IKK2dn转染并负载供体抗原的受体未成熟树突状细胞诱导免疫耐受的实验研究
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摘要
近年来,在所有用来诱导免疫耐受的策略中,树突状细胞(dendritic cell, DC)的潜在作用备受关注。DC是已知的体内功能最强的专职抗原递呈细胞,DC的表型、成熟度和功能的异质性是平衡免疫反应和免疫耐受的重要因素。成熟DC表达高水平的共刺激分子,分泌T细胞刺激因子(如IL-12、IL-15、IL-18等),选择并激活带有相应抗原受体的T细胞,从而诱发免疫反应。未成熟树突状细胞(immature dendritic cells, imDC)表达低水平的共刺激分子,通过T细胞无能、免疫偏离、T细胞凋亡或者诱导调节性T细胞(T-regulatory cells,Treg细胞)产生等诱导特异性免疫耐受,所以又称致耐受DC(tolerogenic DC, Tol DC)。
然而,imDC注入受体体内后会逐渐成熟,且供者DC很可能会被受体自然杀伤(natural killer cells, NK)细胞清除。用供体DC诱导耐受,需提前一周左右从死亡供体中培养DC,限制了其在临床尸肾移植中的应用。
DC的成熟和功能与核因子-κB(nuclear factor-κB, NF-κB)信号通路密切相关。NF-κB活化过程中,抑制性蛋白IκB激酶2(IκB kinases 2, IKK2)的作用最为关键,用腺病毒将IKK2的显性负性突变体(dominant negative form of IKK2, IKK2dn)基因转染至DC后,不仅高效维持其未成熟状态,而且又可以保持机体对细菌感染的免疫能力,有望成为今后移植领域新的治疗靶点。应用受体imDC负载供体抗原后,可以诱导针对供体的特异性免疫耐受,更有过渡到临床应用的前景。
因此,我们设想:通过体外培养获得受体大鼠的骨髓源性DC,转染IKK2dn后负载供体抗原,术前一周输入受体大鼠体内,期望诱导出针对供体大鼠的特异性免疫耐受。为临床应用受体DC诱导免疫耐受寻求一种切实可行的方法。
第一部分Lewis大鼠骨髓源性树突状细胞的诱导分化及功能鉴定
目的探讨建立合适的大鼠骨髓源性未成熟树突状细胞(immature dendritic cells, imDC)的培养方法,并对其生物学特性进行评价。
方法分别以不同剂量的粒-巨噬细胞集落刺激因子(GM-CSF,2.5、5、10、20ng/ml)和IL-4(5ng/ml)诱导分化获得大鼠骨髓源性DC,比较收获率及OX62阳性的DC数量,获得最佳细胞因子浓度。并用流式细胞仪分析DC表型,混合淋巴细胞反应(mixed lymphocyte reaction, MLR)检测其刺激同种T细胞增殖能力,ELISA测定细胞培养上清IL-12、MLR上清IL-12、IFN-γ水平。
结果随GM-CSF剂量增高可以诱导出更高的收获率(依次为20.3±2.41%、48.7±7.56%、57.5±11.48%和71.6±9.24%),但OX62表达依次降低(分别为88.6±6.27%、84.3±6.29%、59.4±13.07%和37.8±9.41%)。GM-CSF 5ng/ml培养7d的DC,表达中等水平的MHCII和低水平的CD86;分泌少量IL-12;刺激同种T细胞增殖能力极低。LPS刺激后MHCII、CD86表达明显增加;分泌IL-12和IFN-γ增加;刺激同种T细胞增殖能力增强。
结论GM-CSF 5ng/ml为诱导大鼠骨髓源性未成熟DC的最佳剂量,培养7~9d可以获得足够数量和纯度的未成熟DC,为通过DC诱导耐受提供可能。
第二部分编码IKK2dn重组腺病毒载体的构建及表达验证
目的构建含有IKK2dn基因的重组腺病毒载体,为转染未成熟树突状细胞(immature dendritc cells, imDC)诱导免疫耐受研究奠定基础。
