转染Ⅱ类分子反式激活因子基因突变体抑制猪SLA的表达

为观察CⅡTA基因突变体对猪SLA表达的影响，用指质体转染法将pcDNA3及CⅡTA突变体pcDNA3mCⅡTA2、pcDNA3mCⅡTA3和pcDNA3mCⅡTA4转染入猪血管内皮细胞系PIEC和猪B淋巴细胞系L23。利用流式细胞术/RT-PCR检测各种突变体对猪SLA-DR、-DQ分子/mRNA组成性和诱导性表达的影响。发现两种突变体pcDNA3mCⅡTA3和pcDNA3mCⅡTA4对PIEC/L23细胞SLAⅡ类分子的表达起明显的抑制作用，而空载体pcDNA3和不具有起始密码子突变体pcDNA3mCⅡTA2无此作用。提示pcDNA3mCⅡTA3和pcDNA3mCⅡTA4能抑制猪SLAⅡ类分子的表达。     

修饰CⅡTA基因抑制异种器官移植中供体猪SLA的表达

目的 构建可抑制猪MHC（SLA）Ⅱ类分子表达的MHCⅡ类分子反式激活因子（CⅡTA）突变体，抑制人CD4^ T细胞对猪细胞株的免疫应答，为异种器官的应用研究奠定基础。方法 用PCR，人工合成寡核苷酸，限制性内切酶等技术构建不含起始密码子的pcDNA3mCⅡTA2，含起始密码子的pcDNA3mCⅡTA3突变体。用脂质体转染法将上述2种突变体及pcDNA3空载体转入猪细胞株PIEC细胞和L23细胞。用流式细胞术和RT-PCR法观察它们对PIEC/L23细胞SLA-DR/DQ分子的诱导性和组成性表达的影响。通过混合淋巴细胞反应观察人CD4^ T细胞对转入突变体及空载体后的猪细胞的免疫应答强度的变化。结果 在细胞和分子水平证实pcDNA3空载体无此作用。SLAⅡ类分子受抑制的猪细胞已基本丧失了对人CD4^ T细胞的刺激能力。结论 转染能抑制SLAⅡ类分子表达的突变体使PIEC细胞丧失了对人CD4^ T细胞的刺激能力，为异种器官的修饰提供了新的思路。     

CIITA反义RNA抑制MHCII类分子表达

为探索利用CIITA (MHCclassIItransactivator)基因反义RNA抑制MHCII类分子表达的可能性 ,为CIITA的应用研究奠定基础。利用RT PCR扩增能与CIITAcDNA第 95至 5 0 0bp互补的片段 ,并构建成pcDNA3/CIITAa 重组质粒 ,脂质体转染法将其转入人HeLa/Raji细胞和猪PIEC/L2 3细胞 ,流式细胞术动态检测反义RNA对人 /猪MHCII类分子的抑制率和抑制程度。结果显示HeLa、Raji、PIEC和L2 3四种细胞MHCII类分子受抑制率分别为 4 6 7% ( 7/ 15 ) ,4 0 % ( 6 / 15 ) ,4 6 7% ( 7/ 15 )和33 3% ( 5 / 15 )。典型受抑克隆细胞MHCII类分子受抑程度高达 85 % ,反义RNA对MHCII类分子的抑制时间持续 35～ 5 0d。CIITA反义RNA能有效抑制人 /猪MHCII类分子的表达 ,它在自身免疫和移植免疫中有一定的应用前景     

两种抑制MHCⅡ类分子表达方法的比较

目的：评价cⅡTA（MHC classⅡ transactivator，CⅡTA）基因突变体和反义RNA对MHCⅡ类分子的抑制效率和胞内稳定性，为改造MHC分子奠定基础。方法：用PCR、酶切及连接技术，构建不含起始密码子的peDNA3mCⅡTA2，含起始密码子的peDNA3mCⅡTA3突变体及cⅡTA基因的反义RNA-pcDNA3／CⅡTAJ亘。用脂质体转染法，将突变体、反义RNA及空载体pcDNA3转入PIEC细胞和123细胞中。持续四个月，流式细胞术观察它们对PIEC／123细胞株MHCⅡ类（sLA-DR）分子的诱导性和组成性表达的影响。结果：转染pcDNA3、pcDNA3mCⅡTA2后，PIEC／123细胞SLA-DR的表达没有受到抑制，转染peDNA3mCⅡTA3后，9／20（45．0％）的PIEc细胞、10／20（50．O％）的L23细胞SLA-DR的表达受到明显抑制，抑制率高达90％以上；转染pcDNA3mCIITA＆后，7／15（46．7％）PIEC细胞、5／15（33．3％）123细胞sLA-DR的表达受到明显抑制，抑制率高达85％以上。持续检测4个月，转染peDNA3mCⅡTA3的PIEC／123细胞SLA-DR的表达受抑率均在90％以上。转染反义RNA的PIEC／123细胞在第65／45天。SLA-DR表达抑制率降至10％以下。结论：构建成功的CⅡTA突变体和反义RNA均能有效抑制sLA-DR表达，但CⅡTA突变体稳定存在于胞内的时间长，构建突变体是和用CⅡTA抑制MHCⅡ类分子的好方法。     

