Foxp3转染CD4~+CD25~-T细胞诱导大鼠同种异体心脏移植免疫耐受的研究
摘要
背景:心脏移植是治疗终未期心脏病的有效方法,但目前受体必须通过终身使用免疫抑制剂,降低受体的全身的免疫功能,减少或抑制对移植物的免疫排斥反应,从而达到维持移植心脏的功能。但长期使用免疫抑制剂易导致感染和肿瘤的高发,而且各种免疫抑制剂的其他毒副作用影响受体的生存质量。因此,如何诱导免疫耐受,摆脱免疫抑制依赖是目前移植领域面临的主要问题。近十年来的研究表明,调节性T细胞具有主动调控反应性T细胞而使机体产生免疫耐受的能力得到学者的广泛关注。但对于调节性T细胞诱导免疫耐受的机理尚不是十分清楚,且可应用的调节性T细胞的数量过少,而限制了其临床应用。本试验通过构建大鼠Foxp3基因,转染CD4+CD25-T细胞,研究CD4+CD25+调节性T细胞诱导免疫耐受的效果和机理。
第一部分携带Foxp3基因慢病毒载体的构建
目的:克隆Foxp3的cDNA,构建Foxp3-cDNA表达载体。
方法:根据Genebank公布的大鼠Foxp3-cDNA的基因序列,用Trizol从大鼠胸腺中提取总RNA,应用RT-PCR技术扩增Foxp3-cDNA基因,并将其克隆到载体pWPXL-MOD,构建Foxp3-cDNA表达载体,然后转染DH5α感受态细菌。选择阳性克隆菌,行双酶切鉴定和测序验证所获得的cDNA。用慢病毒包装的Foxp3-cDNA的穿梭质粒转染包装细胞293T细胞进行扩增后,使用由四质粒组成的慢病毒转染系统进行基因重组。
结果:双酶切鉴定和测序验证所获得的cDNA为1900bp的Foxp3基因,序列测定结果与GenBank报道的完全一致。
结论:使用RT-PCR法成功构建Foxp3-cDNA表达载体;构建Foxp3基因重组慢病毒。
第二部分用携带目的基因的Foxp3慢病毒转染CD4+CD25-T细胞
目的:用免疫磁珠分离CD4+CD25+T细胞及CD4+CD25-T细胞;使用携带目的基因Foxp3的慢病毒感染CD4+CD25-T细胞。
方法:用免疫磁珠分离CD4+CD25+T细胞及CD4+CD25-T细胞,流式细胞仪鉴定分离细胞的纯度,在感染前24h通过添加抗CD3单抗及抗CD28单抗刺激细胞增殖。用携带Foxp3-cDNA的慢病毒,转染经免疫磁珠分离后扩增的CD4+CD25-T细胞,检测其荧光表达。提取蛋白后,行Western blot检测Foxp3蛋白的表达。
结果:分离纯化的CD4+CD25+T细胞的纯度达95.2%,CD4+CD25-T细胞的纯度为99.1%。病毒感染的CD4+CD25-T细胞荧光显微镜下观察,感染率达80%。
结论:成功地用免疫磁珠分离了CD4+CD25+T细胞及CD4+CD25-T细胞;用携带Foxp3的慢病毒能感染大鼠的CD4+CD25-T细胞,并表达Foxp3蛋白。
第三部分转入Foxp3基因的CD4+CD25-T细胞免疫抑制功能的研究
目的:体外检测转入Foxp3基因的CD4+CD25-T细胞免疫抑制功能。
方法:流式细胞仪鉴定转入Foxp3基因的CD4+CD25-T细胞的CD25的表达率;通过混合淋巴细胞反应、增殖抑制试验及上清液中IL-10和IL-6的检测,推断转基因细胞的功能。
结果:转入Foxp3基因的CD4+CD25-T细胞的CD25阳性表达率为80.2%,在体外单向淋巴细胞混合反应及增殖抑制试验中发现转入Foxp3基因的CD4+CD25-T细胞与CD4+CD25+T细胞具有相似的功能。
结论:转入Foxp3基因的CD4+CD25-T细胞在体外具有抑制效应性T细胞的功能;其表面分子发生了变化,CD25呈阳性表达。
第四部分同种异体大鼠异位心脏移植模型的建立
目的:用改良Ono法进行同种异体大鼠腹腔异位心脏移植,建立动物模型。
方法:以体重约为250g的Wistar大鼠为受体,体重为200-250g的SD大鼠为供体,进行大鼠腹部异位心脏移植手术。行供体的主动脉与受体的腹主动脉行端侧吻合,供体的肺动脉与受体的下腔静脉行端侧吻合。
结果:大鼠腹腔异位心脏移植模型成功率达90.91%,总手术时间约49-71min。
结论:以改良的Ono法进行大鼠腹部心脏移植,可靠易行,成功率高,但需要一定的显微外科基础。
第五部分Foxp3基因转染的CD4+CD25-T细胞对同种异体心脏移植的作用及其机制
目的:研究Foxp3基因转染的CD4+CD25-T细胞对同种异体心脏移植术后免疫耐受的影响及其机制。
方法:64只Wistar大鼠为受体,分为四组,每组16只。第一组为空白对照组(术前注入PBS),第二组为术前注入CD4+CD25+调节性T细胞组,第三组为术前一周每日用CsA灌胃,第四组为转入Foxp3基因CD4+CD25-T细胞组。移植术后每日三次腹部触诊1次观察移植心脏心跳的情况;7日后每组取8只大鼠,麻醉后开腹观察移植心脏的情况,同时采血5ml,取下脾脏及双侧腋下淋巴结;用ELISA法测定血清中的IL-6、IL-10、TGF-β1;用流式细胞仪测定脾脏及双侧腋下淋巴结的淋巴细胞CD4、CD8分子的表达。