方法从质粒pACCMVPLPA SR(+)-IKK2dn中酶切出IKK2dn基因,并插入pShuttle-CMV-GFP(-)TEMP载体中构建成腺病毒穿梭质粒,KpnI/HindIII酶切鉴定。将pShuttle-CMV-GFP(-)TEMP-IKK2dn转移到pAdxsi载体上,得到pAdxsi-GFP-IKK2dn病毒质粒,XhoI酶切后鉴定。将鉴定正确的质粒用脂质体法转染293细胞,包装成重组病毒颗粒;并在293细胞中反复扩增并纯化,根据报告基因GFP测定病毒滴度。转染hela细胞后RT-PCR检测目的基因的表达。
结果经酶切和RT-PCR鉴定,得到预期的1060bp条带,证实成功构建了携带IKK2dn基因的重组腺病毒载体,并制备出高滴度(2×1011pfu/ml)的重组腺病毒。
结论成功构建了含IKK2dn-cDNA的重组腺病毒,为进一步研究用IKK2dn基因修饰DC诱导免疫耐受等研究奠定了基础。
第三部分IKK2dn体外转染对大鼠树突状细胞生物学行为的影响目的探讨腺病毒介导IKK2dn(Adv-IKK2dn)基因体外转染大鼠树突状细胞(dendritic cell, DC)对其生物学行为的影响。
方法将表达Adv-IKK2dn腺病毒载体转染大鼠骨髓源性DC,采用Western blotting检测转染基因表达,获得高表达IKK2dn的DC。并用流式细胞仪分析DC表型,混合淋巴细胞反应(mixed lymphocyte reaction, MLR)检测其刺激同种T细胞增殖能力,ELISA测定MLR上清IL-10、IFN-γ水平。
结果Western blotting检测结果显示转染pAdxsi-GFP-IKK2dn病毒质粒的DC稳定高表达IKK2dn基因,和转染前相比,CD86的表达水平无明显改变(P>0.05)。和负载BN大鼠抗原的未转染DC相比,转染IKK2dn并负载抗原的DC刺激同种T细胞增殖能力低下(P<0.01),MLR上清中IL-10水平升高(P<0.01),而IFN-γ水平降低(P<0.01)。
结论转染pAdxsi-GFP-IKK2dn病毒质粒的DC能够稳定高表达IKK2dn基因,并使其维持于未成熟状态。负载BN大鼠抗原后可以抑制同种T细胞增殖反应,为诱导体内免疫耐受提供了实验依据。
第四部分转染IKK2dn的树突状细胞诱导大鼠同种异体肾移植免疫耐受
目的建立大鼠同种异体肾移植的急性排斥反应动物模型,探讨IKK2dn基因转染并负载供体抗原的受体未成熟DC诱导肾移植免疫耐受的作用及机制。寻求一种应用受体DC诱导肾移植免疫耐受更切实可行的方法。
方法用GM-CSF和IL-4培养获得受体Lewis大鼠的骨髓源性DC。以BN大鼠为肾移植供体,Lewis或Wistar大鼠为受体;Lewis大鼠骨髓源性DC转染IKK2dn后负载BN大鼠脾脏细胞抗原。将上述DC于肾移植术前7d经阴茎背静脉注入受体作为治疗组,并设立对照组(急性排斥组);DC组;空载体组(转染Adv-0并负载BN抗原);第三方供体组(转染IKK2dn并负载BN抗原,但供体采用Wistar大鼠)。术后观察各组大鼠生存时间,检测肾功能,ELISA法检测血清IL-2以及IFN-γ水平,术后第5d单向MLR检测供体脾细胞刺激受体脾脏细胞的增殖反应,移植肾的病理学检查,判断排斥反应程度。
结果与对照组以及其它各组相比,治疗组移植肾生存时间显著延长(26.8±1.76d,P<0.01);其受体脾脏细胞对供体抗原刺激的反应明显低于其它各组(0.054±.006,P<0.01);血清IL-2、IFN-γ水平降低;排斥反应轻。
结论成功建立稳定的大鼠同种异体肾移植的急性排斥反应动物模型;IKK2dn基因转染并负载供体抗原的受体未成熟DC可以诱导针对供体的特异性免疫耐受,其机制可能与抑制T细胞增殖能力有关。应用受体DC诱导肾移植免疫耐受为临床尸肾移植提供了一种更切实可行的方法。
Part I: Generation of Lewis Rat Bone Marrow-derived Dendritic Cells in Vitro and Evaluation of Their Biological Characterization Objective:To establish a suitable method for the generation of Lewis rat immature dendritic cells and evaluate their biological characterization.
Methods:Bone marrow cells were developed under the concentration of granulocyte- macrophage colonystimulating factor ranging from 2.5 to 20ng/ml and IL-4(5ng/ml) , and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization were analyzed with flowcytometry,the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction (MLR),and the levels of IL-12 secreted by DC and interferon-γ(IFN-γ) and IL-12 levels in 5-day mixed leukocyte culture supernatants was detected by ELISA.
Results:With the increasing GM-CSF concentrations resulted in a higher number of cell yield(20.3±2.41%、48.7±7.56%、57.5±11.48% and 71.6±9.24%), but at the same time, the percentage of OX62 become lower(88.6±6.27%、84.3±6.29%、59.4±13.07% and 37.8±9.41%)at day 7. Under the condition of GM-CSF(5ng/ml), DC at day 9 expressed an intermediate levels of MHCII and low levels of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHCII and CD86 and a stronger IL-2 and IFN-γproduction and higher stimulatory capacity of allogeneic T cell. Conclusions: GM-CSF at a concentration of 5ng/ml is the best method to generate large number of lewis rat immature dendritic cells, and this make it possible to induce immunological tolerance by DC.
Part II: Construction of Adenoviral Vector Encoding IKK2dn Gene and Identification of Their Expression
Objective: To construct the recombinant adenovirus vector encoding IKK2dn gene for using IKK2dn modified imDC to induce immune tolerance.
Methods: IKK2dn cDNA was cloned into adenovirus transfer vector pShuttle- CMV-GFP(-)TEMP, and analyzed by restriction endonuclease KpnI&HindIII digestion. Then, the obtained plasmid, pShuttle-CMV-GFP(-)TEMP-IKK2dn was transferred into pAdxsi vector to construct pAdxsi-GFP-IKK2dn plasmid, and was identified by XhoI digestion. The correct adenoviral recombinant was then cleaved with PacI and transfected into 293 cells to produce and purify viral particles.The virus was then transfected into hela cells,and the infection titer was monitored by green fluorescence protein(GFP)expression, IKK2dn gene was identified by RT-PCR.
Results: As confirmed by restriction digestion analysis and RT-PCR, an expectant fragment of 1060bp was observed in proper recombinants, proved that we successfully constructed the recombinant adenovirus vector encoding IKK2dn gene. The viral titer checked by GFP was about 2×1011pfu/ml.
Conclusions: The recombinant adenovirus vector encoding IKK2dn gene was constructed successfully, which make it easier to investigate the induction of immune tolerance by imDC transfected by IKK2dn.
Part III: Effect of Biological Characteristics of Rat Bone Marrow-derived Dendritic Cells Transfected by IKK2dn in Vitro Objective:To study the effect of biological characteristics of rat bone marrow-derived dendritic cells transfected by IKK2dn gene in vitro.
Methods: Recombinant adenovirus expression plasmid IKK2dn was transfected into rat bone marrow-derived dendritic cells to arrest their maturation, construct tolerenence DC. The expression of transfected gene was detected by western blotting analysis, surface molecules of Adv-IKK2dn-DC were detected by FCM. Autologous T cell proliferation stimulated by Adv-IKK2dn-DC was detected by MTT assay, and the level of IFN-γand IL-10 secreted by DC was analyzed by ELISA.
Results: Western blotting analysis detected high and stable expression of IKK2dn gene in pAdxsi-GFP-IKK2dn transfected DC, and compared with the untransfected DC, DC transfected by recombinant adenovirus vector encoding IKK2dn didn’t up-regulate the expression of CD86(P>0.05). In contrast to untransfected DC, allogeneic T cells proliferation induced by Adv-IKK2dn-DC was obviously lower(P<0.01), and a stronger IL-10 secretion level(P<0.01) and a weaker IFN-γsecretion level(P<0.01) was detected in the IKK2dn transfected DC.