CIITA反义RNA抑制MHC Ⅱ类分子表达

为探索利用CIITA(MHC eclass Ⅱ transactivator)基因反义RNA抑制MHC Ⅱ类分子表达的可能性，为CIITA的应用研究奠定基础。利用RT-PCR扩增能与CIITA cDNA第95至500bP互补的片段，并构建成pcDNA3/CIITA，重组质粒，脂质体转染法将其转入人HeLa／Raji细胞和猪PIEC／L23细胞，流式细胞术动态检测反义RNA对人／猪MHCⅡ类分子的抑制率和抑制程度。结果显示HeLa、Raji、PIEC和L23四种细胞MHC Ⅱ类分子受抑制率分别为46．7％(7／15)，40％(6／15)，46．7％(7／15)和33．3％(5／15)。典型受抑克隆细胞MHC Ⅱ类分子受抑程度高达85％，反义RNA对MHC Ⅱ类分子的抑制时间持续35-50d。CIITA反义RNA能有效抑制人／猪MHC Ⅱ类分子的表达，它在自身免疫和移植免疫中有一定的应用前景。     

一种新的CIITA显性负向突变体的构建和功能研究

II类反式激活因子(CIITA)参与MHC II类基因转录表达的调控,本研究采用RT-PCR等分子克隆技术构建含起始密码子和NLS序列,但是删除N和P/S/T结构域的CIITA突变体质粒pCDNA3.1(+)mCIITA,用脂质体转染法将上述突变体及pCDNA3.1(+)空载体转入来源于BALB/c小鼠的1B4.B6细胞株,用流式细胞术和RT-PCR观察I-A分子表达的影响,并通过混合淋巴细胞反应观察C3H小鼠CD4+T细胞对转入突变体及空载体的1B4.B6细胞的免疫应答强度。结果在细胞和分子水平证实pCDNA3.1(+)mCIITA对1B4.B6细胞I-A的表达起明显抑制作用,强阳性克隆抑制率为90%以上。细胞已基本丧失了对受者CD4+T细胞的刺激能力。以上结果为减低同种异体/异种器官细胞性排斥提供了新的思路和手段。     

构建MHCⅡ类分子反式激活因子基因突变体抑制转染细胞HLAⅡ类分子的表达

目的：探讨MHCⅡ类分子反式激活因子（CⅡ\TA）突变体抑制HLAⅡ类分子表达的可能性。方法：构建不含起始密码子的pcDNA3mCⅡTA2、含起始密码子的pcDNA3mCⅡTA3的含起始密码子及NLS（nuclear localization signal)的pcDNA3mCⅡTA4突变体。用脂质体转染法将上述3种突变体及pcDNA3空载体转入HeLa细胞和的Raji细胞。用流式细胞术和RT－PCR法观察它们对HeLa/Raji细胞HLA－DR／DQ分子的诱导性和组成性表达的影响。结果：在细胞和基因水平证实pcDNA3mCⅡTA3和pcDNA3mCⅡTA4对HeLa/Raji细胞HLA－DR／DQ的表达起明显抑制作用，而pcDNA3mCⅡTA2和pcDNA3空载体无此作用。结论：成功构建的pcDNA3mCⅡTA3和pcDNA3mⅡTA4能抑制HLAⅡ类分子的表达。     

构建可抑制HLA-DR/DQ表达的MHC-Ⅱ类分子反式激活因子基因的突变体及其机制

目的：构建能抑制MHC—Ⅱ类分子表达的MHC—Ⅱ类分子反式激活因子(MHC class Ⅱ transactivator，CⅡTA)基因的突变体．并探讨其抑制MHC—Ⅱ类分子表达的机制。方法：用PCR、酶切及连接技术，构建不含起始密码子的pcDNA3mCⅡTA2，含起始密码子的pcDNA3mCⅡTA3以及含起始密码子及NLS(nuclear localization signal)的pcDNA3mCⅡTA4突变体。用脂质体转染法，将上述3种突变体及空载体pcDNA3转入Hela细胞和Raji细胞中。用流式细胞术和RT-PCR法，观察他们对Hela／Raji细胞HLA—DR／DQ分子的诱导性和组成性表达的影响。将mCⅡTA4转移到对四环素浓度依赖的质粒pU-HD10-3上，通过改变培养环境中四环素的浓度，调节外源CⅡTA突变体的表达量，观察突变体的表达量与MHC-Ⅱ类分子受抑率的关系。结果：细胞和基因水平证实，pcDNA3mCⅡTA3和pcDNA3mCⅡTA4对Hela／Raji细胞HLA-DR／DQ的表达均有明显的抑制作用；而pcDNA3mCⅡTA2和空载体pcD-NA3无此作用。MHC-Ⅱ类分子被抑制的程度与外源转入CⅡTA突变体(pUHD10-3mCⅡTA4)的量明显相关。结论：成功地构建pcDNA3mCⅡTA3和pcDNA3mCⅡTA4，并能抑制HLA-Ⅱ类分子的表达。初步证实CⅡTA突变体是通过与胞内的野生型CⅡTA竞争性结合反式激活蛋白，来抑制MHC-Ⅱ类分子的转录和表达。     

