PTD-mFoxp3抑制小鼠皮膚移植排斥反應(yīng)的研究
發(fā)布時(shí)間：2019-04-08 19:55
【摘要】：器官移植是治療終末期器官疾病的重要方法之一,但移植后的排斥反應(yīng)仍是影響移植物存活的關(guān)鍵問題。誘導(dǎo)受者產(chǎn)生針對(duì)供者器官的特異性免疫耐受,成為當(dāng)前器官移植研究的重點(diǎn)。CD4~+CD25~(hi)調(diào)節(jié)性T細(xì)胞(regulatory Tcells,Treg)在器官移植中能夠誘導(dǎo)器官特異性免疫耐受,在器官移植領(lǐng)域引起了廣泛的關(guān)注。但要獲得足量的Treg用于臨床治療仍然十分困難。Foxp3在Treg的發(fā)生、發(fā)育及其生物學(xué)功能中起著關(guān)鍵作用,成為Treg細(xì)胞的相對(duì)特異性標(biāo)志。轉(zhuǎn)染了外源性鼠Foxp3(mFoxp3)或人FOXP3(hFOXP3)的CD4~+CD25~-非Treg細(xì)胞可表現(xiàn)出類似Treg細(xì)胞樣的表型或抑制Jurkat T細(xì)胞增殖功能。 HIV-TAT中的蛋白轉(zhuǎn)導(dǎo)結(jié)構(gòu)域(protein transduction domain,PTD)是最常用的蛋白轉(zhuǎn)導(dǎo)肽之一,該功能區(qū)域能將與其融合的蛋白跨膜導(dǎo)入幾乎所有的組織和細(xì)胞中,被其導(dǎo)入細(xì)胞的融合蛋白仍然保持其原有的生物學(xué)活性。 目的:研究基因工程表達(dá)的帶有蛋白穿膜結(jié)構(gòu)域的小鼠Foxp3融合蛋白(fusin protein of transduction domain combining with mouse box P3,PTD-mFoxp3),抑制活化的小鼠脾淋巴細(xì)胞增殖和延長(zhǎng)同種異基因小鼠皮膚移植物生存時(shí)間的可行性。 方法: 1.融合蛋白的表達(dá):在大腸桿菌Rosetta(DE3)中表達(dá)PTD-mFoxp3,經(jīng)Ni~(2+)分離柱純化PTD-mFoxp3。 2.Western-blot分析PTD-mFoxp3在細(xì)胞中的定位:融合蛋白穿膜進(jìn)入小鼠T淋巴細(xì)胞瘤株EL-4細(xì)胞中的能力。 3.淋巴細(xì)胞增殖試驗(yàn)檢測(cè)PTD-mFoxp3對(duì)活化的小鼠脾淋巴細(xì)胞增殖反應(yīng)的影響:取正常C57BL/6小鼠脾淋巴細(xì)胞,在體外給予刀豆蛋白A(ConA)刺激,并分別給予不同濃度的PTD-mFoxp3、環(huán)孢素A(cyclosporine,CsA)、mFoxp3、PTD-GFP、生理鹽水處理,48 h后通過酶標(biāo)儀檢測(cè)細(xì)胞的增殖程度。 4.同種異基因小鼠皮膚移植試驗(yàn)觀察PTD-mFoxp3對(duì)移植物存活時(shí)間的影響:建立從Balb/c小鼠移植到C57BL/6小鼠的皮膚移植模型,各組分別以PTD-mFoxp3、CsA、生理鹽水(NS)、PTD-GFP于手術(shù)當(dāng)天開始連續(xù)腹腔注射8天為干預(yù)手段。術(shù)后第9天各組隨機(jī)取兩只移植小鼠,采集皮片標(biāo)本,進(jìn)行組織學(xué)檢查。各組其余的8只小鼠用于觀察移植皮片的存活時(shí)間。 結(jié)果: 1.成功表達(dá)并純化了PTD-mFoxp3。 2.通過Western-blot分析證實(shí)PTD-mFoxp3能夠穿入EL-4細(xì)胞并主要出現(xiàn)在細(xì)胞核。 3.在淋巴細(xì)胞增殖反應(yīng)檢測(cè)中,PTD-mFoxp3各濃度組(320、640、1280nM)和CsA組(濃度1μg/ml)相似,與各對(duì)照組相比ConA活化的小鼠脾淋巴細(xì)胞增殖指數(shù)明顯降低(P＜0.05)。 4.器官移植后第9天移植皮片病理學(xué)檢查顯示:NS組和PTD-GFP組皮膚移植物中可見大量淋巴細(xì)胞浸潤(rùn)。PTD-mFoxp3組、CsA組移植物中淋巴細(xì)胞浸潤(rùn)明顯輕于NS組、PTD-GFP組。 5.各組皮膚移植物存活時(shí)間:PTD-mFoxp3組為13.6±1.50天,CsA組為14.2±1.28天,NS組為10.0±1.07天,PTD-GFP組為10.5±1.31天。經(jīng)單因素方差分析表明四組之間有顯著性差異。兩兩比較,PTD-mFoxp3組和CsA組移植物的存活時(shí)間較NS組和PTD-GFP組顯著延長(zhǎng)(P＜0.05)。 結(jié)論: 1 PTD-mFoxp3能抑制活化的小鼠脾淋巴細(xì)胞的增殖。 2 PTD-mFoxp3具有延長(zhǎng)同種異基因小鼠皮膚移植物存活時(shí)間,減輕移植物周圍炎癥反應(yīng)的能力。
[Abstract]:Organ transplantation is one of the most important methods for the treatment of end-stage organ disease. Induction of the recipient's specific immune tolerance to the donor organ is the focus of the present study on organ transplantation. CD4 ~ + CD25 ~ (hi) regulatory T cells (Treg) can induce organ-specific immune tolerance in organ transplantation. However, it is still difficult to obtain a sufficient amount of Treg for clinical treatment. Foxp3 plays a key role in the genesis, development and biological function of Treg, and becomes the relative specific marker of Treg cells. CD4 ~ + CD25 ~-non-Treg cells transfected with exogenous mouse Foxp3 (mFoxp3) or human FOXP3 (hFOXP3) can show a similar Treg cell-like phenotype or inhibit the proliferation of Jurkat T cells. The protein transduction domain (PTD) in the HIV-TAT is one of the most commonly used protein transduction peptides, which can introduce the protein fused with it into almost all tissues and cells, and the fusion protein that is