【摘要】目的:利用RTPCR的方法检测EAU时Lewis大鼠视网膜和葡萄膜Foxp3的mRNA表达情况,探讨EAU时转录因子Foxp3的变化及TGFβ2对其的诱导调控作用,为研究葡萄膜炎的病变机制提供理论支持,为临床治疗葡萄膜炎探索新方向。方法:将IRBP R16多肽注入Lewis大鼠左后足底部制备EAU动物模型。按照随机数字表法将36只Lewis大鼠分为正常对照组、EAU未治疗组和EAU TGFβ2治疗组。TGFβ2治疗组于EAU动物模型制备后第1,4,7,10,13,16,19d给予TGFβ2 10μL(浓度为5mg/L)玻璃体内注射治疗。分别于IRBP免疫后7,10,14,21d处死Lewis大鼠,刮取视网膜和葡萄膜,利用RTPCR法检测Foxp3的表达,所得数据应用SPSS 10.0 统计分析软件进行处理。结果:随着EAU病程的进展,Foxp3的表达逐渐增加,但同正常对照组相比,差异不显著,各时间组之间差异不显著。随着EAU病程的进展,TGFβ2治疗组7dFoxp3的表达增加,同正常对照组和未治疗组相比无显著差异。TGFβ2治疗组10,14,21d Foxp3的表达增加,同正常对照组相比有显著差异(P<0.01),同TGFβ2未治疗组相比Foxp3的表达增加,差异显著(P<0.05)。结论:探讨Foxp3在EAU疾病过程中的表达变化,为进一步研究Foxp3在EAU中的作用机制提供了理论支持。研究了TGFβ2在EAU病程中对Foxp3的表达调控,发现TGFβ2能够增加Foxp3的表达,从而调控Treg细胞,启动免疫抑制功能,为临床治疗葡萄膜炎开辟新方向。
【关键词】 EAU;转化生长因子β2;Foxp3
Regulation of TGFβ2 on the expression of Foxp3 in experimental autoimmune uveoretinitis by injected into vitreous cavity
Xue Li, Qi Hu
Foundation item: Science Foundation of Education Office of Heilongjiang Province, China (No. 11521163)
Department of Ophthalmology, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China
Abstract AIM: To detect the expression of Foxp3 mRNA in different time during experimental autoimmune uveoretinitis (EAU) and to investigate the regulation of TGFβ2 to Foxp3. METHODS: EAU was induced in 32 Lewis rats by immunization with Interphotoreceptor retinoidbinding protein (IRBP) R16 (30μg) and complete Freunds adjuvant, and another 4 rats were in the normal control group. Thirtytwo Lewis rats were divided into EAU group and therapeutic group. The rats of therapeutic group were injected into vitreous cavity with TGFβ2 10μL (5mg/L) at the 1st, 4th, 7th , 10th , 13th ,16th and 19th day and sacrificed at 7th , 10th , 14th and 21st day after immunization, respectively. The eyeballs were excised and total RNA was isolated from the retina, uvea and reversetranscribed into cDNA to design the specific primer of Foxp3. Amplification of PCR reactions was conducted. The resulting PCR products were separated by electrophoresis in 2% agarose gels and analyzed by a Pharmacia Biotech Image Master VDS Video Documentation system using image analysis software. The optical intensity of each PCR product was normalized to that of βactin from the same animal and electrophoresed. Data was expressed at the mean normalized values ± standard error of the mean. RESULTS: During the course of EAU, the expression of Foxp3 increased gradually, but changed less compared to the normal control group and had no significant