized water to obtain the decellularized liver scaffold. The liver scaffold was transferred into 0.1% peracetic acid solution and stirred for 2.0-3.0 hours, placed in 1×PBS solution for 15 minutes, stirred in deionized water for twice, rinsed in 1×PBS for 15 minutes, and then lyophilized and liquid nitrogen-milled. Digested into a liver matrix solution, and after subsequent trimming, the polypeptide molecules self-assemble into a liver matrix hydrogel. The degree of decellularization, DNA content, composition, turbidity dynamics and microstructure of the liver matrix hydrogel were examined.
RESULTS AND CONCLUSION: (1) The decellularized liver scaffold was obtained by mild decellularization technique, and the decellularization was complete. (2) The DNA content of the decellularized liver scaffold was significantly lower than that of normal liver tissue (P < 0.001). (3) The liver matrix hydrogel precursor solution retained many components of the pig liver extracellular matrix, such as collagen, elastin, glycan and its precursors. (4) The turbidity kinetics experiment showed that the absorbance value of the plateau increased with the increase of the mass concentration of the liver matrix gel. (5) Scanning electron microscope showed that the liver matrix hydrogel possessed a fibrous network porous structure, and the nano-scale extracellular matrix fibers were interlaced with each other. As the mass concentration of the liver matrix hydrogel increased, the fiber density increased relatively and the diameter revealed no change. (6) These results indicate that the liver matrix hydrogel is prepared by gentle decellularization technique and exhibits a three-dimensional network structure, which provides a structural basis for cell adhesion and growth.

Key words: biomaterial, decellularized scaffold, hydrogel, turbidity kinetics, mass spectrum, microstructure, extracellular matrix, cells

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