转染MHCII类基因对卵巢癌细胞株免疫特性的影响

建立稳定表达CIITA基因的人卵巢癌细胞株HO CIITA ,分析转染CIITA基因对T细胞体外抗肿瘤免疫应答的影响。以CIITA基因逆转录病毒 (pLXSN/CIITA )转染并筛选获得HO CIITA。用FACS和RT PCR分析HLA以及抗原加工递呈基因表达水平。以磁珠法分离得到正常人外周血CD4 +/CD8+T细胞 ,分别进行混合淋巴细胞反应及细胞因子测定 (ELISA和RT PCR )。结果显示 ,转染CIITA基因后 ,使HO细胞HLAII类分子和LMP7基因表达增高 ,而Ii基因表达由阳性转为阴性 ,未检测到TAP1表达 ;HO和HO CIITA细胞刺激CD4 +T细胞分泌IL 4含量两者有显著差异。刺激 4 8h后达到顶峰 ,前者分泌量约为后者的 1/2 ,但分泌IFN γ无差异 ,RT PCR与ELISA两种测定结果一致。表明转染CIITA基因可增加肿瘤细胞表面MHCII类分子的表达 ,该作用与IFN γ具有协同效应 ;并能诱导CD4 +T细胞表达IL 4。     

转染MHCⅡ类基因对卵巢癌细胞株免疫特性的影响

建立稳定表达CIITA基因的人卵巢癌细胞株HO-CIITA，分析转染CIITA基因对T细胞体外抗肿瘤免疫应答的影响。以CIITA基因逆转录病毒(pLXSN/CIITA)转染并筛选获得HO-CIITA。用FACS和RT-PCR分析HLA以及抗原加工递呈基因表达水平。以磁珠法分离得到正常人外周血CD4 ／CD8 T细胞，分别进行混合淋巴细胞反应及细胞因子测定(ELISA和RT-PCR)。结果显示，转染CIITA基因后，使HO细胞HLA Ⅱ类分子和LMP7基因表达增高，而Ⅱ基因表达由阳性转为阴性，未检测到TAP1表达；HO和HO-CIITA细胞刺激CD4 T细胞分泌IL-4含量两者有显著差异。刺激48h后达到顶峰，前者分泌量约为后者的1／2，但分泌IFN-γ无差异，RT-PCR与ELISA两种测定结果一致。表明转染CIITA基因可增加肿瘤细胞表面MHC Ⅱ类分子的表达，该作用与IFN-γ具有协同效应；并能诱导CD4 T细胞表达IL-4。     

Enhanced anti-tumor effect of dendritic cells modified by CIITA gene in hepatocellular carcinoma-bearing mice]

AIM: To study the effect of MHC class II transactivator(CIITA ) on the expression of MHC class I/II molecules, CD80 and CD86 molecules on dendritic cells(DCs), and explor the use of DCs modified by CIITA gene in the biotherapy of tumors. METHODS: We transfected the mouse CIITA gene by lipofectamine into DCs drived from mouse bone marrow. The expression of MHC class I/II, CD80, and CD86 molecules was detected by flow cytometry. 40 mice bearing hepatcellular carcinoma were divided into 4 groups: group 1 were treated with a para-tumor injection of PBS, group 2 DCs, group 3 modified DCs, and group 4 modified DCs pulsed with H22 tumor antigen. Then the anti-tumor effect of DCs modified by mCIITA was evaluated by measuring the tumor size. RESULTS: After transfection of mCIITA, the expression rate of MHC class I/II molecules of DCs increased from 74.2%/66.7% to 93.6%/91.4%, and that of CD80/CD86 increased from 52.3%/60.5% to 89.7%/91.5%. After immunization, the growth of H22 tumors in group 4 mice was significantly inhibited compared with that in other three groups (P<0.05 at 3, 4, 5, 6, and 7 weeks versus groups 1, 2; P<0.05 at 5, 6, 7 weeks versus group 3). CONCLUSION: The expression of MHC I/II, CD80 and CD86 on DCs was regulated markedly by transfection of mCIITA. mCIITA transfection also enhanced the anti-tumor effect of DCs. Our results provided a new method for biotherapy of tumors with DCs.     

CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes

Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes.