本文選題：樹(shù)突狀細(xì)胞 + 骨髓��； 參考：《蘇州大學(xué)》2008年博士論文

【摘要】： 近年來(lái),在所有用來(lái)誘導(dǎo)免疫耐受的策略中,樹(shù)突狀細(xì)胞(dendritic cell, DC)的潛在作用備受關(guān)注。DC是已知的體內(nèi)功能最強(qiáng)的專(zhuān)職抗原遞呈細(xì)胞,DC的表型、成熟度和功能的異質(zhì)性是平衡免疫反應(yīng)和免疫耐受的重要因素。成熟DC表達(dá)高水平的共刺激分子,分泌T細(xì)胞刺激因子(如IL-12、IL-15、IL-18等),選擇并激活帶有相應(yīng)抗原受體的T細(xì)胞,從而誘發(fā)免疫反應(yīng)。未成熟樹(shù)突狀細(xì)胞(immature dendritic cells, imDC)表達(dá)低水平的共刺激分子,通過(guò)T細(xì)胞無(wú)能、免疫偏離、T細(xì)胞凋亡或者誘導(dǎo)調(diào)節(jié)性T細(xì)胞(T-regulatory cells,Treg細(xì)胞)產(chǎn)生等誘導(dǎo)特異性免疫耐受,所以又稱(chēng)致耐受DC(tolerogenic DC, Tol DC)。 然而,imDC注入受體體內(nèi)后會(huì)逐漸成熟,且供者DC很可能會(huì)被受體自然殺傷(natural killer cells, NK)細(xì)胞清除。用供體DC誘導(dǎo)耐受,需提前一周左右從死亡供體中培養(yǎng)DC,限制了其在臨床尸腎移植中的應(yīng)用。 DC的成熟和功能與核因子-κB(nuclear factor-κB, NF-κB)信號(hào)通路密切相關(guān)。NF-κB活化過(guò)程中,抑制性蛋白IκB激酶2(IκB kinases 2, IKK2)的作用最為關(guān)鍵,用腺病毒將IKK2的顯性負(fù)性突變體(dominant negative form of IKK2, IKK2dn)基因轉(zhuǎn)染至DC后,不僅高效維持其未成熟狀態(tài),而且又可以保持機(jī)體對(duì)細(xì)菌感染的免疫能力,有望成為今后移植領(lǐng)域新的治療靶點(diǎn)。應(yīng)用受體imDC負(fù)載供體抗原后,可以誘導(dǎo)針對(duì)供體的特異性免疫耐受,更有過(guò)渡到臨床應(yīng)用的前景。 因此,我們?cè)O(shè)想:通過(guò)體外培養(yǎng)獲得受體大鼠的骨髓源性DC,轉(zhuǎn)染IKK2dn后負(fù)載供體抗原,術(shù)前一周輸入受體大鼠體內(nèi),期望誘導(dǎo)出針對(duì)供體大鼠的特異性免疫耐受。為臨床應(yīng)用受體DC誘導(dǎo)免疫耐受尋求一種切實(shí)可行的方法。 第一部分Lewis大鼠骨髓源性樹(shù)突狀細(xì)胞的誘導(dǎo)分化及功能鑒定 目的探討建立合適的大鼠骨髓源性未成熟樹(shù)突狀細(xì)胞(immature dendritic cells, imDC)的培養(yǎng)方法,并對(duì)其生物學(xué)特性進(jìn)行評(píng)價(jià)。 方法分別以不同劑量的粒-巨噬細(xì)胞集落刺激因子(GM-CSF,2.5、5、10、20ng/ml)和IL-4(5ng/ml)誘導(dǎo)分化獲得大鼠骨髓源性DC,比較收獲率及OX62陽(yáng)性的DC數(shù)量,獲得最佳細(xì)胞因子濃度。并用流式細(xì)胞儀分析DC表型,混合淋巴細(xì)胞反應(yīng)(mixed lymphocyte reaction, MLR)檢測(cè)其刺激同種T細(xì)胞增殖能力,ELISA測(cè)定細(xì)胞培養(yǎng)上清IL-12、MLR上清IL-12、IFN-γ水平。 結(jié)果隨GM-CSF劑量增高可以誘導(dǎo)出更高的收獲率(依次為20.3±2.41%、48.7±7.56%、57.5±11.48%和71.6±9.24%),但OX62表達(dá)依次降低(分別為88.6±6.27%、84.3±6.29%、59.4±13.07%和37.8±9.41%)。GM-CSF 5ng/ml培養(yǎng)7d的DC,表達(dá)中等水平的MHCII和低水平的CD86;分泌少量IL-12;刺激同種T細(xì)胞增殖能力極低。LPS刺激后MHCII、CD86表達(dá)明顯增加;分泌IL-12和IFN-γ增加;刺激同種T細(xì)胞增殖能力增強(qiáng)。 結(jié)論GM-CSF 5ng/ml為誘導(dǎo)大鼠骨髓源性未成熟DC的最佳劑量,培養(yǎng)7~9d可以獲得足夠數(shù)量和純度的未成熟DC,為通過(guò)DC誘導(dǎo)耐受提供可能。 第二部分編碼IKK2dn重組腺病毒載體的構(gòu)建及表達(dá)驗(yàn)證 目的構(gòu)建含有IKK2dn基因的重組腺病毒載體,為轉(zhuǎn)染未成熟樹(shù)突狀細(xì)胞(immature dendritc cells, imDC)誘導(dǎo)免疫耐受研究奠定基礎(chǔ)。 方法從質(zhì)粒pACCMVPLPA SR(+)-IKK2dn中酶切出IKK2dn基因,并插入pShuttle-CMV-GFP(-)TEMP載體中構(gòu)建成腺病毒穿梭質(zhì)粒,KpnI/HindIII酶切鑒定。