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

发布时间：2018-07-02 08:37

本文选题：树突状细胞 + 骨髓　；参考：《苏州大学》2008年博士论文

【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(P0.05)。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(P0.01),MLR上清中IL-10水平升高(P0.01),而IFN-γ水平降低(P0.01)。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,P0.01);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,P0.01);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。
[Abstract]:In recent years, in all strategies used to induce immune tolerance, the potential role of dendritic cell (DC) has attracted much attention..DC is known as the most powerful specific antigen presenting cell in the body. The phenotype, maturity and function heterogeneity of DC are important factors for balancing immune response and immune tolerance. The high level of mature DC is expressed. Co stimulators, secreting T cell stimulating factors (such as IL-12, IL-15, IL-18, etc.), select and activate T cells with corresponding antigen receptors to induce immune responses. Immature dendritic cells (immature dendritic cells, imDC) express low levels of CO stimulators, through T cell incompetence, immune deviation, T cell apoptosis or induction of regulation. T cells (T-regulatory cells, Treg cells) are induced to induce specific immune tolerance, so it is also known as tolerance DC (tolerogenic DC, Tol DC).
However, imDC injected into the receptor will gradually mature, and donor DC is likely to be removed by the natural killer (natural killer cells, NK) cells of the receptor. It is necessary to induce tolerance by donor DC, and it is necessary to train DC from the dead donor in a week or so, limiting its application in the clinical cadaver kidney transplantation.
The maturation and function of DC are closely related to the activation process of nuclear factor kappa B (nuclear factor- kappa B, NF- kappa B) in the activation process of.NF- kappa B, which is the most important factor for the inhibitory protein I kappa B kinase 2 (I kappa 2). To maintain its immature state and maintain the immune ability of the organism to bacterial infection, it is expected to be a new therapeutic target in the field of transplantation in the future. After the application of receptor imDC to donor antigen, it can induce specific immune tolerance to the donor and the prospect of transition to clinical application.
Therefore, we conceive that the bone marrow derived DC of rat was obtained by culture in vitro, and the donor antigen was loaded after transfection of IKK2dn, and the recipient rats were entered in the recipient body a week before the operation to induce specific immune tolerance for donor rats. A practical method was sought for the clinical application of receptor DC to induce immune tolerance.
Part one: induction, differentiation and functional identification of bone marrow derived dendritic cells from Lewis rats
Objective to establish a suitable method for the cultivation of immature dendritic cells (imDC) of rat bone marrow derived immature dendritic cells (imDC), and to evaluate its biological characteristics.
Methods the bone marrow derived DC of rats was obtained by different doses of GM-CSF (2.5,5,10,20ng/ml) and IL-4 (5ng/ml), and the optimum cytokine concentration was obtained by comparing the harvest rate and the DC number of OX62 positive. The phenotype of DC and the mixed lymphocyte reaction (mixed lymphocyte reactio) were analyzed by flow cytometry. N (MLR) was used to detect the proliferation ability of the same T cells stimulated. ELISA was used to detect IL-12, MLR supernatant IL-12 and IFN- gamma in cell culture supernatant.
The results could induce higher harvest rate (20.3 + 2.41%, 48.7 + 7.56%, 57.5 + 11.48% and 71.6 +), but the expression of OX62 decreased in sequence (88.6 + 6.27%, 84.3 + 6.29%, 59.4 + 48.7) and.GM-CSF 5ng/ml for 7d DC, which expressed moderate level MHCII and low level CD86; secreted a small amount of IL-12. The proliferation of T cells was very low. The expression of MHCII and CD86 increased significantly after.LPS stimulation, the secretion of IL-12 and IFN- IFN- increased, and the proliferation ability of homologous T cells was enhanced.
Conclusion GM-CSF 5ng/ml is the best dose to induce immature DC of rat bone marrow, and the cultivation of 7~9d can obtain sufficient quantity and purity of immature DC, which may provide the possibility of inducing tolerance through DC.
Construction and expression verification of the second part encoding IKK2dn recombinant adenovirus vector
Objective to construct recombinant adenovirus vector containing IKK2dn gene and lay a foundation for the study of immune tolerance induced by immature dendritc cells (imDC).
