不同诱导方法所得Treg样细胞的多方面比较

发布时间：2018-01-02 21:38

本文关键词：不同诱导方法所得Treg样细胞的多方面比较　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

更多相关文章： CD4+CD25+Treg细胞 FOXP3 TGF-β 免疫耐受

【摘要】： 目的建立MSCV-FOXP3转导法和TGF-β诱导法获取调节性T淋巴细胞(regulatory T cells, Treg)样细胞的方法体系,比较两种方法获得Treg样细胞的产率及其免疫抑制功能的差异,并初步探讨两种Treg样细胞发挥免疫抑制作用相关机制的差异。方法 1.构建重组逆转录病毒质粒pMSCV-FOXP3,转染PT67细胞,建立可稳定产毒单克隆细胞株；采用差速离心法对病毒进行浓缩。 2.免疫磁珠分选法获取CD4+CD25-T淋巴细胞；以CD4+CD25-T淋巴细胞为研究对象,采用优化后实验方法进行MSCV-FOXP3转导和TGF-β诱导获取Treg样细胞,以流式细胞术和Real time PCR技术对两种方法获得Treg样细胞的效率进行检测和比较。 3.应用活性染料CFSE染色和流式细胞术观察两种Treg样细胞和天然Treg细胞对自体CD4+T淋巴细胞增殖活性的影响,用ModFit软件分析CD4+T淋巴细胞增殖动力学模型。 4.应用ELISA技术,检测两种Treg样细胞培养上清IL-10和TGF-β的分泌水平；应用Real time PCR技术检测两种Treg样细胞颗粒酶A和颗粒酶B mRNA的转录水平差异。结果 1.成功构建pMSCV-FOXP3重组质粒,转染PT67细胞,筛选建立了稳定产毒单克隆细胞株,最高病毒滴度为6×105cfu/ml。浓缩后病毒滴度可达1.2×107cfu／ml。 2.免疫磁珠分离获得CD4+CD25-T淋巴细胞,流式细胞术检测其纯度为92.46%,CD4+CD25+T淋巴细胞纯度为90.82%,台盼蓝染色检测细胞存活率高于90%。当病毒滴度为1×107cfu/ml时,诱导获得的Treg样细胞产率最高,可达49.12%；当TGF-β为5ng/ml,诱导时间为第6天时,诱导获得的Treg样细胞产率最高,可达41.08%；MSCV-FOXP3转导组CD4+T淋巴细胞的FOXP3 mRNA表达水平高于TGF-β组(P0.05),MSCV-FOXP3转导组CD4+T淋巴细胞的FOXP3蛋白阳性率高于TGF-β组(P0.05)。 3.流式细胞术检测结果显示,MSCV-FOXP3转导CD4+T淋巴细胞获得的Treg样细胞、TGF-β诱导获得的Treg样细胞以及天然型Treg细胞对自体CD4+T淋巴细胞的增殖均具有明显抑制作用,与对照组相比均具有显著性差异(P0.05)。 4.MSCV-FOXP3转导CD4+T淋巴细胞获得的Treg样细胞与TGF-β诱导获得的Treg样细胞分泌IL-10水平明显上升,与对照组相比均具有显著性差异(P0.05)。TGF-β诱导组和MSCV-FOXP3转导组TGF-β分泌水平较对照组无明显差异(P0.05)。颗粒酶B在TGF-β诱导获得Treg样细胞表达水平较对照组明显升高(P0.05)。颗粒酶B在TGF-β诱导获得Treg样细胞表达水平较显著高于MSCV-FOXP3转导组(P0.05)。结论 MSCV-FOXP3转导法和TGF-β诱导法均可在体外获得具有一定免疫抑制功能的Treg样细胞,但MSCV-FOXP3转导法的Treg样细胞产率要高于TGF-β诱导法,其获得的Treg样细胞免疫抑制活性可能高于TGF-β诱导法。在两种Treg样细胞发挥免疫抑制功能的过程中,IL-10可能均参与其中,而颗粒酶B可能只参与了TGF-β诱导的Treg样细胞发挥免疫抑制功能的过程。
[Abstract]:objective
The establishment of MSCV-FOXP3 transduction method and TGF- method to obtain the beta induced regulatory T cells (regulatory T cells, Treg) method system like cells, the difference between the two methods to obtain immune and yield Treg like cell suppression function, and discusses two kinds of Treg like immune cells play differences inhibition mechanism.
Method
1. the recombinant retrovirus plasmid pMSCV-FOXP3 was constructed and transfected into PT67 cells to establish a stable and toxic monoclonal cell line, and the virus was concentrated by differential centrifugation.
2. Macs to obtain CD4+CD25-T lymphocytes; CD4+CD25-T lymphocytes as the research object, using the optimized experimental method for MSCV-FOXP3 transduction and induced by TGF- to obtain Treg like cells by flow cytometry and Real time PCR of the two methods were analyzed and compared for efficiency of Treg like cells.
3. the effects of two Treg like cells and natural Treg cells on the proliferation of autologous CD4+T lymphocytes were observed by CFSE staining and flow cytometry. The proliferation kinetics model of CD4+T lymphocytes was analyzed by ModFit software.
