【摘要】 目的：定量检测转化生长因子β（Transforming Growth Factorβ,TGFβ）及其受体在翼状胬肉和正常球结膜组织中的表达水平，探讨其在翼状胬肉病变发生发展过程中的作用。方法：随机选取翼状胬肉患者30例，手术切除翼状胬肉组织，并留取患眼角膜上缘上方的正常球结膜组织作为自身对照，实时荧光定量PCR技术分析翼状胬肉和正常球结膜组织中TGFβ1、TGFβ2及其I型和II型受体相对于内参照基因18S的相对mRNA含量。结果：TGFβ1在正常球结膜和翼状胬肉组织中平均表达水平分别为4.26×107±1.45×107和10.67×107±7.47×107，TGFβ2在正常球结膜和翼状胬肉组织中表达水平分别为1.08×1010±0.68×1010和8.23×1011±6.63×1011。TGFβⅠ型受体在正常球结膜和胬肉组织中平均表达水平分别为0.003015±0.0036和0.000379±0.000281， TGFβⅡ型受体在正常球结膜和胬肉组织中表达水平分别为5.33×105±5.05×105和1.002×105±9.04×106。TGFβ1在胬肉组织中表达水平比正常结膜中表达水平升高倍数为2.9±2.8，差异有统计学意义（P<0.01）。TGFβ2在胬肉组织中表达水平比正常结膜中表达水平升高倍数为7.5±1.4，差异有统计学意义（P<0.01）。TGFβⅠ型受体在胬肉组织中表达水平比正常球结膜中明显降低，差异有统计学意义（P<0.05）。TGFβⅡ型受体在胬肉组织中表达水平比正常球结膜中表达水平明显降低，差异有统计学意义（P<0.01）。结论：TGFβ1和TGFβ2在胬肉组织中表达水平均高于正常球结膜组织。TGFβ1在正常球结膜和胬肉组织中表达水平均远高于相应组织中的TGFβ2表达，但是TGFβ2在胬肉中表达水平升高变化幅度高于TGFβ1。TGFβⅠ型和Ⅱ型受体在胬肉组织中表达水平均低于正常球结膜组织。TGFβⅠ型受体在正常球结膜和胬肉组织中表达水平均高于相应组织中的TGFβⅡ型受体表达，提示胬肉组织中Ⅰ型、Ⅱ型受体表达的降低可能导致TGFβ分泌的增加，从而导致翼状胬肉病变的发生发展，提示转化生长因子β及其受体在翼状胬肉病变发生发展中可能起着重要的作用。
【关键词】 转化生长因子 β 受体 翼状胬肉 实时荧光定量PCR 基因表达
INTRODUCTION
The pterygium is one common disease frequentlyoccurring in ophthalmology department, which until now still had no consensus to its pathogenesis. Clinic also lacks ideal methods for permanent therapy. Therefore it is urgently needed to study its pathological change essence and seek the effective measures for prevention. The pathology research indicated that the principal constituents of pterygium become the fibroblasts by the massive proliferations, causing the corresponding pathological. In recent years, it is indicated that the growth factors play an important role in inflammation and the proliferation of fibroblasts [1]. Transforming growth factorβ (TGFβ) is such kind of multipeptides that are able to regulate the cell growth and differentiation, which is reported to participate in pterygium pathology process [2]. But its concrete mechanism is still not discovered. We used quantitative realtime PCR (QRTPCR) to determine the expression of TGFβ1 and TGFβ2 as well as their receptors in pterygium and normal bulbar conjunctiva tissues, exploring their roles in the process of pterygium lesions.
MATERIALS AND METHODS
Materials Thirty cases of pterygium patients were randomly selected with surgical resection of pterygium tissues, collecting 2mm×5mm from the top of the normal margin of bulbar conjunctival tissues as control. All the patients have no other lesions of cornea and conjunctiva.
Methods Specimen Preparation: under aseptic conditions, normal bulbar conjunctival tissues and pterygium tissues were obtained through surgery, drawing rapidly into liquid nitrogen immediately, using RNA Extraction Kit Trizol Reagent (Invitrogen) to extract the total RNA, UV spectrophotometry to measure its purity and content, 4g/L formaldehyde denaturing gel electrophoresis test its integrity. Primer design: In accordance with QRTPCR primer design principles, TaqMan probe fluorescence was chosen as a fluorescent FAM group report, TAMRA as quenching groups. PCR primers and TaqMan probe were designed according to TGFβ1 and TGFβ2 and its type I and II receptors and the refer to 18 S cDNA sequence . Primer sequences were as follows:
TGFβ1F：5>AAC TAC TGC TTC AGC TCC AC<3
TGFβ1R：5>TGT GTC CAG GCT CCA AAT GTA<3
TGFβ1TM：FAM 5>CAG AAG TTG GCA TGG TAG CCC TTG GG<3TAMRA
TGFβ2F：5>ATG TGC AGG ATA ATT GCT GCC<3
TGFβ2R ：5>TGG TGT TGT ACA GGC TGA GG<3
TGFβ2TM：FAM 5>TGT TGT GTG TCT GAA CTC CAC AGA T<3TAMRA
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