结果:移植的心脏第一组和第三组的生存时间明显低于第二组和第四组,第四组与第二组生存时间相比无明显差异;第一组外周血的IL-6显者高于其他组,第二、三、四组间比较无显著性差异;第一组外周血的IL-10显著低于其他组,第二、三、四组间比较无显著性差异;第二、四组外周血的TGF-β1显著高于第一、三组,第二、四组间比较无显著性差异。
结论:转入Foxp3基因CD4+CD25-T细胞具有诱导移植免疫耐受功能与CD4+CD25+T细胞相比差异无显著性。
Background: Heart transplantation has become the preferred theraphy for the end-stage cardiopathic diseases. To depress recipient systematic immunologic function and suppress the immunological rejection to graft, since immunosuppressive agent must be used to maintain the cardiac function of the patients. Nevertheless, clinical transplantation still faces some problems such as lifelong immunosuppression associated with toxicity, opportunistic infection and a high incidence of cancer. So it is a key issue in transplant territory how to induce immune tolerance to take the place of immunosuppressive dependence. In recent 10 years, CD4+CD25+ regulatory T cell has attracted wide attention for its ability of initiative regulation of reactive T cell, and considered as one of effective methods in inducing immune tolerance. The mechanism is not very clear that CD4+CD25+ regulatory T cells induce immune tolerance. But so far there isn’t any report about its clinical application, the most important reason may be its quantity limitation. In this experimental, we constructed Foxp3 Gene, and transferred into CD4+CD25- T cell, then invest the effect and mechanism of immune tolerance induced by CD4+CD25+ regulatory T cell.
PartⅠConstruction of lentiviral vector encoding Foxp3 gene
Objective: To clone the Foxp3 gene and construct the plasmid.
Methods: According to the nucleotide sequence of the rat Foxp3-cDNA announced by Genebank, total RNA was acquired from rat thymus by Trizol, Foxp3-cDNA fragment was amplified using RT-PCR and the PCR product was then ligated into pWPXL-MOD vector to construct the plasmid. The bacterial strain DH5αwas used as the host cell to amplify reconstructed plasmid. The amplified product was lysed to get the supernatant. Double Enzymes EcoRⅠand Bam H1 verified approach and analysising the sequence were used to confirm Foxp3-cDNA. Lentivirus Packaging Kit with four plasmids was used for gene recombination after constructing with Foxp3-cDNA shuttling plasmid which amplified with 293 T cells.