4., ELISA technology was applied to detect the secretion level of IL-10 and TGF- beta in two kinds of Treg like cell culture supernatants. Real time PCR technology was applied to detect the difference of Treg transcript level between two Treg like cells and A granzyme B.
Result
1., pMSCV-FOXP3 recombinant plasmid was successfully constructed, transfected into PT67 cells, and a stable cytotoxic monoclonal cell line was screened. The highest titer of virus was 6 * 105cfu/ml., and the titer of virus reached 1.2 * 107cfu / ml. after concentrated.
2. immunomagnetic separation CD4+CD25-T lymphocyte detection, the purity was 92.46% by flow cytometry, the purity of CD4+CD25+T cells was 90.82%, trypan blue staining to detect cell survival rate was higher than that of 90%. when the virus titer was 1 * 107cfu/ml, obtained by Treg cells showed the highest yield up to 49.12%; when the TGF- beta 5ng/ml, induction time the sixth day, obtained by Treg cells showed the highest yield up to 41.08%; the expression level of MSCV-FOXP3 CD4+T FOXP3 mRNA transduction of lymphocytes is higher than that of TGF- group (P0.05), beta FOXP3 protein positive rate in MSCV-FOXP3 group was higher than that of TGF- transduction CD4+T lymphocyte beta group (P0.05).
3. flow cytometry showed that Treg cells, MSCV-FOXP3 lymphocytes transduced CD4+T, TGF- beta induced proliferation of Treg cells and Treg cells to obtain natural type of autologous CD4+T lymphocytes were significantly inhibited, compared with the control group had significant difference (P0.05).
4.MSCV-FOXP3 transduction CD4+T lymphocytes obtained Treg cells induced by TGF- and IL-10 secretion levels increased obviously from Treg cells, compared with the control group had significant difference (P0.05) induced by.TGF- group and MSCV-FOXP3 group TGF- beta secretion transduction levels than the control group showed no significant difference (P0.05). Granzyme B induced the expression level of Treg cells was significantly increased compared with the control group in the TGF- beta (P0.05). Granzyme B induced Treg cells the expression level was significantly higher than that of MSCV-FOXP3 group in the transduction of TGF- beta (P0.05).
conclusion
Method can obtain Treg like cells with immunosuppressive function in vitro MSCV-FOXP3 transduction method and induced by TGF-, but Treg like cell yield MSCV-FOXP3 transduction was higher than that of TGF- beta induced method, the Treg cells may be higher than the immunosuppressive activity induced by TGF- method. In the two kinds of Treg like cells exert immunosuppressive process the function of IL-10, may be involved, while granzyme B may only be involved in TGF- induced Treg cells play immune suppression function.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】