大鼠尾腱细胞外基质制备方法及其用于肌腱损伤修复的前期研究

Preparation method of extracellular matrix of rat tail tendon and its preliminary study on repair of tendon injury

目的探究大鼠尾腱细胞外基质制备方法及其复合脂肪间充质干细胞构建微组织,为组织工程法修复肌腱损伤所需生物材料提供实验基础。方法取20只150~200 g SD大鼠鼠尾作为实验材料,分别抽取出大鼠尾腱,经PBS彻底清洗后反复冻融3次,然后经过1%SDS于室温下处理24 h,并用大量PBS清洗1周除去细胞残渣,最后^(60)Co消毒备用。对制备的大鼠尾腱细胞外基质进行病理学染色,观察细胞残留情况。同时,检测该来源的生物源性材料残留α-Gal含量。此外,观察制备的大鼠尾腱细胞外基质复合脂肪间充质干细胞对细胞增殖的影响。同时,用CCK-8试剂盒检测该生物材料的细胞毒性。结果大体观察可见,经脱细胞处理过的尾腱体积增大,呈乳白色匀浆状。HE以及DAPI染色结果显示,经脱细胞处理后的尾腱细胞核大量减少,细胞数量显著降低,每个高倍镜视野内细胞残余量不大于5个。脱细胞尾腱残留α-Gal含量与正常尾腱相比有统计学差异,脱细胞尾腱残留DNA极少。复合脂肪间充质干细胞培养7 d相比培养1 d活细胞数量明显增多。CCK-8毒性测试结果显示这种脱细胞大鼠尾腱的生物相容性较好,没有细胞毒性。结论经过脱细胞方法制备的大鼠尾腱细胞外基质可有效除去细胞成分,并且与脂肪间充质干细胞有较好的生物相容性,可作为一种生物材料用于肌腱损伤修复。

Objective To explore the preparation method of extracellular matrix of rat tail tendon, and to construct micro-tissue with adipose mesenchymal stem cells to provide the experimental basis for the repair of tendon injury. Methods 20 SD rats （150 - 200 g） tails were used as experimental materials, the rat＇s tail tendons were extracted, washed thoroughly with PBS, frozen and thawed three times, then treated with 1% SDS at room temperature for 24 h. The cell debris was removed by washing with a large amount of PBS for 1 week, and finally 60Co was disinfected for use. The pathological analysis of the material was carried out to observe the cell debris. Then, its α-Gal content was detected. In addition, the effects of complexed rat tail tendon extracellular matrix with adipose mesenchymal stem cells on cell proliferation were observed. At last, the cytotoxicity of the biomaterials was detected by CCK-8 kit.Results The gross observation showed that the volume of the treated tail tendon was increased, and its shape was milky white homogenate. The results of HE and DAPI staining showed that the nucleus of the treated tail tendon was significantly reduced, the number of cells was decreased markedly, and the cells residue in each high magnification lens was not more than 5. The residual α-Gal content of the treated tail tendon was statistically different from that of the normal tail tendon. The number of living cells significantly increased after 7 days of co-culture. And the results of CCK-8 toxicity test showed that the biocompatibility of this treated tail tendon was good and no cytotoxicity was found. Conclusion The extracellular matrix of rat tail tendon prepared by this method can effectively remove the cell components, and have good biocompatibility with adipose mesenchymal stem cells, and can be used as a biomaterial to repair tendon injury.