Conclusions:IKK2dn gene can be highly and stably expressed in pAdxsi-GFP-IKK2dn transfected DC, and it can restrain their maturation. DC transfected by pAdxsi-GFP-IKK2dn and loaded with BN antigen can lower the allogeneic MLR, which provided an experimental base for immunological tolerance induction by DC.
Part IV: Tolerance in Rat Allograft renal transplantation Induced by Dendritic Cells Transfected by IKK2dn Gene
Objective: To establish rat allograft renal transplantation model of acute rejective reaction and investigate the effect and mechanisms of recipient-derived imDC transfected by IKK2dn and loaded with donor antigen to induce immune tolerance. To develop a more applicable approach that uses recipient-derived dendritic cells to induce tolerance for clinical renal transplantation.
Methods: Renal transplantations were performed from BN or Wistar donors to Lewis recipients. DC were cultured from recipient rats (Lewis) bone marrow with low dose GM-CSF and IL-4. At day 7, they were transfected by IKK2dn to arrest maturation, and at day 9, they were plused with BN splenocyte lysate for another 2 days. Then, this modified recipient bone marrow derived DC were injected into recipient rats 7 days before transplantation. Null treatment, DC-treatment, Adv-0 transfected and plused BN spleen cell lysate, and a third party donor (Wistar) were served as controls. The survival of renal allografts were observed, cytokine levels were analyzed by ELISA. Their allostimulatory activity was assessed in vitro by one-way MLR. Pathological examination was performed to identified the grade of rejection.
Results: Compared with Null treatment, DC- treatment, and the third party donor(Wistar) controls, IKK2dn transfected DC plused with BN splenocyte lysate markedly prolonged the survival of renal allografts in an antigen-specific manner(26.8±1.76d, P<0.01). IKK2dn-DC loaded with BN antigen elicited markedly lower proliferative responses and reduced IL-2 and IFN-γproduction. In contrast to other groups, IKK2dn-DC loaded with BN antigen show a lower grade as estimated by pathological examination.
Conclusions: Successfully and surely established rat allograft renal transplantation model of acute rejective reaction. Recipient-derived imDC transfected by IKK2dn and loaded with donor splenocyte lysate could induce immune tolerance in a donor-specific manner, it might be associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Our successful induction of tolerance by DC in a recipient manner provides a more feasible strategy for deceased-donor renal transplantation.