將pShuttle-CMV-GFP(-)TEMP-IKK2dn轉(zhuǎn)移到pAdxsi載體上,得到pAdxsi-GFP-IKK2dn病毒質(zhì)粒,XhoI酶切后鑒定。將鑒定正確的質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染293細(xì)胞,包裝成重組病毒顆粒;并在293細(xì)胞中反復(fù)擴(kuò)增并純化,根據(jù)報(bào)告基因GFP測(cè)定病毒滴度。轉(zhuǎn)染hela細(xì)胞后RT-PCR檢測(cè)目的基因的表達(dá)。 結(jié)果經(jīng)酶切和RT-PCR鑒定,得到預(yù)期的1060bp條帶,證實(shí)成功構(gòu)建了攜帶IKK2dn基因的重組腺病毒載體,并制備出高滴度(2×1011pfu/ml)的重組腺病毒。 結(jié)論成功構(gòu)建了含IKK2dn-cDNA的重組腺病毒,為進(jìn)一步研究用IKK2dn基因修飾DC誘導(dǎo)免疫耐受等研究奠定了基礎(chǔ)。 第三部分IKK2dn體外轉(zhuǎn)染對(duì)大鼠樹(shù)突狀細(xì)胞生物學(xué)行為的影響目的探討腺病毒介導(dǎo)IKK2dn(Adv-IKK2dn)基因體外轉(zhuǎn)染大鼠樹(shù)突狀細(xì)胞(dendritic cell, DC)對(duì)其生物學(xué)行為的影響。 方法將表達(dá)Adv-IKK2dn腺病毒載體轉(zhuǎn)染大鼠骨髓源性DC,采用Western blotting檢測(cè)轉(zhuǎn)染基因表達(dá),獲得高表達(dá)IKK2dn的DC。并用流式細(xì)胞儀分析DC表型,混合淋巴細(xì)胞反應(yīng)(mixed lymphocyte reaction, MLR)檢測(cè)其刺激同種T細(xì)胞增殖能力,ELISA測(cè)定MLR上清IL-10、IFN-γ水平。 結(jié)果Western blotting檢測(cè)結(jié)果顯示轉(zhuǎn)染pAdxsi-GFP-IKK2dn病毒質(zhì)粒的DC穩(wěn)定高表達(dá)IKK2dn基因,和轉(zhuǎn)染前相比,CD86的表達(dá)水平無(wú)明顯改變(P0.05)。和負(fù)載BN大鼠抗原的未轉(zhuǎn)染DC相比,轉(zhuǎn)染IKK2dn并負(fù)載抗原的DC刺激同種T細(xì)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)。 結(jié)論轉(zhuǎn)染pAdxsi-GFP-IKK2dn病毒質(zhì)粒的DC能夠穩(wěn)定高表達(dá)IKK2dn基因,并使其維持于未成熟狀態(tài)。負(fù)載BN大鼠抗原后可以抑制同種T細(xì)胞增殖反應(yīng),為誘導(dǎo)體內(nèi)免疫耐受提供了實(shí)驗(yàn)依據(jù)。 第四部分轉(zhuǎn)染IKK2dn的樹(shù)突狀細(xì)胞誘導(dǎo)大鼠同種異體腎移植免疫耐受 目的建立大鼠同種異體腎移植的急性排斥反應(yīng)動(dòng)物模型,探討IKK2dn基因轉(zhuǎn)染并負(fù)載供體抗原的受體未成熟DC誘導(dǎo)腎移植免疫耐受的作用及機(jī)制。尋求一種應(yīng)用受體DC誘導(dǎo)腎移植免疫耐受更切實(shí)可行的方法。 方法用GM-CSF和IL-4培養(yǎng)獲得受體Lewis大鼠的骨髓源性DC。以BN大鼠為腎移植供體,Lewis或Wistar大鼠為受體;Lewis大鼠骨髓源性DC轉(zhuǎn)染IKK2dn后負(fù)載BN大鼠脾臟細(xì)胞抗原。將上述DC于腎移植術(shù)前7d經(jīng)陰莖背靜脈注入受體作為治療組,并設(shè)立對(duì)照組(急性排斥組);DC組;空載體組(轉(zhuǎn)染Adv-0并負(fù)載BN抗原);第三方供體組(轉(zhuǎn)染IKK2dn并負(fù)載BN抗原,但供體采用Wistar大鼠)。術(shù)后觀察各組大鼠生存時(shí)間,檢測(cè)腎功能,ELISA法檢測(cè)血清IL-2以及IFN-γ水平,術(shù)后第5d單向MLR檢測(cè)供體脾細(xì)胞刺激受體脾臟細(xì)胞的增殖反應(yīng),移植腎的病理學(xué)檢查,判斷排斥反應(yīng)程度。 結(jié)果與對(duì)照組以及其它各組相比,治療組移植腎生存時(shí)間顯著延長(zhǎng)(26.8±1.76d,P0.01);其受體脾臟細(xì)胞對(duì)供體抗原刺激的反應(yīng)明顯低于其它各組(0.054±.006,P0.01);血清IL-2、IFN-γ水平降低;排斥反應(yīng)輕。 結(jié)論成功建立穩(wěn)定的大鼠同種異體腎移植的急性排斥反應(yīng)動(dòng)物模型;IKK2dn基因轉(zhuǎn)染并負(fù)載供體抗原的受體未成熟DC可以誘導(dǎo)針對(duì)供體的特異性免疫耐受,其機(jī)制可能與抑制T細(xì)胞增殖能力有關(guān)。應(yīng)用受體DC誘導(dǎo)腎移植免疫耐受為臨床尸腎移植提供了一種更切實(shí)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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本文編號(hào)：2089550