Results: The acquired cDNA is Foxp3 gene with 1900bp. The sequence was comfimed by comparison with the published one.
Conclusion: Foxp3 had been successfully cloned and the plasmid was reconstructed. Foxp3 gene was constructed to recombinate lentivirus.
PartⅡTtransfect CD4+CD25- T cells with Foxp3 gene packaged by lentivirus
Objective: CD4+CD25- T cells and CD4+CD25+ regulatory T cells were isolated by magnetic cell sorting system. Foxp3 gene packaged by lentivirus was uses to transfect CD4+CD25- T cells.
Methods: CD4+CD25- T cells and CD4+CD25+ T cells were isolated by magnetic cell sorting system. The purity was detected by flow cytometry. Anti-CD3 and anti-CD28 were added to stimulate the cell proliferation 24 hours before infection. Foxp3-cDNA shuttling plasmid packaged by lentivirus was used to transfect amplified CD4+CD25- T cells isolated by MACS. GFP was detected by the fluorescence microscope. Protein was extracted for Western blot analysis.
Results: The purity of CD4+CD25+ T cell was 95.2%. The purity of CD4+CD25- T cell was 99.1%. The rate of CD4+CD25- T cell infected by lentivirus was 80%.
Conclusion: CD4+CD25- T cells and CD4+CD25+ T cells were successfully isolated by magnetic cell sorting system and using lentivirus vector with Foxp3 gene to transfecte rat CD4+CD25- T cells.
PartⅢResearch on immune suppression of Foxp3 gene-transferred CD4+CD25- T cells
Objective: Ddetect the immune suppression of Foxp3 gene-transferred CD4+CD25- T cells ex vivo.
Methods: The CD25 expression of Foxp3 gene-transferred CD4+CD25- T cell was detected by flow cytometry; the optical density(OD) of mixed lymphocyte reaction (MLR), lymphproliferation response, the expression of IL-2、IL-6 in supernatant were observed.
Results: The rate of CD25 is 80.2%. The experiment of MLR and lymphproliferation response comfirms that Foxp3 gene-transferred CD4+CD25- T cell and isolated CD4+CD25+ T cell has the similar ability in suppressing the lymphocyte proliferation.
Conclusion: Foxp3 gene-transferred CD4+CD25- T cell can suppress the proliferation of responsive cell. The surface molecule changed with the positive expression of CD25.
PartⅣEstablishment the model of heterotopic heart transplantation with rat
Objective: According to the surgical procedures of improved Ono,s pattern , establish rat model of abdomen heterotopic heart transplantation.
Methods: SD rat weighed about 250g was used as donor and Wistar rat weighed about 200 to 250g was used as recipient to establish rat model of abdomen heterotopic heart transplantation . The ascending artery and pulmonary artery of the donor’s heart was end-to-side anastomosed with abdominal aorta and vena cava inferior of the recipient,s heart respectively.
Results: The mean operational time was 58.4±21.1min.The successful rate for rat cardiac transplantation was 90.91%.
PartⅤThe effect and of Foxp3 gene-transferred CD4+CD25- T cell on rat cardiac allografts and the mechanism behind
Objective: To investigate the effect of Foxp3 gene-transferred CD4+CD25- T cell on acute rejection after transplantation to rat cardiac allografts and mechanism.
Methods: 64 recipients (Wistar rat) were divided randomly into four groups. Every group contain 16 rats. Group 1: 1 ml PBS was injected before transplantation. Group 2: 1x 106 fresh CD4+CD25+ regulatory T cells were injected intravenously into the vein of each Wistar rat before transplantation. Group 3: CSA was used for intragastric administration one week beore transplantation. Group 4: 1x 106 fresh CD4+CD25+ regulatory T cells were injected intravenously before transplantation. Abdominal touch was carried out 3 times one day to observe cardiopalmus. 8 Wistar rats from every group were operated to observe the abdominal graft condition after anesthesia, simultaneously 5 ml blood、spleen and oxter lymph nodes were collectted. The level of IL-6 and TGF-β1 of peripheral blood were detected with ELISA; the expression of CD4 and CD8 of spleen and oxter lymph nodes was test by flow cytometry.
