医用小型猪骨加工工艺与免疫原性的检测与分析

The Machining Process of Medical Minipig-bone and the Detection and Analysis of Immunogenicity

目的制备医用小型猪骨的浸出性异种蛋白,检测和分析其皮质和松质混合骨(以下简称混合骨)与单纯皮质骨(以下简称皮质骨)浸出性异种蛋白刺激和诱导人淋巴细胞增殖的应答反应。方法应用化学和物理学方法制备获得医用小型猪混合骨与皮质骨粉,以定量RPMI-1640搅拌和浸泡4℃72h后高速离心获取上清液且进行蛋白定量检测;随即应用CCK-8方法检测和分析混合骨与皮质骨浸出性异种蛋白刺激和诱导人外周血淋巴细胞增殖的差异性;以及人外周血淋巴细胞增殖培养上清中四种细胞因子表达的异同。结果 CCK-8实验结果表明:CCK-8实验未见混合骨与皮质骨浸出性异种蛋白具有刺激和诱导人淋巴细胞增殖的应答反应的统计学差异;而应用ELISA酶联免疫吸附实验对人淋巴细胞培养上清中IFN-r、IL-1、IL-10和TNF-a的检测结果提示:与对照组相比,经工艺处理后小型猪骨组的四种炎症相关因子均较处理前组具有统计学的显著性差异(P<0.05),而皮质骨组较混合骨组其炎症相关因子的表达同样具有统计学差异。结论本实验结果提示,应用已建立的小型猪骨加工工艺可有效处理和抑制异种蛋白对人淋巴细胞增殖的刺激和诱导作用,表明小型猪混合骨与皮质骨其异种蛋白的抗原性存在一定的差异;本小型猪骨加工工艺和免疫原性的检测和分析系统的建立为医用性异种骨的使用提供了理论与实验数据的参考。

Objective To prepare the heterogeneous protein of medical minipig-bone,and detect and analyze the lymphocyte proliferation responses stimulated and inducted by the heterologous protein of cortical and cancellous mixed bone（hereinafter referred to as mixed bone）and cortical bone alone（hereinafter referred to as cortical bone）.Methods Medical minipig mixed bone and cortical bone meal were prepared by chemical and physical methods,stirred them with quantitative RPMI-1640 and soaked for 72 hours in 4℃,then supernatant was obtained by high-speed centrifugation followed by protein quantitative detection.Immediately the lymphocyte proliferation responses of human peripheral blood stimulated and induced by mixed and cortical bone heterogeneous protein were detected by CCK-8 kit,as well as the four kinds of cytokine expression in cultured serum.Results CCK-8 results showed that：the lymphocyte proliferation responses stimulated and induced by mixed bone and cortical bone had no statistical difference.Whereas ELISA（enzyme-linked immunosorbent assay） for IFN-r,IL-1,IL-10 and TNF-a in human lymphocyte culture supernatant results showed that：compared with the control group,these four inflammation-related factors of minipig-bone treated group showed statistically significant difference after treatment（P 0.05）;meanwhile compared with mixed bone group,the expression of inflammation-related factors of cortical bone group also had significant difference.Conclusion The results suggest that the application minipig-bone machining process which has been established can effectively handle and inhibit the role of heterologous proteins in stimulating and inducing lymphocyte proliferation response,and the heterogeneous protein antigenicity of minipig mixed bone and cortical bone have some differences.The establishment of the minipig-bone machining process and immunogenicity detection and analysis system can be a reference of theoretical and experimental data for the use of medical xenograft bone.