PTD-mFoxp3抑制小鼠皮肤移植排斥反应的研究
发布时间：2019-04-08 19:55
【摘要】：器官移植是治疗终末期器官疾病的重要方法之一,但移植后的排斥反应仍是影响移植物存活的关键问题。诱导受者产生针对供者器官的特异性免疫耐受,成为当前器官移植研究的重点。CD4~+CD25~(hi)调节性T细胞(regulatory Tcells,Treg)在器官移植中能够诱导器官特异性免疫耐受,在器官移植领域引起了广泛的关注。但要获得足量的Treg用于临床治疗仍然十分困难。Foxp3在Treg的发生、发育及其生物学功能中起着关键作用,成为Treg细胞的相对特异性标志。转染了外源性鼠Foxp3(mFoxp3)或人FOXP3(hFOXP3)的CD4~+CD25~-非Treg细胞可表现出类似Treg细胞样的表型或抑制Jurkat T细胞增殖功能。 HIV-TAT中的蛋白转导结构域(protein transduction domain,PTD)是最常用的蛋白转导肽之一,该功能区域能将与其融合的蛋白跨膜导入几乎所有的组织和细胞中,被其导入细胞的融合蛋白仍然保持其原有的生物学活性。 目的:研究基因工程表达的带有蛋白穿膜结构域的小鼠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细胞增殖和延长同种异基因小鼠皮肤移植物生存时间的可行性。 方法: 1.融合蛋白的表达:在大肠杆菌Rosetta(DE3)中表达PTD-mFoxp3,经Ni~(2+)分离柱纯化PTD-mFoxp3。 2.Western-blot分析PTD-mFoxp3在细胞中的定位:融合蛋白穿膜进入小鼠T淋巴细胞瘤株EL-4细胞中的能力。 3.淋巴细胞增殖试验检测PTD-mFoxp3对活化的小鼠脾淋巴细胞增殖反应的影响:取正常C57BL/6小鼠脾淋巴细胞,在体外给予刀豆蛋白A(ConA)刺激,并分别给予不同浓度的PTD-mFoxp3、环孢素A(cyclosporine,CsA)、mFoxp3、PTD-GFP、生理盐水处理,48 h后通过酶标仪检测细胞的增殖程度。 4.同种异基因小鼠皮肤移植试验观察PTD-mFoxp3对移植物存活时间的影响:建立从Balb/c小鼠移植到C57BL/6小鼠的皮肤移植模型,各组分别以PTD-mFoxp3、CsA、生理盐水(NS)、PTD-GFP于手术当天开始连续腹腔注射8天为干预手段。术后第9天各组随机取两只移植小鼠,采集皮片标本,进行组织学检查。各组其余的8只小鼠用于观察移植皮片的存活时间。 结果: 1.成功表达并纯化了PTD-mFoxp3。 2.通过Western-blot分析证实PTD-mFoxp3能够穿入EL-4细胞并主要出现在细胞核。 3.在淋巴细胞增殖反应检测中,PTD-mFoxp3各浓度组(320、640、1280nM)和CsA组(浓度1μg/ml)相似,与各对照组相比ConA活化的小鼠脾淋巴细胞增殖指数明显降低(P＜0.05)。 4.器官移植后第9天移植皮片病理学检查显示:NS组和PTD-GFP组皮肤移植物中可见大量淋巴细胞浸润。PTD-mFoxp3组、CsA组移植物中淋巴细胞浸润明显轻于NS组、PTD-GFP组。 5.各组皮肤移植物存活时间:PTD-mFoxp3组为13.6±1.50天,CsA组为14.2±1.28天,NS组为10.0±1.07天,PTD-GFP组为10.5±1.31天。经单因素方差分析表明四组之间有显著性差异。两两比较,PTD-mFoxp3组和CsA组移植物的存活时间较NS组和PTD-GFP组显著延长(P＜0.05)。 结论: 1 PTD-mFoxp3能抑制活化的小鼠脾淋巴细胞的增殖。 2 PTD-mFoxp3具有延长同种异基因小鼠皮肤移植物存活时间,减轻移植物周围炎症反应的能力。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.4
本文编号：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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.4
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