Results: Mean survival time of the grafted heart of group 1 and group 2 was shorter obviously than that of group 2 and group 4. There was no manifest difference between group 2 and group 4. The level of IL-6 of group 1 was higher than that of other groups, yet there is no difference in group 2、group 3 and group 4. The level of TGF-β1 of group 2 and group 4 was much higher than group 1 and group 3, but there were no difference between group 2 and group 4.
Conclusions: Foxp3 gene-transferred CD4+CD25- T cell was successfully used to induce immune tolerance in rat cardiac allografts. There was no apparent difference comparing with CD4+CD25+ T cell.
第一部分携带Foxp3基因慢病毒载体的构建
目的:克隆Foxp3的cDNA,构建Foxp3-cDNA表达载体。
方法:根据Genebank公布的大鼠Foxp3-cDNA的基因序列,用Trizol从大鼠胸腺中提取总RNA,应用RT-PCR技术扩增Foxp3-cDNA基因,并将其克隆到载体pWPXL-MOD,构建Foxp3-cDNA表达载体,然后转染DH5α感受态细菌。选择阳性克隆菌,行双酶切鉴定和测序验证所获得的cDNA。用慢病毒包装的Foxp3-cDNA的穿梭质粒转染包装细胞293T细胞进行扩增后,使用由四质粒组成的慢病毒转染系统进行基因重组。
结果:双酶切鉴定和测序验证所获得的cDNA为1900bp的Foxp3基因,序列测定结果与GenBank报道的完全一致。
结论:使用RT-PCR法成功构建Foxp3-cDNA表达载体;构建Foxp3基因重组慢病毒。
第二部分用携带目的基因的Foxp3慢病毒转染CD4+CD25-T细胞
目的:用免疫磁珠分离CD4+CD25+T细胞及CD4+CD25-T细胞;使用携带目的基因Foxp3的慢病毒感染CD4+CD25-T细胞。
方法:用免疫磁珠分离CD4+CD25+T细胞及CD4+CD25-T细胞,流式细胞仪鉴定分离细胞的纯度,在感染前24h通过添加抗CD3单抗及抗CD28单抗刺激细胞增殖。用携带Foxp3-cDNA的慢病毒,转染经免疫磁珠分离后扩增的CD4+CD25-T细胞,检测其荧光表达。提取蛋白后,行Western blot检测Foxp3蛋白的表达。
结果:分离纯化的CD4+CD25+T细胞的纯度达95.2%,CD4+CD25-T细胞的纯度为99.1%。病毒感染的CD4+CD25-T细胞荧光显微镜下观察,感染率达80%。
结论:成功地用免疫磁珠分离了CD4+CD25+T细胞及CD4+CD25-T细胞;用携带Foxp3的慢病毒能感染大鼠的CD4+CD25-T细胞,并表达Foxp3蛋白。
第三部分转入Foxp3基因的CD4+CD25-T细胞免疫抑制功能的研究
目的:体外检测转入Foxp3基因的CD4+CD25-T细胞免疫抑制功能。
方法:流式细胞仪鉴定转入Foxp3基因的CD4+CD25-T细胞的CD25的表达率;通过混合淋巴细胞反应、增殖抑制试验及上清液中IL-10和IL-6的检测,推断转基因细胞的功能。
结果:转入Foxp3基因的CD4+CD25-T细胞的CD25阳性表达率为80.2%,在体外单向淋巴细胞混合反应及增殖抑制试验中发现转入Foxp3基因的CD4+CD25-T细胞与CD4+CD25+T细胞具有相似的功能。
结论:转入Foxp3基因的CD4+CD25-T细胞在体外具有抑制效应性T细胞的功能;其表面分子发生了变化,CD25呈阳性表达。
第四部分同种异体大鼠异位心脏移植模型的建立
目的:用改良Ono法进行同种异体大鼠腹腔异位心脏移植,建立动物模型。
方法:以体重约为250g的Wistar大鼠为受体,体重为200-250g的SD大鼠为供体,进行大鼠腹部异位心脏移植手术。行供体的主动脉与受体的腹主动脉行端侧吻合,供体的肺动脉与受体的下腔静脉行端侧吻合。
结果:大鼠腹腔异位心脏移植模型成功率达90.91%,总手术时间约49-71min。
结论:以改良的Ono法进行大鼠腹部心脏移植,可靠易行,成功率高,但需要一定的显微外科基础。
第五部分Foxp3基因转染的CD4+CD25-T细胞对同种异体心脏移植的作用及其机制
目的:研究Foxp3基因转染的CD4+CD25-T细胞对同种异体心脏移植术后免疫耐受的影响及其机制。
方法:64只Wistar大鼠为受体,分为四组,每组16只。第一组为空白对照组(术前注入PBS),第二组为术前注入CD4+CD25+调节性T细胞组,第三组为术前一周每日用CsA灌胃,第四组为转入Foxp3基因CD4+CD25-T细胞组。移植术后每日三次腹部触诊1次观察移植心脏心跳的情况;7日后每组取8只大鼠,麻醉后开腹观察移植心脏的情况,同时采血5ml,取下脾脏及双侧腋下淋巴结;用ELISA法测定血清中的IL-6、IL-10、TGF-β1;用流式细胞仪测定脾脏及双侧腋下淋巴结的淋巴细胞CD4、CD8分子的表达。
结果:移植的心脏第一组和第三组的生存时间明显低于第二组和第四组,第四组与第二组生存时间相比无明显差异;第一组外周血的IL-6显者高于其他组,第二、三、四组间比较无显著性差异;第一组外周血的IL-10显著低于其他组,第二、三、四组间比较无显著性差异;第二、四组外周血的TGF-β1显著高于第一、三组,第二、四组间比较无显著性差异。
