京尼平交联大鼠肾去细胞生物支架提高支架生物学性能的实验研究

目的　通过京尼平交联大鼠肾去细胞生物支架，提高支架的生物学性能。 方法　取250 g左右的健康SD大鼠80只，分为正常组、未交联支架组、戊二醛交联支架组和京尼平交联支架组。游离大鼠肾、肾动脉，连接蠕动泵，经PBS灌注去血后得到的肾作为正常组。其余大鼠肾依次灌入肝素化PBS溶液、1% TritonX-100、1%十二烷基硫酸钠（SDS）、去离子水，完成大鼠肾去细胞生物支架制备。戊二醛交联支架组继续灌入0.625%戊二醛（GA）1500 mL；京尼平交联支架组浸入0.5%的京尼平溶液于37 ℃恒温箱中行化学交联24 h；未经戊二醛或京尼平交联的肾去细胞支架作为未交联支架组。对4组肾分别作HE、Masson、免疫荧光染色及电子显微镜扫描，观察支架组织形态学及超微结构改变；力学拉伸试验检测机械力学性能。 结果　SD大鼠肾支架经京尼平交联后，HE、Masson染色显示胶原纤维排列更加紧密有序，肾小球处的纤维呈聚集状，Collagen I和 Collagen IV荧光染色增强，电镜扫描可见交联后的去细胞支架内蜂窝状孔洞结构更加立体，并可见典型的肾小球龛样结构轮廓更加清晰；交联组肾弹性模量较支架组明显增强。 结论　京尼平交联大鼠肾支架能提高支架的生物学性能有助于为后期细胞植入和器官再生。

Experimental study on genipin crosslinking improved the biological performance of rat kidney decellularized matrices

Objective　To improve the biological properties of the rat kidney acellular biological scaffold by genipin crosslinking.　Methods　Eighty healthy SD rats were divided into a normal group, an uncrosslinking group, a glutaraldehyde crosslinking group and a genipin crosslinking group. Rat kidneys and renal arteries were isolated and connected with peristaltic pumps. Rat kidneys were successively infused with heparinized PBS solution, 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), deionized water to complete the preparation of rat kidney acellular biological scaffolds. The glutaraldehyde crosslinking group was infused with 1500 ml 0.625% glutaraldehyde (GA)，and the genipin crosslinking group was immersed in 0.5% genipin solution at 37 ℃ for 24 hours. Non-glutaraldehyde and non-genipincrosslinking renal acellular biological scaffolds served as the uncrosslinking group. Four groups of kidneys were stained with HE, Masson and immunofluorescence, respectively. The morphology and ultrastructure of scaffolds were observed by immunofluorescence and electron microscopy, and mechanical properties were tested by tensile test. Results After crosslinking with genipin, HE and Masson staining showed that collagen fibers arranged more tightly and orderly, glomerular fibers were aggregated, Collagen I and Collagen IV fluorescence staining enhanced, and the honeycomb-like pore structure in the cross-linked acellular scaffold was more stereoscopic and typical. The structure of glomerular niche was clearer, and the elastic modulus of kidney in crosslinking group was significantly higher than uncrosslinking group.　Conclusion　Genipin crosslinking rat renal decellularized scaffold can greatly improve the biological properties of the scaffold and lay a foundation for cell implantation and organ regeneration.