Methods the IKK2dn gene was cut from the plasmid pACCMVPLPA SR (+) -IKK2dn and inserted into the pShuttle-CMV-GFP (-) TEMP vector to construct the adenovirus shuttle plasmid, and the KpnI/HindIII enzyme was cut. The pShuttle-CMV-GFP (-) TEMP-IKK2dn was transferred to the pAdxsi vector. The pAdxsi-GFP-IKK2dn virus plasmid was obtained and the XhoI enzyme was identified. The correct plasmid will be identified. 293 cells were transfected into 293 cells by liposome method and packaged into recombinant virus particles. They were repeatedly amplified and purified in 293 cells. The virus titer was determined according to the reported gene GFP. The expression of the target gene was detected by RT-PCR transfection after transfection of HeLa cells.
Results the expected 1060bp bands were obtained by enzyme digestion and RT-PCR identification, which proved that recombinant adenovirus carrying IKK2dn gene was successfully constructed and a recombinant adenovirus with high titer (2 x 1011pfu/ml) was prepared.
Conclusion the recombinant adenovirus containing IKK2dn-cDNA was successfully constructed, which laid a foundation for further study of IKK2dn gene modified DC inducing immune tolerance.
The effect of the third part of IKK2dn in vitro transfection on the biological behavior of rat dendritic cells in order to explore the effect of adenovirus mediated IKK2dn (Adv-IKK2dn) gene transfection of rat dendritic cells (dendritic cell, DC) on their biological behavior in vitro.
Methods the Adv-IKK2dn adenovirus vector was transfected into rat bone marrow derived DC. The transfection gene expression was detected by Western blotting. The DC. with high expression of IKK2dn was obtained and the DC phenotype was analyzed by flow cytometry. The proliferation ability of the allogeneic T cells was detected by the mixed lymphocyte reaction (mixed lymphocyte reaction, MLR). IFN- gamma level.
Results the results of Western blotting detection showed that the DC of transfected pAdxsi-GFP-IKK2dn virus plasmid was stable and high expression of IKK2dn gene. Compared with before transfection, the expression level of CD86 was not significantly changed (P0.05). The proliferation of T cells transfected with IKK2dn and loaded antigen was lower than that of the antigen loaded BN rats. The level of IL-10 increased (P0.01), while the level of IFN- gamma decreased (P0.01).
Conclusion DC transfected with pAdxsi-GFP-IKK2dn virus plasmids can stabilize the high expression of IKK2dn gene and maintain it in the immature state. It can inhibit the proliferation reaction of the same T cells and provide the experimental basis for inducing immune tolerance in vivo.
In the fourth part, dendritic cells transfected with IKK2dn induce immune tolerance in rat allograft kidney transplantation.
Objective to establish an animal model of acute rejection in renal allograft in rats, and to explore the role and mechanism of IKK2dn gene transfection and the induction of donor antigen receptor immature DC in renal transplantation immune tolerance, and to seek a more practical method to induce renal transplantation tolerance by receptor DC.
Methods the bone marrow derived DC. of Lewis rats was cultured with GM-CSF and IL-4, and BN rats were used as renal transplantation donors, Lewis or Wistar rats were used as receptors. The spleen cell antigen of BN rats was loaded after Lewis rats transfected to IKK2dn after IKK2dn, and the receptor was injected into the dorsal vein of the penis before the kidney transplantation as the treatment group, and the control group was set up. Acute rejection group); DC group; DC group (transfected with Adv-0 and load BN antigen); the donor group (transfected with IKK2dn and loaded BN antigen, but the donor used Wistar rat). After the operation, the survival time of the rats was observed, the renal function was detected, the serum IL-2 and IFN- gamma were detected by ELISA method, and the spleen cells of donor spleen cells were tested for the spleen cells to stimulate the recipient spleen after the operation. The proliferative response of the cells, the pathological examination of the transplanted kidney, and the degree of rejection.
Results compared with the control group and other groups, the survival time of the transplanted kidney in the treatment group was significantly longer (26.8 + 1.76d, P0.01), and the response of the recipient spleen cells to donor antigen stimulation was significantly lower than that of the other groups (0.054 +.006, P0.01), and the serum IL-2, IFN- gamma level decreased, and the repulsion reaction was light.
Conclusion a stable animal model of acute rejection of renal allograft in rats was established successfully. The immature DC of IKK2dn gene transfected and loaded with donor antigen could induce specific immune tolerance to the donor. The mechanism may be related to the inhibition of the proliferation of T cells. The renal transplantation tolerance should be induced by receptor DC as a clinical corpse. Renal transplantation provides a more practical approach.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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