HLA-A2表达沉默的hBMSCs对人异体T淋巴细胞分泌功能及其诱导成骨能力的影响

发布时间：2018-03-03 06:06

  本文选题：骨髓间充质干细胞　切入点：RNAi技术　出处：《辽宁医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的 探讨应用RNA干扰(RNA interference, RNAi)技术沉默HLA-A2表达的人骨髓间充质干细胞(human bone marrow derived mesenchymal stem cells, hBMSCs)对人异体T淋巴细胞分泌功能的调节作用及诱导成骨能力的影响。为进一步研究应用RNAi技术降低人骨髓间充质干细胞免疫原性后修复骨缺损提供实验依据。 方法 从成人骨髓中分离和培养hBMSCs,并且通过免疫细胞化学法检测其表面标记。利用人工合成的HLA-A2靶向小分子干扰RNA(small interference RNA, siRNA)转染至第3代hBMSCs,然后将转染前后各组的hBMSCs,与经植物血凝素(phytohaemagglutinin,PHA)刺激的人异体T淋巴细胞共同培养,72h后用ELISA分别检测各培养组上清液中干扰素(interferon, IFN)γ、白细胞介素(interleuKin, IL)2的表达量;转染siRNA后的hBMSCs为实验组,未转染的hBMSCs为对照组,免疫细胞化学染色、Western Blot法检测两组细胞HLA-A2的表达;对两组细胞进行成骨诱导分化培养,用含成骨诱导剂的DMEM/F12(低糖)培养液(含0.1μmol/L地塞米松、10mmol/Lβ-甘油磷酸钠、50mg/L维生素C)诱导分化,观察细胞形态变化;碱性磷酸酶染色、计数和Von-Kossa染色、计数检测成骨能力。 结果 原代培养3天后,约70%hBMSCs贴壁生长;7天后细胞迅速增殖,进入对数生长期;14天左右进入平台期。第3代细胞多呈梭形生长,形态比较均一,hBMSCs表面抗原CD44、CD166呈阳性。免疫细胞化学染色显示,转染前hBMSCs的HLA-A2表达为阳性,转染后hBMSCs的HLA-A2表达为弱阳性;Western Blot法检测显示,转染前hBMSCs的HLA-A2的蛋白表达量明显高于转染后的HLA-A2的蛋白表达量,前后比较有显著性差异(p0.05)。转染前后的hBMSCs均能抑制人T淋巴细胞分泌IFN-γ、IL-2,转染前后比较有显著性差异(p0.05)。转染后的hBMSCs可见少量细胞死亡,生长速度变缓,一周后细胞间融合生长。ALP染色显示,转染前后的hBMSCs均出现胞浆呈棕黑色的阳性反应;ALP活性检测结果显示,两组之间无显著性差异。Von-Kossa染色显示,转染前后的hBMSCs均有钙结节形成,经计数统计两组之间无显著性差异。 结论 应用RNAi技术沉默HLA-A2表达的hBMSCs可以抑制PHA刺激人T淋巴细胞分泌因子IFN-γ、IL-2的表达量,转染后的hBMSCs比未转染的hBMSCs抑制作用更强。应用RNAi技术沉默HLA-A2表达的hBMSCs,不影响其成骨能力。
[Abstract]:Purpose. To investigate the effect of human bone marrow derived mesenchymal stem cells (hBMSCs) silencing the expression of human bone marrow derived mesenchymal stem cells (hBMSCs) by RNA interference RNAi on the secretion function of human T lymphocytes and the osteogenic induction ability of human bone marrow mesenchymal stem cells (hBMSCs). RNAi technique is used to reduce the immunogenicity of human bone marrow mesenchymal stem cells and repair bone defect. Method. HBMSCs were isolated and cultured from adult bone marrow, and their surface markers were detected by immunocytochemistry. The hBMSCs were transfected into the 3rd passage hBMSCs by synthetic HLA-A2 targeting small interfering RNA(small interference (siRNAs). The hBMSCs of each group before and after transfection were compared with that of hBMSCs before and after transfection. Human allogeneic T lymphocytes stimulated by phytohaemagglutinin (PHA) were cultured for 72 hours. The expression of interferon interferon (IFN) 纬, interleukin interleukin-interleukin (IL)2) in supernatant of each culture group was detected by ELISA. After transfection of siRNA, hBMSCs was used as experimental group and untransfected hBMSCs as control group. The expression of HLA-A2 was detected by immunocytochemical staining and Western Blot. The differentiation was induced by DMEM / F12 (low glucose) medium (containing 0.1 渭 mol/L dexamethasone 10 mmol / L 尾 -glycerophosphate sodium phosphate 50 mg / L vitamin C), and the cell morphology was observed by alkaline phosphatase staining, counting and Von-Kossa staining. Results. After 3 days of primary culture, after 7 days of adherent growth of 70 hBMSCs, the cells proliferated rapidly, and entered the logarithmic growth phase about 14 days into the platform phase. Immunocytochemical staining showed that the HLA-A2 expression of hBMSCs was positive before transfection, and the HLA-A2 expression of hBMSCs was weakly positive by Western Blot after transfection. The protein expression of HLA-A2 in hBMSCs before transfection was significantly higher than that in HLA-A2 after transfection. Before and after transfection, hBMSCs could inhibit the secretion of IFN- 纬 IL-2 by human T lymphocytes, and there was a significant difference before and after transfection. A small number of hBMSCs cells died and the growth rate slowed down after transfection. One week after transfection, the positive reaction of cytoplasm of hBMSCs was brown and black. The results showed that there was no significant difference between the two groups. Von-Kossa staining showed that calcium nodules were formed in hBMSCs before and after transfection. There was no significant difference between the two groups by counting. Conclusion. HBMSCs silenced by RNAi technique could inhibit the expression of IFN- 纬 -IL-2 stimulated by PHA in human T lymphocytes. The inhibitory effect of hBMSCs transfection was stronger than that of untransfected hBMSCs. RNAi technique could not affect the osteogenic ability of hBMSCs expressed by HLA-A2.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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