结论:转入Foxp3基因CD4+CD25-T细胞具有诱导移植免疫耐受功能与CD4+CD25+T细胞相比差异无显著性。
Background: Heart transplantation has become the preferred theraphy for the end-stage cardiopathic diseases. To depress recipient systematic immunologic function and suppress the immunological rejection to graft, since immunosuppressive agent must be used to maintain the cardiac function of the patients. Nevertheless, clinical transplantation still faces some problems such as lifelong immunosuppression associated with toxicity, opportunistic infection and a high incidence of cancer. So it is a key issue in transplant territory how to induce immune tolerance to take the place of immunosuppressive dependence. In recent 10 years, CD4+CD25+ regulatory T cell has attracted wide attention for its ability of initiative regulation of reactive T cell, and considered as one of effective methods in inducing immune tolerance. The mechanism is not very clear that CD4+CD25+ regulatory T cells induce immune tolerance. But so far there isn’t any report about its clinical application, the most important reason may be its quantity limitation. In this experimental, we constructed Foxp3 Gene, and transferred into CD4+CD25- T cell, then invest the effect and mechanism of immune tolerance induced by CD4+CD25+ regulatory T cell.
PartⅠConstruction of lentiviral vector encoding Foxp3 gene
Objective: To clone the Foxp3 gene and construct the plasmid.
Methods: According to the nucleotide sequence of the rat Foxp3-cDNA announced by Genebank, total RNA was acquired from rat thymus by Trizol, Foxp3-cDNA fragment was amplified using RT-PCR and the PCR product was then ligated into pWPXL-MOD vector to construct the plasmid. The bacterial strain DH5αwas used as the host cell to amplify reconstructed plasmid. The amplified product was lysed to get the supernatant. Double Enzymes EcoRⅠand Bam H1 verified approach and analysising the sequence were used to confirm Foxp3-cDNA. Lentivirus Packaging Kit with four plasmids was used for gene recombination after constructing with Foxp3-cDNA shuttling plasmid which amplified with 293 T cells.
Results: The acquired cDNA is Foxp3 gene with 1900bp. The sequence was comfimed by comparison with the published one.
Conclusion: Foxp3 had been successfully cloned and the plasmid was reconstructed. Foxp3 gene was constructed to recombinate lentivirus.
PartⅡTtransfect CD4+CD25- T cells with Foxp3 gene packaged by lentivirus
Objective: CD4+CD25- T cells and CD4+CD25+ regulatory T cells were isolated by magnetic cell sorting system. Foxp3 gene packaged by lentivirus was uses to transfect CD4+CD25- T cells.