change among the different time groups. The expression of Foxp3 at 10th, 14th and 21st day of treated group increased significantly compared to the normal control group (P<0.01) and untreated group (P<0.05). CONCLUSION: It is the first time to find the expression of Foxp3 mRNA in different time during EAU. And It is also the first time to find TGFβ2 can upregulate the synthesis and expression of Foxp3 following EAU and regulate the immune suppression. KEYWORDS: experimental autoimmune uveoretinitis; transforming growth factorβ2; Foxp3
0引言 葡萄膜炎的发病机制复杂,是一类T细胞介导的自身反应性疾病,其中自身反应性T细胞的活化是致病的关键,而调节性T细胞(Tregulatory cells,TR细胞或Treg)可以主动抑制自身反应性T细胞的活化与增殖,从而控制了T细胞对自身抗原或异体抗原的过度反应。这一发现不仅引起了免疫基础理论的更新,也为临床上一些免疫性疾病的治疗提供了新的思路[1]。最近的研究表明,转录因子Foxp3特异表达在调节性T细胞上,在调控调节性T细胞的发育和功能上起很重要的作用[2],目前已认为Foxp3可能是调节性T细胞启动免疫抑制功能的“开关”,而TGFβ、T细胞受体(TCR)都可能通过不同途径影响其表达。因此本研究的目的是探讨EAU时转录因子Foxp3的变化及TGFβ对其的诱导调控作用,为研究葡萄膜炎的病变机制提供理论支持,为临床治疗葡萄膜炎开辟一个新方向。
1材料和方法
1.1材料 近交系雌性Lewis大鼠36只,6~8wk,体质量150~180g,购自北京维通利华实验动物技术有限公司。Freund完全佐剂(CFA)购自美国Sigma公司,Ex TaqTM、反转录酶(AMV)、RNA酶抑制剂、dNTP混合物等均购自TaKaRa BIOTECH大连宝生物工程公司,Trizol Reagent购自Invitrogen公司。IRBPR16多肽片段的合成:IRBP第1177~1191氨基酸残基(ADGSSWEGVGVVPDV),即R16多肽片段[3],由上海生工生物技术有限公司合成,纯度为95.6%。
1.2方法
1.2.1实验分组 按照随机数字表法分为正常对照组4只、EAU未治疗组16只、EAU TGFβ2治疗组16只。TGFβ2治疗组于EAU动物模型制备后第1,4,7,10,13,16,19d给予TGFβ2 10μL(浓度为5mg/L)玻璃体内注射治疗。EAU未治疗组和EAU TGFβ2治疗组再根据IRBP免疫后7,10,14,21d分为4组,每组4只。
1.2.2 Lewis大鼠EAU动物模型的建立 Lewis大鼠戊巴比妥钠腹腔注射麻醉后,将IRBPR16多肽30μg+CFA总体积为0.1mL充分研磨成乳剂,注入大鼠左后足底部。
1.2.3 TGFβ2玻璃体内注射 在手术显微镜下用微量注射器透过结膜在鼻上或颞上象限距角膜缘后2mm、以40°~60°朝向赤道区刺入玻璃体腔,注入TGFβ2或PBS 10μL。
1.2.4标本采集 分别于IRBP注射后7,10,14,21d处死Lewis大鼠,无菌条件下于视神经根部摘除眼球,用无菌手术刀剖开,去除晶状体和玻璃体,刮取视网膜和葡萄膜,置于DEPC处理的研磨离心管中,标明组别和编号,于70℃保存。
1.2.5 RTPCR法检测Foxp3的表达 根据已发表的文献[4,5]和Gene Bank的基因序列设计引物,Foxp3(218bp):5’GCTTGTTTGCTGTGCGGAGAC3’,5’GTTTCTGAAGTAGGCGAACAT3’和βactin(212bp)5’TGGAGAAGAGCTACGAGCTGCCTG3’,5’GTGCCGCCAGACAGCACTGTGTTG3’,引物均由TaKaRa BIOTECH大连宝生物工程公司合成。每一组样品进行PCR反应时,用βactin作为内参照,用同一样品的cDNA和同样的PCR反应条件进行扩增。琼脂糖电泳。凝胶扫描仪扫描凝胶。分析结果:利用ImageMasterTM VDS Software ( Pharmacia Biotech )软件,根据DNA条带的光密度值确定DNA含量。结果以粘附分子OD/βactin OD β标准差来表示。
表1TGFβ2治疗组和未治疗组视网膜Foxp3的表达情况(略)
统计学处理:所得数据应用SPSS 10.0统计分析软件进行处理,统计学方法采用配对样品T检验(T检验)和单因素方差分析(F检验)。
2结果
2.1 EAU时视网膜Foxp3的表达变化 随着EAU病程的进展,Foxp3的表达逐渐增加,但同正常对照组相比,差异不显著,各时间组之间差异不显著。
2.2 TGFβ2治疗组对视网膜Foxp3表达的诱导调控 随着EAU病程的进展,TGFβ2治疗组7d Foxp3的表达增加,同正常对照组和未治疗组相比无显著差异。TGFβ2治疗组10,14,21d Foxp3的表达增加,同正常对照组相比有显著差异(P<0.01),同TGFβ2未治疗组相比有显著差异。
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