【摘要】：目的:探究系統(tǒng)性紅斑狼瘡(Systemic Lupus Erythematosus,SLE)患者血漿對(duì)健康者骨髓來源的間充質(zhì)干細(xì)胞(Mesenchymal Stem Cells,MSCs)免疫抑制作用的影響。方法:實(shí)驗(yàn)采用添加SLE患者富集血漿或胎牛血清的細(xì)胞培養(yǎng)體系(胎牛血清作為對(duì)照組),在體外共同培養(yǎng)SLE患者來源的B淋巴細(xì)胞與健康者骨髓來源的MSCs,檢測(cè)MSCs對(duì)B淋巴細(xì)胞的增殖能力和分化成熟的影響。采用細(xì)胞增殖試驗(yàn)檢測(cè)分析B淋巴細(xì)胞的增殖能力情況;使用流式細(xì)胞儀檢測(cè)B淋巴細(xì)胞的表面標(biāo)志物,統(tǒng)計(jì)分析B淋巴細(xì)胞的分化成熟情況。實(shí)驗(yàn)數(shù)據(jù)統(tǒng)計(jì)分析:采用SPSS 12.0軟件進(jìn)行學(xué)生t檢驗(yàn)統(tǒng)計(jì)學(xué)分析,P0.05視為兩組差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1、MSCs的分離、擴(kuò)增培養(yǎng)和誘導(dǎo)分化:實(shí)驗(yàn)成功培養(yǎng)獲得健康骨髓捐獻(xiàn)者來源的MSCs;(1)MSCs經(jīng)過流式細(xì)胞儀檢測(cè)結(jié)果顯示,高表達(dá)CD44、CD73、CD90、和CD105,不表達(dá)CD34、CD45、CD14和CD106;(2)通過添加特殊誘導(dǎo)劑誘導(dǎo)培養(yǎng)MSCs后,成功分化為成脂細(xì)胞和成骨細(xì)胞。2、正常MSCs可以抑制SLE患者來源B淋巴細(xì)胞的增殖(脂多糖為增殖刺激分子)。3、SLE患者富集血漿能夠抵制正常MSCs對(duì)SLE患者來源B淋巴細(xì)胞的增殖抑制作用(脂多糖為增殖刺激分子)。4、正常MSCs可以抑制SLE患者來源B淋巴細(xì)胞的成熟:在胎牛血清培養(yǎng)系統(tǒng)中,加入MSC干預(yù)后,SLE患者來源B淋巴細(xì)胞表面CD27與CD38的表達(dá)量下調(diào),CD19的表達(dá)水平無明顯影響。5、SLE患者富集血漿能夠抵制正常MSCs對(duì)SLE患者來源B淋巴細(xì)胞成熟的抑制作用:在SLE患者富集血漿培養(yǎng)系統(tǒng)中,加入MSCs干預(yù)后,SLE患者來源B淋巴細(xì)胞表面CD27與CD38的表達(dá)量明顯上調(diào),CD19的表達(dá)水平受到抑制作用。結(jié)論:正常MSCs可以抑制脂多糖刺激的SLE患者B淋巴細(xì)胞的增殖與成熟,調(diào)節(jié)B淋巴細(xì)胞亞群所占比例。而SLE患者富集血漿卻能夠抵制正常MSCs對(duì)B淋巴細(xì)胞的免疫抑制作用,這將負(fù)向干擾正常MSCs移植對(duì)SLE的治療效果。
[Abstract]:Objective: to investigate the effect of plasma on bone marrow derived mesenchymal stem cells (Mesenchymal Stem Cells,MSCs) immunosuppressive effect in patients with systemic lupus erythematosus (Systemic Lupus Erythematosus,SLE). Methods: the cell culture system supplemented with SLE patients to enrich plasma or fetal bovine serum (fetal bovine serum as control group) was used to co-culture B lymphocytes from SLE patients and MSCs, from healthy bone marrow in vitro. The effects of MSCs on the proliferation and differentiation and maturation of B lymphocytes were detected. Cell proliferation test was used to detect the proliferation ability of B lymphocytes, flow cytometry was used to detect the surface markers of B lymphocytes, and the differentiation and maturation of B lymphocytes were statistically analyzed. Statistical analysis of experimental data: SPSS 12.0 software was used to analyze the t test of students, and the difference between the two groups was statistically significant (P 0.05). Results: 1. Isolation, amplification, culture and differentiation of MSCs: MSCs; from healthy bone marrow donors was successfully cultured. The main results were as follows: (1) the results of flow cytometry showed that high expression of CD44,CD73,CD90, and CD105, did not express CD34,CD45,CD14 and CD106;. (2) after MSCs was induced by adding special inducer, it was successfully differentiated into adipocytes and osteoblasts. 2, normal MSCs could inhibit the proliferation of B lymphocytes from SLE patients (lipopolysaccharide was a proliferation stimulator). The enrichment of plasma in SLE patients can resist the inhibitory effect of normal MSCs on the proliferation of B lymphocytes from SLE patients (lipopolysaccharide is a proliferation stimulator). Normal MSCs could inhibit the maturation of B lymphocytes from SLE patients: in fetal bovine serum culture system, the expression of CD27 and CD38 on the surface of B lymphocytes from SLE patients was down-regulated after adding MSC intervention, but the expression level of CD19 was not significantly affected. The enrichment of plasma in SLE patients can resist the inhibitory effect of normal MSCs on the maturation of B lymphocytes from SLE patients: in the enrichment plasma culture system of SLE patients, MSCs intervention was added. The expression of CD27 and CD38 on the surface of B lymphocytes from SLE patients was significantly up-regulated, and the expression level of CD19 was inhibited. Conclusion: normal MSCs can inhibit the proliferation and maturation of B lymphocytes and regulate the proportion of B lymphocytes subsets in SLE patients stimulated by lipopolysaccharide. However, the enrichment of plasma in patients with SLE can resist the immunosuppressive effect of normal MSCs on B lymphocytes, which will negatively interfere with the therapeutic effect of normal MSCs transplantation on SLE.
【學(xué)位授予單位】：貴州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.241

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