中国实用口腔科杂志

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宿主T淋巴细胞对以脱落乳牙干细胞为基础的骨组织再生能力影响研究

刘雪梅杨宁陈旭刘尧   

  1. 中国医科大学口腔医学院儿童口腔科,辽宁省口腔疾病重点实验室,辽宁省口腔疾病转化医学研究中心,辽宁  沈阳  110002
  • 出版日期:2018-11-15 发布日期:2018-12-12
  • 基金资助:

    国家自然科学基金青年科学基金(81600825);军事口腔医学国家重点实验室开放课题(2017KA04);2018大学生创新创业训练计划(201810159063)

  • Online:2018-11-15 Published:2018-12-12

摘要:

目的 研究宿主T淋巴细胞对以脱落乳牙干细胞(stem cells from exfoliated deciduous teeth,SHED)为基础的骨组织再生能力的影响以及可能的机制,为揭示宿主免疫微环境在组织再生过程中的作用及机制奠定基础。方法 原代分离培养人SHED和小鼠脾脏来源CD4+ T淋巴细胞。将SHED与羟基磷灰石支架材料(HA/TCP)复合后种植于裸鼠背部皮下。实验组术前2 d,经尾静脉系统性回输小鼠CD4+ T细胞,以回输PBS的裸鼠作为对照组。分别于术后2、5、14 d收取标本,对SHED细胞种植复合物中促炎症细胞因子水平进行检测;术后8周收取标本,制备组织学切片进行HE染色观察。采用 SPSS20.0 软件对两组的检测数据进行统计学分析。结果 原代SHED细胞形态呈梭形,表达间充质干细胞表面标记物CD73、CD90、CD105、CD146,体外诱导培养具有成骨、成脂向分化潜能。免疫磁珠分选CD4+ T淋巴细胞纯度达95.7%。与对照组相比,实验组细胞种植复合物中促炎症细胞因子干扰素-γ(interferon-γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白介素-17(interleukin-17,IL-17)表达水平显著升高(均P < 0.05),其中IFN-γ变化较为明显,在术后5 d表达水平达峰值。细胞种植复合物种植术后8周,实验组SHED种植复合物新生骨组织面积显著降低(P < 0.05)。结论 宿主T淋巴细胞明显抑制以SHED为基础的骨组织再生,且与促炎症细胞因子的分泌水平有关,其中IFN-γ可能在宿主T淋巴细胞抑制骨组织再生中发挥重要作用。

关键词: 宿主T淋巴细胞, 脱落乳牙干细胞, 骨组织再生, 免疫微环境

Abstract:

Objective To clarify the role of recipient T lymphocytes in stem cells from exfoliated deciduous teeth(SHED)-based bone regeneration and further explore the underlying mechanisms,and provide the experimental evidence for revealing the mechanisms of host immune microenvironment in tissue regeneration. Methods Human SHED and mouse CD4+ T lymphocytes were isolated and cultured respectively. SHED mixed with HA/TCP were implanted into immunocompromised mice subcutaneously,and CD4+ T cells were infused intravenously via tail vein 2 days before operation. The mice with PBS infusion were used as control. The samples were harvested 2 days,5 days and 14 days,and the levels of pro-inflammatory cytokines in implantations were examined by ELISA analysis;the samples were collected 8 weeks after operation,and new bone formation were evaluated by HE staining. SPSS 20.0 was used to perform the statistical analysis. Results SHED showed spindle-like morphology,expressed markers of mesenchymal stem cells (CD73,CD90,CD105 and CD146),and possessed multilineage differentiation potential. The purity of CD4+ T cells was up to 95.7%. Compared with the control group,the levels of IFN-γ,TNF-α and IL-17 in the implantation were significantly increased in experiment group (P < 0.05). Furthermore,the IFN-γ level was dramatically increased and reached the peak 5 days after operation. At 8 weeks after operation,the area of new bone formation of the implantation was remarkably decreased in experiment group (P < 0.05). Conclusion T lymphocytes inhibit SHED-based bone regeneration,which is related to the secretion of proinflammatory factor,and IFN-γ plays an important role in this process.

Key words: recipient T lymphocytes;stem cells from exfoliated deciduous teeth, SHED;bone regeneration;immuno-environment