本文關(guān)鍵詞：不同誘導(dǎo)方法所得Treg樣細胞的多方面比較　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CD4+CD25+Treg細胞 FOXP3 TGF-β 免疫耐受

【摘要】： 目的 建立MSCV-FOXP3轉(zhuǎn)導(dǎo)法和TGF-β誘導(dǎo)法獲取調(diào)節(jié)性T淋巴細胞(regulatory T cells, Treg)樣細胞的方法體系,比較兩種方法獲得Treg樣細胞的產(chǎn)率及其免疫抑制功能的差異,并初步探討兩種Treg樣細胞發(fā)揮免疫抑制作用相關(guān)機制的差異。 方法 1.構(gòu)建重組逆轉(zhuǎn)錄病毒質(zhì)粒pMSCV-FOXP3,轉(zhuǎn)染PT67細胞,建立可穩(wěn)定產(chǎn)毒單克隆細胞株；采用差速離心法對病毒進行濃縮。 2.免疫磁珠分選法獲取CD4+CD25-T淋巴細胞；以CD4+CD25-T淋巴細胞為研究對象,采用優(yōu)化后實驗方法進行MSCV-FOXP3轉(zhuǎn)導(dǎo)和TGF-β誘導(dǎo)獲取Treg樣細胞,以流式細胞術(shù)和Real time PCR技術(shù)對兩種方法獲得Treg樣細胞的效率進行檢測和比較。 3.應(yīng)用活性染料CFSE染色和流式細胞術(shù)觀察兩種Treg樣細胞和天然Treg細胞對自體CD4+T淋巴細胞增殖活性的影響,用ModFit軟件分析CD4+T淋巴細胞增殖動力學(xué)模型。 4.應(yīng)用ELISA技術(shù),檢測兩種Treg樣細胞培養(yǎng)上清IL-10和TGF-β的分泌水平；應(yīng)用Real time PCR技術(shù)檢測兩種Treg樣細胞顆粒酶A和顆粒酶B mRNA的轉(zhuǎn)錄水平差異。 結(jié)果 1.成功構(gòu)建pMSCV-FOXP3重組質(zhì)粒,轉(zhuǎn)染PT67細胞,篩選建立了穩(wěn)定產(chǎn)毒單克隆細胞株,最高病毒滴度為6×105cfu/ml。濃縮后病毒滴度可達1.2×107cfu／ml。 2.免疫磁珠分離獲得CD4+CD25-T淋巴細胞,流式細胞術(shù)檢測其純度為92.46%,CD4+CD25+T淋巴細胞純度為90.82%,臺盼藍染色檢測細胞存活率高于90%。當(dāng)病毒滴度為1×107cfu/ml時,誘導(dǎo)獲得的Treg樣細胞產(chǎn)率最高,可達49.12%；當(dāng)TGF-β為5ng/ml,誘導(dǎo)時間為第6天時,誘導(dǎo)獲得的Treg樣細胞產(chǎn)率最高,可達41.08%；MSCV-FOXP3轉(zhuǎn)導(dǎo)組CD4+T淋巴細胞的FOXP3 mRNA表達水平高于TGF-β組(P0.05),MSCV-FOXP3轉(zhuǎn)導(dǎo)組CD4+T淋巴細胞的FOXP3蛋白陽性率高于TGF-β組(P0.05)。 3.流式細胞術(shù)檢測結(jié)果顯示,MSCV-FOXP3轉(zhuǎn)導(dǎo)CD4+T淋巴細胞獲得的Treg樣細胞、TGF-β誘導(dǎo)獲得的Treg樣細胞以及天然型Treg細胞對自體CD4+T淋巴細胞的增殖均具有明顯抑制作用,與對照組相比均具有顯著性差異(P0.05)。 4.MSCV-FOXP3轉(zhuǎn)導(dǎo)CD4+T淋巴細胞獲得的Treg樣細胞與TGF-β誘導(dǎo)獲得的Treg樣細胞分泌IL-10水平明顯上升,與對照組相比均具有顯著性差異(P0.05)。TGF-β誘導(dǎo)組和MSCV-FOXP3轉(zhuǎn)導(dǎo)組TGF-β分泌水平較對照組無明顯差異(P0.05)。顆粒酶B在TGF-β誘導(dǎo)獲得Treg樣細胞表達水平較對照組明顯升高(P0.05)。顆粒酶B在TGF-β誘導(dǎo)獲得Treg樣細胞表達水平較顯著高于MSCV-FOXP3轉(zhuǎn)導(dǎo)組(P0.05)。 結(jié)論 MSCV-FOXP3轉(zhuǎn)導(dǎo)法和TGF-β誘導(dǎo)法均可在體外獲得具有一定免疫抑制功能的Treg樣細胞,但MSCV-FOXP3轉(zhuǎn)導(dǎo)法的Treg樣細胞產(chǎn)率要高于TGF-β誘導(dǎo)法,其獲得的Treg樣細胞免疫抑制活性可能高于TGF-β誘導(dǎo)法。在兩種Treg樣細胞發(fā)揮免疫抑制功能的過程中,IL-10可能均參與其中,而顆粒酶B可能只參與了TGF-β誘導(dǎo)的Treg樣細胞發(fā)揮免疫抑制功能的過程。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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本文編號：1371044