introduced into the cell still retains its original biological activity. Objective: To study the expression of gene-engineered mouse Foxp3 fusion protein with protein-penetrating domain (PTD-mFoxp3), to inhibit the proliferation of the activated mouse splenocytes and to prolong the survival time of the allogenic mouse skin graft. feasibility Methods:1. Expression of fusion protein: PTD-mFoxp3 was expressed in E. coli Rosetta (DE3) and PTD was purified by Ni ~ (2 +) separation column. -mFoxp3.2. Western-blot analysis of the localization of PTD-mFoxp3 in the cells: the fusion protein penetrates into the mouse T-lymphocyte tumor strain E 3. The effect of PTD-mFoxp3 on the proliferation of activated mouse splenocytes: A normal C57BL/6 mouse spleen cell was used to stimulate the proliferation of splenocytes in mice with normal C57BL/6 mice, and the different concentrations of PTD-mFoxp3, ciclosporine, CsA and mFo were given respectively. xp3, PTD-GFP, physiological saline treatment, through enzyme after 48 h The effect of PTD-mFoxp3 on the survival time of the graft was observed by the skin graft test of the allogenic mouse. The model of skin transplantation from Balb/ c mice to C57BL/6 mice was established, and the groups were treated with PTD-mFoxp3, CsA, NS, and PTD-GFP at the same day of the operation. The eight-day intervention method was used for continuous abdominal cavity injection. Two transplanted mice were randomly divided into two groups on the 9th day after operation. The specimens were collected for histological examination. The remaining 8 mice in each group to view The survival time of the graft skin was observed. PTD-mFoxp3 was expressed and purified by Western-blot analysis. In the detection of lymphocyte proliferation, the concentration groups of PTD-mFoxp3 (320,640,1280 nM) and CsA (concentration 1. mu.g/ ml) were similar to the control group, and ConA activated mice The proliferation index of splenic lymphocytes was significantly lower (P <0.05). Large number of lymphocytes infiltrates in the skin grafts of the TD-GFP group. The PTD-mFoxp3 group, the CsA group graft, The survival time of the skin grafts in each group was from 13.6 to 1.50 days in the PTD-mFoxp3 group, 14.2 to 1.28 days in the CsA group and 10.0 in the NS group. 1.07 days, the PTD-GFP group was 10.5%1 ...................................................................................................... NS The group and the PTD-GFP group were significantly prolonged (P <0.05). 1PTD-mFoxp3 could inhibit the proliferation of activated mouse spleen lymphocytes.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.4
本文編號(hào)：2454863
[Abstract]:Organ transplantation is one of the most important methods for the treatment of end-stage organ disease. Induction of the recipient's specific immune tolerance to the donor organ is the focus of the present study on organ transplantation. CD4 ~ + CD25 ~ (hi) regulatory T cells (Treg) can induce organ-specific immune tolerance in organ transplantation. However, it is still difficult to obtain a sufficient amount of Treg for clinical treatment. Foxp3 plays a key role in the genesis, development and biological function