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1、类抗原反式激活因子与HLADR抗原的关系及其意义_医学论文 【摘要】 本研究探讨类抗原反式激活因子（CIITA）和人类白细胞抗原（HLADR）表达时相的关系和差异，及STAT1反义寡核苷酸（STAT1 AS）对CIITA和HLADR的抑制作用。分离健康志愿者外周血T淋巴细胞，给予不同剂量干扰素（IFN）后，用RTPCR法检测CIITA mRNA，Western blot分析HLADR抗原表达，然后给予不同浓度STAT1 AS和STAT1寡核苷酸有义链（STAT1 S），再次检测CIITA mRNA和HLADR的表达。结果表明： CIITA mRNA在IFN 作用后5小时开始表达，14小时达峰值
2、；HLADR在28小时后可被检测出，52小时达高峰。5、10和20 mol/L STAT1 AS作用于细胞后，CIITA mRNA的表达显著低于对照组（P0.01），而在S组明显高于AS处理组（P0.01），S组与对照组间无显著差异；HLADR的表达可被STAT1 AS抑制，AS组仅为对照组的64.3%（P0.01），S组与对照组间仍无差异； STAT1 AS作用后，HLADR变化同CIITA。结论： CIITA mRNA表达与HLADR表达呈正相关且早于后者； STAT1 AS可特异性抑制CIITA和HLADR的表达，并能预防T淋巴细胞激活， CIITA在移植免疫病因中起重要作用。 【关键词
3、】 类抗原反式激活因子 HLADR STAT1 反义寡核苷酸 干扰素This work was supported by National Natural Science Fundation of China（国家 自然 科学 基金）（No 30170389）and the 135 project fundation of Jiangsu province The class II transactivator (CIITA) was initially cloned using genetic complementation of the in vitro derived HLADRnega
4、tive mutant B cell line, RJ2.2.51. All current evidence shows that CIITA is absolutely essential for MHC class II gene expression. In addition to regulating the transcription of class II genes, CIITA transfected into nonprofessional antigenpresenting cells (APCs) results in the expression of HLADM a
5、nd invariant chain 2. CIITA is thus a key regulatory transcription factor to coordinate expression of genes required for HLA class IImediated immune responses3. We investigate the relationship between CIITA and HLADR expression in T cells, and explore its potential effect and significance.Human peri
6、pheral blood mononuclear cells (PBMNC) were isolated from heparinized peripheral blood of normal donors using FicollHypaque densitygradient centrifugation and washed in Hanks balanced salt solution and used for experiments. To obtain a monocyteenriched cell population, PBMNC were incubated at 37 for
7、 2 hours in plastic Petri dishes in RPMI 1640 culture medium (Gibco) containing 100 U/ml penicillin, 100 U/ml streptomycin and 10% heatinactivated fetal calf serum (FCS). Nonadherent cells were removed by washing the dishes with prewarmed (37) culture medium. Then adherent cells were rinsed off with
8、 cold PBS and resuspended in culture medium. CD3positive cells accounted for 70% of the nonadherent cell population. Interferon (IFN, Shanghai Clone HighTech Company, China) was added to T cells (6106/ml) cultures at different final concentration of 50 U/ml to 1200 U/ml for induction of HLADR antige
9、n at 37, 5% CO2. T cells were harvested respectively at 2, 5, 8, 11, 14, 17, 20, 23 hours to test the expression of CIITA mRNA, and HLADR protein were detected respectively at 12, 20, 28, 36, 44, 52, 60, 68, 76 hours after IFN treatment. Samples were stored at -70 and were thawed only once.Reverse t
10、ranscriptionpolymerase chain reaction (RTPCR)Total RNA was isolated using TRIZOL reagent (Shanghai BioProject Company, China) following instructions supplied by the manufacturer. Singlestranded cDNA was synthesized from 2 g of total RNA in a 40 l reaction mixture containing 0.2 g random primer and 3
11、00 U reverse transcriptase (Promega, Madison, WI, USA). A 471bp CIITA fragment and a 306bp GAPDH fragment were amplified as follows. Five microliters of cDNA was subjected to PCR for 30 cycles, 30 sec of denaturation at 94, 1 min of annealing at 61 and 1 min of extension at 72. Upon completion of th
12、e PCR reaction, PCR products were analyzed by ethidium bromide staining in 1.7% agarose gel. Amplification of GAPDH mRNA was used as a control. The sequences of oligonucleotide primers used in PCR amplifications were as follows: CIITA sense, 5CAA GTC CCT GAA GGA TGT GGA3; CIITA antisense, 5 ACG TCC
13、ATC ACC CGG AGG GAC3; GAPDH sense, 5CGG AGT CAA CGG ATT GGT CGT AT3; and GAPDH antisense, 5AGC CTT CTC CAT GGT GGT GAA GAC3 (Synthesized by Invitrogen, USA). In certain cases, serial dilutions of input cDNA were examined to ensure that any effects of CIITA on mRNA induction were not obscured due to
14、a plateau effect in the PCR reaction.Restriction analysis PCR products were purified by Wizard DNA purification kit(Promega, USA). PCRpurified products were digested by restriction endonuclease BamHat 37 for 4-5 hours. Then the products were analyzed by ethidium bromide staining in 1.7% agarose gel,
15、 and selfquantitation was performed by total lab software.Western blot analysisFollowing treatment with IFN, cells were solubilized on ice for 15 minutes in lysis buffer containing 10 mmol/L HEPES（pH 7.9）, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol (DTT), 1 mg/L leupept
16、in, 1 mg/L apronitin, and 10 NP40. Lysates were vortexed and centrifuged at 1110g for 10 minutes at 4. The supernatants were boiled at 100 for 5 minutes in SDSPAGE sample buffer. The proteins were separated on 7.5 % SDSPAGE and were electrotransferred onto nitrocellulose membrane (Amersham, Buckinghamshire, UK). The membrane was probed with the mouse monocl
