摘要
目的探讨液氮冻融联合胰蛋白酶消化脱细胞法构建猪心脏瓣膜支架的效果。方法将新鲜的猪心脏瓣膜剪成约1 cm×1 cm的小块,按随机数字表法分为实验组、对照组和未处理组,每组设5个重复标本。3组标本经液氮冻融后,实验组采用0.6%胰蛋白酶和5%十二烷基硫酸钠(SDS)各处理3 d;对照组采用5%SDS处理6 d;未处理组置于无菌0.9%氯化钠溶液中常温静置6 d。通过HE染色和丽春红-苦味酸染色、残留DNA定量及α-Gal抗原检测确定脱细胞效率。将脱细胞的猪心脏瓣膜在大鼠皮下包埋2周,通过茜素红染色观察其抗钙化潜能。结果实验组的猪心脏瓣膜标本细胞成分基本去除,细胞外基质结构完整;残留DNA含量、α-Gal抗原分别为(55.6±16.8)μg/g、0.02±0.01,明显低于对照组的(750.4±178.1)μg/g、0.14±0.02(均P<0.01)。大鼠皮下包埋2周后,实验组钙化结节明显少于对照组(P<0.01)。结论应用液氮冻融联合胰蛋白酶消化构建的脱细胞猪心脏瓣膜基质可能是组织工程瓣膜的良好支架。
Objective To construction of acellular porcine heart valve scaffold by liquid nitrogen freezing and thawing combined with trypsin digestion method.Methods The fresh porcine heart valve leaflets were trimmed to pieces sized 1 cm×1 cm,and the pieces were randomly distributed to the experimental group,control group and untreated group with 5 samples in each group.After freezing and thawing in liquid nitrogen,samples in the experimental group were treated with 0.6%trypsin for 3 d,and 5%sodium dodecyl sulfate for 3 d,samples in the control group were treated with 5%sodium dodecyl sulfate for 6 d,and samples in the untreated group were soaked in sterile saline for 6 d at room temperature.The decellularization efficiency was determined by hematoxylin-eosin staining and Van Gieson staining,residual DNA quantification,andα-Gal antigen detection.The decellularized valve scaffolds were stained with Alizarin red to observe the anti-calcification potential after 2-week subcutaneous embedding in rats.Results The cell components of samples in the experimental group were basically removed,and the extracellular matrix structure remained integrated.The residual DNA content andα-Gal antigen were(55.6±16.8)μg/g,0.02±0.01,which were significantly lower than those in the control group(750.4±178.1)μg/g,0.14±0.02(all P<0.01).There were significantly fewer calcified nodules in the experimental group than those in the control or untreated groups,suggesting a certain anti-calcification ability(P<0.01).Conclusion The acellular porcine heart valve matrix constructed by liquid nitrogen freeze-thaw combined with trypsin digestion may be a good scaffold for tissue engineering valves.
作者
崔勇
梅富杨
胡志斌
葛根贤
孙高忠
CUI Yong;MEI Fuyang;HU Zhibin;GE Genxian;SUN Gaozhong(Department of Cardiovascular Surgery,People's Hospital of Hangzhou Medical College(Zhejiang Provincial People's Hospital),Hangzhou 310014,China)
出处
《浙江医学》
CAS
2022年第10期1021-1024,I0003,共5页
Zhejiang Medical Journal
基金
浙江省自然科学基金资助项目(LY18H020009)。
关键词
脱细胞
胰蛋白酶
猪心脏瓣膜
生物材料支架
Decellularization
Trypsin
Porcine heart valve
Biomaterial scaffold
