两种方法制备脱细胞血管材料在动物体内免疫反应的比较

Comparison of immune responses of acellular vascular materials prepared by the two methods in animals

背景:去垢剂作为目前较成熟的脱细胞方法,具有操作简便、破坏小、实验条件容易控制等优点,有研究将不同的去垢剂联合应用于脱细胞中取得了不错的效果。目的:对比两种去垢剂联合方案脱细胞的效果,以及两种脱细胞血管材料植入动物皮下后的免疫反应。方法:采用两种方案对猪颈动脉进行脱细胞处理,一组置于1%十二烷基硫酸钠与1%脱氧胆酸钠混合溶液内处理72h(SDS+SDC组),另一组置于1%十二烷基硫酸钠与1%TritonX-100混合溶液内处理72h(SDS+Triton X-100组),脱细胞后分别进行组织学分析、扫描电镜分析、力学分析与DNA含量检测。将两组脱细胞血管材料分别植入SD大鼠背部皮下,术后1,2,4,8周取出移植血管材料,进行苏木精-伊红染色与免疫荧光染色。实验已通过江南大学实验动物伦理委员会批准。结果与结论:①组织学分析显示,SDS+SDC组方案有效去除了细胞成分,较完整的保留了细胞外基质结构,脱细胞效果优于SDS+Triton X-100组;②SDS+SDC组DNA含量明显低于SDS+Triton X-100组(P<0.05),两组爆裂强度与缝合耐受强度比较差异无显著性意义(P>0.05);③扫描电镜显示,SDS+TritonX-100组细胞外基质破坏程度大于较SDS+SDC组;④皮下植入实验中,术后1周时两组颈动脉材料周围均可见大量炎性细胞浸润,术后8周时SDS+SDC组脱细胞材料周围已无炎性细胞浸润,SDS+Triton X-100组脱细胞材料周围仍可见明显的淋巴细胞浸润;SDS+TritonX-100组脱细胞材料主要诱导巨噬细胞向M1型分化,SDS+SDC组脱细胞材料主要诱导巨噬细胞向M2型分化;⑤结果表明相对比1%十二烷基硫酸钠联合1%Triton X-100脱细胞处理方案,1%十二烷基硫酸钠联合1%脱氧胆酸钠脱是较有前景的脱细胞细胞处理方案。

BACKGROUND: As a mature method of decellularization at present, descaling agent has the advantages of simple operation, small damage, and easy control of experimental conditions. Some studies have combined different descaling agents to achieve good results in decellularization. OBJECTIVE: To compare the effect of two descaling agents and the immune response of two kinds of acellular vascular materials subcutaneously implanted into animals. METHODS: Two methods were used to treat the porcine carotid artery for descaling agent. One group was treated in the mixed solution of 1% sodium dodecyl sulfate and 1% sodium deoxycholate for 72 hours;the other group was treated in the mixed solution of 1% sodium dodecyl sulfate and 1% Triton X-100 for 72 hours. After cell removal, histological analysis, scanning electron microscope analysis, mechanical analysis and DNA content detection were carried out. Two groups of acellular vascular materials were implanted subcutaneously in the back of SD rats. After 1, 2, 4 and 8 weeks, the grafted vascular materials were taken out for hematoxylin and eosin and immunofluorescence staining. This study was approved by the Animal Ethics Committee of Jiangnan University. RESULTS AND CONCLUSION:(1) Histological analysis showed that 1% sodium dodecyl sulfate combined with 1% sodium deoxycholate effectively removed the cell components and retained the extracellular matrix structure, and the effect of cell removal was better than 1% sodium dodecyl sulfate combined with 1% Triton X-100.(2) The DNA content of 1% sodium dodecyl sulfate combined with 1% sodium deoxycholate group was significantly lower than that of 1% sodium dodecyl sulfate combined with 1% Triton X-100 group(P < 0.05). There were no significant differences in bursting strength and suture tolerance between the two schemes(P > 0.05).(3) Scanning electron microscopy showed that 1% sodium dodecyl sulfate combined with 1% Triton X-100 significantly damaged the extracellular matrix compared with 1% sodium dodecyl sulfate combined with 1% sodium deoxycholate.(4) In the subcutaneous implantation experiment, a large number of inflammatory cells infiltrated around the carotid artery materials of the two groups at 1 week after operation. No inflammatory cells infiltrated around the acellular materials of 1% sodium dodecyl sulfate and 1% sodium deoxycholate at 8 weeks after operation. Lymphocyte infiltrated around the acellular materials of 1% sodium dodecyl sulfate and 1% Triton X-100 group. 1% sodium dodecyl sulfate combined with 1% Triton X-100 acellular material mainly induced macrophages to differentiate into M1 type. 1% sodium dodecyl sulfate combined with 1% sodium deoxycholate acellular material mainly induced macrophages to differentiate into M2 type.(5) Compared with 1% sodium dodecyl sulfate and 1% Triton X-100, 1% sodium dodecyl sulfate and 1% sodium deoxycholate are more promising cell-free treatment.