异种皮质骨粉与淋巴细胞体外共培养后的细胞增殖

背景：异种骨材料在临床上的使用受到限制，主要是其较强的免疫原性，但目前对于异种骨材料的免疫原性的评价多是以临床观察，或是用组织病理观察来间接测定的，没有量化的指标，也没有行业的“金标准”。目的：在人外刷血淋巴细胞与骨材料共培养情况下，观察淋巴细胞体外活化和增殖水平的改变，为下。步的特异性免疫原性实验做准备。方法：以羟基磷灰石2g／mL为阴性埘照，以植物血凝索为阳性对照，CCK-8法枪测0．5，1，2g／mL实验皮质骨粉（经过脱脂、脱细胞、多方位除抗原等处理）、1g／mL消毒皮质骨骨粉等骨材料列人淋巴细胞增碴的影响：流式细胞仪检测CD69标记后的骨材料对淋巴绌胞体外活化的影响。结果与结论：0．5，1，2g／mL实验皮质骨粉组淋巴细胞增殖水平与羟幕磷灰石组相比差异无显著性意义（P〉0．05），而消毒骨材料组、植物血凝素阳性对照组淋巴细胞增殖水平较羟基磷灰石组明显升高（P〈0．05，0．01）。羟基磷灰石组的淋巴细胞CD69表达牢很低，表明T细胞处于静息状念：0．5，1，2g／mL实验皮质骨粉组淋巴细胞CD69表达葺夏与羟基磷灰石组相比差异无显著性意义（P〉0．05）；而与消毒骨共培养的淋巴细胞其CD69表达明显上调：植物血凝素阳性对照组淋巴细胞CD69表达最为明显。提示经过处理的实验骨粉未出现对淋巴细胞刺激的免疫原性作用。

Proliferation of lymphocytes co-cultured with xenograft cortical bone meal in vitro

BACKGROUND： Xenograft bone materials have been limited in clinical application, mainly due to their strong immunogenicity, but immunogenicity evaluation on the xenograft bone materials is based on the clinical observation or through indirect determination by histopathological observation. Up to now, there is no quantitative indicator or industry ＂gold standard＂. OBJECTIVE： To observe the in vitro activation and proliferation of peripheral blood lymphocytes during co-culturing with bone material and to prepare for the next specific immunogenicity experiments. METHODS： The hydroxyapatite （2 g/mL） was selected as negative control, the phytohemagglutinin was considered as positive control. The CCK-8 method was used to detect the effect of 0.5, 1 and 2 g/mL cortical bone meal （treated with degreasing, acellular and multi-faceted antigen processing） and 1 g/mL disinfected cortical bone meal on the proliferation of lymphocytes; flow cytometry was used to detect the effect of CD69 labeled bone materials on the in vitro activation of lymphocytes. RESULTS AND CONCLUSION： There was no significant difference of proliferation of lymphocytes between 0.5, 1 and 2 g/mL cortical bone meal groups and hydroxyapatite group （P 〉 0.05）, while the proliferation of lymphocytes in disinfected cortical bone meal group and phytohemagglutinin positive group was higher than that in the hydroxyapatite group （P 〈 0.05, 0.01 ）. The low expression rate of CD69 of lymphocytes in the hydroxyapatite group indicated that the T cells were in the static status; there was no significant difference in expression rate of CD69 of lymphocytes between 0.5, 1 and 2 g/mL cortical bone meal groups and hydroxyapatite group （P 〉 0.05）; but the expression rate of CD69 of lymphocytes co-cultured with disinfected cortical bone meal was significantly increased; the expression of CD69 of lymphocytes in the phytohemagglutinin positive group was most obvious. The processed bone meal has no immunogenic effect on lymphocytes.