Methods: CD4+CD25- T cells and CD4+CD25+ T cells were isolated by magnetic cell sorting system. The purity was detected by flow cytometry. Anti-CD3 and anti-CD28 were added to stimulate the cell proliferation 24 hours before infection. Foxp3-cDNA shuttling plasmid packaged by lentivirus was used to transfect amplified CD4+CD25- T cells isolated by MACS. GFP was detected by the fluorescence microscope. Protein was extracted for Western blot analysis.
Results: The purity of CD4+CD25+ T cell was 95.2%. The purity of CD4+CD25- T cell was 99.1%. The rate of CD4+CD25- T cell infected by lentivirus was 80%.
Conclusion: CD4+CD25- T cells and CD4+CD25+ T cells were successfully isolated by magnetic cell sorting system and using lentivirus vector with Foxp3 gene to transfecte rat CD4+CD25- T cells.
PartⅢResearch on immune suppression of Foxp3 gene-transferred CD4+CD25- T cells
Objective: Ddetect the immune suppression of Foxp3 gene-transferred CD4+CD25- T cells ex vivo.
Methods: The CD25 expression of Foxp3 gene-transferred CD4+CD25- T cell was detected by flow cytometry; the optical density(OD) of mixed lymphocyte reaction (MLR), lymphproliferation response, the expression of IL-2、IL-6 in supernatant were observed.
Results: The rate of CD25 is 80.2%. The experiment of MLR and lymphproliferation response comfirms that Foxp3 gene-transferred CD4+CD25- T cell and isolated CD4+CD25+ T cell has the similar ability in suppressing the lymphocyte proliferation.
Conclusion: Foxp3 gene-transferred CD4+CD25- T cell can suppress the proliferation of responsive cell. The surface molecule changed with the positive expression of CD25.
PartⅣEstablishment the model of heterotopic heart transplantation with rat
Objective: According to the surgical procedures of improved Ono,s pattern , establish rat model of abdomen heterotopic heart transplantation.
Methods: SD rat weighed about 250g was used as donor and Wistar rat weighed about 200 to 250g was used as recipient to establish rat model of abdomen heterotopic heart transplantation . The ascending artery and pulmonary artery of the donor’s heart was end-to-side anastomosed with abdominal aorta and vena cava inferior of the recipient,s heart respectively.
Results: The mean operational time was 58.4±21.1min.The successful rate for rat cardiac transplantation was 90.91%.
PartⅤThe effect and of Foxp3 gene-transferred CD4+CD25- T cell on rat cardiac allografts and the mechanism behind
Objective: To investigate the effect of Foxp3 gene-transferred CD4+CD25- T cell on acute rejection after transplantation to rat cardiac allografts and mechanism.
Methods: 64 recipients (Wistar rat) were divided randomly into four groups. Every group contain 16 rats. Group 1: 1 ml PBS was injected before transplantation. Group 2: 1x 106 fresh CD4+CD25+ regulatory T cells were injected intravenously into the vein of each Wistar rat before transplantation. Group 3: CSA was used for intragastric administration one week beore transplantation. Group 4: 1x 106 fresh CD4+CD25+ regulatory T cells were injected intravenously before transplantation. Abdominal touch was carried out 3 times one day to observe cardiopalmus. 8 Wistar rats from every group were operated to observe the abdominal graft condition after anesthesia, simultaneously 5 ml blood、spleen and oxter lymph nodes were collectted. The level of IL-6 and TGF-β1 of peripheral blood were detected with ELISA; the expression of CD4 and CD8 of spleen and oxter lymph nodes was test by flow cytometry.
Results: Mean survival time of the grafted heart of group 1 and group 2 was shorter obviously than that of group 2 and group 4. There was no manifest difference between group 2 and group 4. The level of IL-6 of group 1 was higher than that of other groups, yet there is no difference in group 2、group 3 and group 4. The level of TGF-β1 of group 2 and group 4 was much higher than group 1 and group 3, but there were no difference between group 2 and group 4.
Conclusions: Foxp3 gene-transferred CD4+CD25- T cell was successfully used to induce immune tolerance in rat cardiac allografts. There was no apparent difference comparing with CD4+CD25+ T cell.
引文
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