然而,imDC注入受体体内后会逐渐成熟,且供者DC很可能会被受体自然杀伤(natural killer cells, NK)细胞清除。用供体DC诱导耐受,需提前一周左右从死亡供体中培养DC,限制了其在临床尸肾移植中的应用。
DC的成熟和功能与核因子-κB(nuclear factor-κB, NF-κB)信号通路密切相关。NF-κB活化过程中,抑制性蛋白IκB激酶2(IκB kinases 2, IKK2)的作用最为关键,用腺病毒将IKK2的显性负性突变体(dominant negative form of IKK2, IKK2dn)基因转染至DC后,不仅高效维持其未成熟状态,而且又可以保持机体对细菌感染的免疫能力,有望成为今后移植领域新的治疗靶点。应用受体imDC负载供体抗原后,可以诱导针对供体的特异性免疫耐受,更有过渡到临床应用的前景。
因此,我们设想:通过体外培养获得受体大鼠的骨髓源性DC,转染IKK2dn后负载供体抗原,术前一周输入受体大鼠体内,期望诱导出针对供体大鼠的特异性免疫耐受。为临床应用受体DC诱导免疫耐受寻求一种切实可行的方法。
第一部分Lewis大鼠骨髓源性树突状细胞的诱导分化及功能鉴定
目的探讨建立合适的大鼠骨髓源性未成熟树突状细胞(immature dendritic cells, imDC)的培养方法,并对其生物学特性进行评价。
方法分别以不同剂量的粒-巨噬细胞集落刺激因子(GM-CSF,2.5、5、10、20ng/ml)和IL-4(5ng/ml)诱导分化获得大鼠骨髓源性DC,比较收获率及OX62阳性的DC数量,获得最佳细胞因子浓度。并用流式细胞仪分析DC表型,混合淋巴细胞反应(mixed lymphocyte reaction, MLR)检测其刺激同种T细胞增殖能力,ELISA测定细胞培养上清IL-12、MLR上清IL-12、IFN-γ水平。
结果随GM-CSF剂量增高可以诱导出更高的收获率(依次为20.3±2.41%、48.7±7.56%、57.5±11.48%和71.6±9.24%),但OX62表达依次降低(分别为88.6±6.27%、84.3±6.29%、59.4±13.07%和37.8±9.41%)。GM-CSF 5ng/ml培养7d的DC,表达中等水平的MHCII和低水平的CD86;分泌少量IL-12;刺激同种T细胞增殖能力极低。LPS刺激后MHCII、CD86表达明显增加;分泌IL-12和IFN-γ增加;刺激同种T细胞增殖能力增强。
结论GM-CSF 5ng/ml为诱导大鼠骨髓源性未成熟DC的最佳剂量,培养7~9d可以获得足够数量和纯度的未成熟DC,为通过DC诱导耐受提供可能。
第二部分编码IKK2dn重组腺病毒载体的构建及表达验证
目的构建含有IKK2dn基因的重组腺病毒载体,为转染未成熟树突状细胞(immature dendritc cells, imDC)诱导免疫耐受研究奠定基础。
方法从质粒pACCMVPLPA SR(+)-IKK2dn中酶切出IKK2dn基因,并插入pShuttle-CMV-GFP(-)TEMP载体中构建成腺病毒穿梭质粒,KpnI/HindIII酶切鉴定。将pShuttle-CMV-GFP(-)TEMP-IKK2dn转移到pAdxsi载体上,得到pAdxsi-GFP-IKK2dn病毒质粒,XhoI酶切后鉴定。将鉴定正确的质粒用脂质体法转染293细胞,包装成重组病毒颗粒;并在293细胞中反复扩增并纯化,根据报告基因GFP测定病毒滴度。转染hela细胞后RT-PCR检测目的基因的表达。
结果经酶切和RT-PCR鉴定,得到预期的1060bp条带,证实成功构建了携带IKK2dn基因的重组腺病毒载体,并制备出高滴度(2×1011pfu/ml)的重组腺病毒。
结论成功构建了含IKK2dn-cDNA的重组腺病毒,为进一步研究用IKK2dn基因修饰DC诱导免疫耐受等研究奠定了基础。
第三部分IKK2dn体外转染对大鼠树突状细胞生物学行为的影响目的探讨腺病毒介导IKK2dn(Adv-IKK2dn)基因体外转染大鼠树突状细胞(dendritic cell, DC)对其生物学行为的影响。
方法将表达Adv-IKK2dn腺病毒载体转染大鼠骨髓源性DC,采用Western blotting检测转染基因表达,获得高表达IKK2dn的DC。并用流式细胞仪分析DC表型,混合淋巴细胞反应(mixed lymphocyte reaction, MLR)检测其刺激同种T细胞增殖能力,ELISA测定MLR上清IL-10、IFN-γ水平。
结果Western blotting检测结果显示转染pAdxsi-GFP-IKK2dn病毒质粒的DC稳定高表达IKK2dn基因,和转染前相比,CD86的表达水平无明显改变(P>0.05)。和负载BN大鼠抗原的未转染DC相比,转染IKK2dn并负载抗原的DC刺激同种T细胞增殖能力低下(P<0.01),MLR上清中IL-10水平升高(P<0.01),而IFN-γ水平降低(P<0.01)。
结论转染pAdxsi-GFP-IKK2dn病毒质粒的DC能够稳定高表达IKK2dn基因,并使其维持于未成熟状态。负载BN大鼠抗原后可以抑制同种T细胞增殖反应,为诱导体内免疫耐受提供了实验依据。
第四部分转染IKK2dn的树突状细胞诱导大鼠同种异体肾移植免疫耐受
目的建立大鼠同种异体肾移植的急性排斥反应动物模型,探讨IKK2dn基因转染并负载供体抗原的受体未成熟DC诱导肾移植免疫耐受的作用及机制。寻求一种应用受体DC诱导肾移植免疫耐受更切实可行的方法。
方法用GM-CSF和IL-4培养获得受体Lewis大鼠的骨髓源性DC。以BN大鼠为肾移植供体,Lewis或Wistar大鼠为受体;Lewis大鼠骨髓源性DC转染IKK2dn后负载BN大鼠脾脏细胞抗原。将上述DC于肾移植术前7d经阴茎背静脉注入受体作为治疗组,并设立对照组(急性排斥组);DC组;空载体组(转染Adv-0并负载BN抗原);第三方供体组(转染IKK2dn并负载BN抗原,但供体采用Wistar大鼠)。术后观察各组大鼠生存时间,检测肾功能,ELISA法检测血清IL-2以及IFN-γ水平,术后第5d单向MLR检测供体脾细胞刺激受体脾脏细胞的增殖反应,移植肾的病理学检查,判断排斥反应程度。
