本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：RNAi技術(shù)　出處：《遼寧醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 探討應(yīng)用RNA干擾(RNA interference, RNAi)技術(shù)沉默HLA-A2表達(dá)的人骨髓間充質(zhì)干細(xì)胞(human bone marrow derived mesenchymal stem cells, hBMSCs)對(duì)人異體T淋巴細(xì)胞分泌功能的調(diào)節(jié)作用及誘導(dǎo)成骨能力的影響。為進(jìn)一步研究應(yīng)用RNAi技術(shù)降低人骨髓間充質(zhì)干細(xì)胞免疫原性后修復(fù)骨缺損提供實(shí)驗(yàn)依據(jù)。 方法 從成人骨髓中分離和培養(yǎng)hBMSCs,并且通過(guò)免疫細(xì)胞化學(xué)法檢測(cè)其表面標(biāo)記。利用人工合成的HLA-A2靶向小分子干擾RNA(small interference RNA, siRNA)轉(zhuǎn)染至第3代hBMSCs,然后將轉(zhuǎn)染前后各組的hBMSCs,與經(jīng)植物血凝素(phytohaemagglutinin,PHA)刺激的人異體T淋巴細(xì)胞共同培養(yǎng),72h后用ELISA分別檢測(cè)各培養(yǎng)組上清液中干擾素(interferon, IFN)γ、白細(xì)胞介素(interleuKin, IL)2的表達(dá)量;轉(zhuǎn)染siRNA后的hBMSCs為實(shí)驗(yàn)組,未轉(zhuǎn)染的hBMSCs為對(duì)照組,免疫細(xì)胞化學(xué)染色、Western Blot法檢測(cè)兩組細(xì)胞HLA-A2的表達(dá);對(duì)兩組細(xì)胞進(jìn)行成骨誘導(dǎo)分化培養(yǎng),用含成骨誘導(dǎo)劑的DMEM/F12(低糖)培養(yǎng)液(含0.1μmol/L地塞米松、10mmol/Lβ-甘油磷酸鈉、50mg/L維生素C)誘導(dǎo)分化,觀察細(xì)胞形態(tài)變化;堿性磷酸酶染色、計(jì)數(shù)和Von-Kossa染色、計(jì)數(shù)檢測(cè)成骨能力。 結(jié)果 原代培養(yǎng)3天后,約70%hBMSCs貼壁生長(zhǎng);7天后細(xì)胞迅速增殖,進(jìn)入對(duì)數(shù)生長(zhǎng)期;14天左右進(jìn)入平臺(tái)期。第3代細(xì)胞多呈梭形生長(zhǎng),形態(tài)比較均一,hBMSCs表面抗原CD44、CD166呈陽(yáng)性。免疫細(xì)胞化學(xué)染色顯示,轉(zhuǎn)染前hBMSCs的HLA-A2表達(dá)為陽(yáng)性,轉(zhuǎn)染后hBMSCs的HLA-A2表達(dá)為弱陽(yáng)性;Western Blot法檢測(cè)顯示,轉(zhuǎn)染前hBMSCs的HLA-A2的蛋白表達(dá)量明顯高于轉(zhuǎn)染后的HLA-A2的蛋白表達(dá)量,前后比較有顯著性差異(p0.05)。轉(zhuǎn)染前后的hBMSCs均能抑制人T淋巴細(xì)胞分泌IFN-γ、IL-2,轉(zhuǎn)染前后比較有顯著性差異(p0.05)。轉(zhuǎn)染后的hBMSCs可見(jiàn)少量細(xì)胞死亡,生長(zhǎng)速度變緩,一周后細(xì)胞間融合生長(zhǎng)。ALP染色顯示,轉(zhuǎn)染前后的hBMSCs均出現(xiàn)胞漿呈棕黑色的陽(yáng)性反應(yīng);ALP活性檢測(cè)結(jié)果顯示,兩組之間無(wú)顯著性差異。Von-Kossa染色顯示,轉(zhuǎn)染前后的hBMSCs均有鈣結(jié)節(jié)形成,經(jīng)計(jì)數(shù)統(tǒng)計(jì)兩組之間無(wú)顯著性差異。 結(jié)論 應(yīng)用RNAi技術(shù)沉默HLA-A2表達(dá)的hBMSCs可以抑制PHA刺激人T淋巴細(xì)胞分泌因子IFN-γ、IL-2的表達(dá)量,轉(zhuǎn)染后的hBMSCs比未轉(zhuǎn)染的hBMSCs抑制作用更強(qiáng)。應(yīng)用RNAi技術(shù)沉默HLA-A2表達(dá)的hBMSCs,不影響其成骨能力。
[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) 緯, 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- 緯 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- 緯 -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.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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本文編號(hào)：1559821