of Treg, and becomes the relative specific marker of Treg cells. CD4 ~ + CD25 ~-non-Treg cells transfected with exogenous mouse Foxp3 (mFoxp3) or human FOXP3 (hFOXP3) can show a similar Treg cell-like phenotype or inhibit the proliferation of Jurkat T cells. The protein transduction domain (PTD) in the HIV-TAT is one of the most commonly used protein transduction peptides, which can introduce the protein fused with it into almost all tissues and cells, and the fusion protein that is introduced into the cell still retains its original biological activity. Objective: To study the expression of gene-engineered mouse Foxp3 fusion protein with protein-penetrating domain (PTD-mFoxp3), to inhibit the proliferation of the activated mouse splenocytes and to prolong the survival time of the allogenic mouse skin graft. feasibility Methods:1. Expression of fusion protein: PTD-mFoxp3 was expressed in E. coli Rosetta (DE3) and PTD was purified by Ni ~ (2 +) separation column. -mFoxp3.2. Western-blot analysis of the localization of PTD-mFoxp3 in the cells: the fusion protein penetrates into the mouse T-lymphocyte tumor strain E 3. The effect of PTD-mFoxp3 on the proliferation of activated mouse splenocytes: A normal C57BL/6 mouse spleen cell was used to stimulate the proliferation of splenocytes in mice with normal C57BL/6 mice, and the different concentrations of PTD-mFoxp3, ciclosporine, CsA and mFo were given respectively. xp3, PTD-GFP, physiological saline treatment, through enzyme after 48 h The effect of PTD-mFoxp3 on the survival time of the graft was observed by the skin graft test of the allogenic mouse. The model of skin transplantation from Balb/ c mice to C57BL/6 mice was established, and the groups were treated with PTD-mFoxp3, CsA, NS, and PTD-GFP at the same day of the operation. The eight-day intervention method was used for continuous abdominal cavity injection. Two transplanted mice were randomly divided into two groups on the 9th day after operation. The specimens were collected for histological examination. The remaining 8 mice in each group to view The survival time of the graft skin was observed. PTD-mFoxp3 was expressed and purified by Western-blot analysis. In the detection of lymphocyte proliferation, the concentration groups of PTD-mFoxp3 (320,640,1280 nM) and CsA (concentration 1. mu.g/ ml) were similar to the control group, and ConA activated mice The proliferation index of splenic lymphocytes was significantly lower (P <0.05). Large number of lymphocytes infiltrates in the skin grafts of the TD-GFP group. The PTD-mFoxp3 group, the CsA group graft, The survival time of the skin grafts in each group was from 13.6 to 1.50 days in the PTD-mFoxp3 group, 14.2 to 1.28 days in the CsA group and 10.0 in the NS group. 1.07 days, the PTD-GFP group was 10.5%1 ...................................................................................................... NS The group and the PTD-GFP group were significantly prolonged (P <0.05). 1PTD-mFoxp3 could inhibit the proliferation of activated mouse spleen lymphocytes.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.4
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