结果与对照组以及其它各组相比,治疗组移植肾生存时间显著延长(26.8±1.76d,P<0.01);其受体脾脏细胞对供体抗原刺激的反应明显低于其它各组(0.054±.006,P<0.01);血清IL-2、IFN-γ水平降低;排斥反应轻。
结论成功建立稳定的大鼠同种异体肾移植的急性排斥反应动物模型;IKK2dn基因转染并负载供体抗原的受体未成熟DC可以诱导针对供体的特异性免疫耐受,其机制可能与抑制T细胞增殖能力有关。应用受体DC诱导肾移植免疫耐受为临床尸肾移植提供了一种更切实可行的方法。
Part I: Generation of Lewis Rat Bone Marrow-derived Dendritic Cells in Vitro and Evaluation of Their Biological Characterization Objective:To establish a suitable method for the generation of Lewis rat immature dendritic cells and evaluate their biological characterization.
Methods:Bone marrow cells were developed under the concentration of granulocyte- macrophage colonystimulating factor ranging from 2.5 to 20ng/ml and IL-4(5ng/ml) , and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization were analyzed with flowcytometry,the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction (MLR),and the levels of IL-12 secreted by DC and interferon-γ(IFN-γ) and IL-12 levels in 5-day mixed leukocyte culture supernatants was detected by ELISA.
Results:With the increasing GM-CSF concentrations resulted in a higher number of cell yield(20.3±2.41%、48.7±7.56%、57.5±11.48% and 71.6±9.24%), but at the same time, the percentage of OX62 become lower(88.6±6.27%、84.3±6.29%、59.4±13.07% and 37.8±9.41%)at day 7. Under the condition of GM-CSF(5ng/ml), DC at day 9 expressed an intermediate levels of MHCII and low levels of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHCII and CD86 and a stronger IL-2 and IFN-γproduction and higher stimulatory capacity of allogeneic T cell. Conclusions: GM-CSF at a concentration of 5ng/ml is the best method to generate large number of lewis rat immature dendritic cells, and this make it possible to induce immunological tolerance by DC.
Part II: Construction of Adenoviral Vector Encoding IKK2dn Gene and Identification of Their Expression
Objective: To construct the recombinant adenovirus vector encoding IKK2dn gene for using IKK2dn modified imDC to induce immune tolerance.
Methods: IKK2dn cDNA was cloned into adenovirus transfer vector pShuttle- CMV-GFP(-)TEMP, and analyzed by restriction endonuclease KpnI&HindIII digestion. Then, the obtained plasmid, pShuttle-CMV-GFP(-)TEMP-IKK2dn was transferred into pAdxsi vector to construct pAdxsi-GFP-IKK2dn plasmid, and was identified by XhoI digestion. The correct adenoviral recombinant was then cleaved with PacI and transfected into 293 cells to produce and purify viral particles.The virus was then transfected into hela cells,and the infection titer was monitored by green fluorescence protein(GFP)expression, IKK2dn gene was identified by RT-PCR.
