中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (43): 7995-7999.doi: 10.3969/j.issn.2095-4344.2012.43.004

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇    下一篇

异种皮质骨粉与淋巴细胞体外共培养后的细胞增殖

谭新宇1，张惠碧2，林茂群3，章 莹1，唐先高4   

  1. 1解放军广州军区广州总医院创伤骨科，广东省广州市 510010
    2广州市黄埔区中医院功能科，广东省广州市 510700
    3广州市天河区红十字会医院骨科，广东省广州市 510600
    4广州冠浩生物科技有限公司，广东省广州市 510000
  • 收稿日期:2012-04-17 修回日期:2012-06-12 出版日期:2012-10-21 发布日期:2012-10-21
  • 通讯作者: 章莹，解放军广州军区广州总医院创伤骨科，广东省广州市 510110 zhangying_doc@yahoo.com.cn
  • 作者简介:谭新宇★，男，1980年生，湖南省益阳市人，汉族，2010年广州医学院毕业，硕士，主治医师，主要从事骨与关节损伤及新型骨材料研究。 TANXY888@126.com

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

Tan Xin-yu1, Zhang Hui-bi2, Lin Mao-qun3, Zhang Ying1, Tang Xian-gao4   

  1. 1Department of Orthopedic Trauma, General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, Guangdong Province, China
    2Department of Function, Traditional Chinese Medicine Hospital of Guangdong Huangpu District, Guangzhou 510700, Guangdong Province, China
    3Department of Orthopedics, the Red Cross Hospital of Tianhe District, Guangzhou 510600, Guangdong Province, China
    4Guangzhou Guanhao Biological Technology Co., Ltd., Guangzhou 510000, Guangdong Province, China
  • Received:2012-04-17 Revised:2012-06-12 Online:2012-10-21 Published:2012-10-21
  • Contact: Zhang Ying, Department of Orthopedic Trauma, General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, Guangdong Province, China zhangying_doc@yahoo.com.cn
  • About author:Tan Xin-yu★, Master, Attending physician, Department of Orthopedic Trauma, General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, Guangdong Province, China TANXY888@126.com

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摘要：

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：在人外周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，为下一步的特异性免疫原性实验做准备。
方法：以羟基磷灰石2 g/mL为阴性对照，以植物血凝素为阳性对照，CCK-8法检测0.5，1，2 g/mL实验皮质骨粉(经过脱脂、脱细胞、多方位除抗原等处理)、1 g/mL消毒皮质骨骨粉等骨材料对人淋巴细胞增殖的影响；流式细胞仪检测CD69标记后的骨材料对淋巴细胞体外活化的影响。
结果与结论：0.5，1，2 g/mL实验皮质骨粉组淋巴细胞增殖水平与羟基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表达率很低，表明T细胞处于静息状态；0.5，1，2 g/mL实验皮质骨粉组淋巴细胞CD69表达率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。

关键词: 淋巴细胞, 体外培养, 细胞增殖, 异种皮质骨, 免疫原性, 脱脂, 脱细胞, 除抗原, 诱导活性修饰, CD69, 生物材料

Abstract:

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.

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