Results: As confirmed by restriction digestion analysis and RT-PCR, an expectant fragment of 1060bp was observed in proper recombinants, proved that we successfully constructed the recombinant adenovirus vector encoding IKK2dn gene. The viral titer checked by GFP was about 2×1011pfu/ml.
Conclusions: The recombinant adenovirus vector encoding IKK2dn gene was constructed successfully, which make it easier to investigate the induction of immune tolerance by imDC transfected by IKK2dn.
Part III: Effect of Biological Characteristics of Rat Bone Marrow-derived Dendritic Cells Transfected by IKK2dn in Vitro Objective:To study the effect of biological characteristics of rat bone marrow-derived dendritic cells transfected by IKK2dn gene in vitro.
Methods: Recombinant adenovirus expression plasmid IKK2dn was transfected into rat bone marrow-derived dendritic cells to arrest their maturation, construct tolerenence DC. The expression of transfected gene was detected by western blotting analysis, surface molecules of Adv-IKK2dn-DC were detected by FCM. Autologous T cell proliferation stimulated by Adv-IKK2dn-DC was detected by MTT assay, and the level of IFN-γand IL-10 secreted by DC was analyzed by ELISA.
Results: Western blotting analysis detected high and stable expression of IKK2dn gene in pAdxsi-GFP-IKK2dn transfected DC, and compared with the untransfected DC, DC transfected by recombinant adenovirus vector encoding IKK2dn didn’t up-regulate the expression of CD86(P>0.05). In contrast to untransfected DC, allogeneic T cells proliferation induced by Adv-IKK2dn-DC was obviously lower(P<0.01), and a stronger IL-10 secretion level(P<0.01) and a weaker IFN-γsecretion level(P<0.01) was detected in the IKK2dn transfected DC.
Conclusions:IKK2dn gene can be highly and stably expressed in pAdxsi-GFP-IKK2dn transfected DC, and it can restrain their maturation. DC transfected by pAdxsi-GFP-IKK2dn and loaded with BN antigen can lower the allogeneic MLR, which provided an experimental base for immunological tolerance induction by DC.
Part IV: Tolerance in Rat Allograft renal transplantation Induced by Dendritic Cells Transfected by IKK2dn Gene
Objective: To establish rat allograft renal transplantation model of acute rejective reaction and investigate the effect and mechanisms of recipient-derived imDC transfected by IKK2dn and loaded with donor antigen to induce immune tolerance. To develop a more applicable approach that uses recipient-derived dendritic cells to induce tolerance for clinical renal transplantation.
Methods: Renal transplantations were performed from BN or Wistar donors to Lewis recipients. DC were cultured from recipient rats (Lewis) bone marrow with low dose GM-CSF and IL-4. At day 7, they were transfected by IKK2dn to arrest maturation, and at day 9, they were plused with BN splenocyte lysate for another 2 days. Then, this modified recipient bone marrow derived DC were injected into recipient rats 7 days before transplantation. Null treatment, DC-treatment, Adv-0 transfected and plused BN spleen cell lysate, and a third party donor (Wistar) were served as controls. The survival of renal allografts were observed, cytokine levels were analyzed by ELISA. Their allostimulatory activity was assessed in vitro by one-way MLR. Pathological examination was performed to identified the grade of rejection.
Results: Compared with Null treatment, DC- treatment, and the third party donor(Wistar) controls, IKK2dn transfected DC plused with BN splenocyte lysate markedly prolonged the survival of renal allografts in an antigen-specific manner(26.8±1.76d, P<0.01). IKK2dn-DC loaded with BN antigen elicited markedly lower proliferative responses and reduced IL-2 and IFN-γproduction. In contrast to other groups, IKK2dn-DC loaded with BN antigen show a lower grade as estimated by pathological examination.
Conclusions: Successfully and surely established rat allograft renal transplantation model of acute rejective reaction. Recipient-derived imDC transfected by IKK2dn and loaded with donor splenocyte lysate could induce immune tolerance in a donor-specific manner, it might be associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Our successful induction of tolerance by DC in a recipient manner provides a more feasible strategy for deceased-donor renal transplantation.
引文
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