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TGFβRIF：5>ACC TTC TGA TCC ATC CGT T <3
TGFβRIR:5>CGC AAA GCT GTC AGC CTA G <3
TGFβRITM：FAM 5>CAG AGC TGT GAG GCC TTG AGA GTG <3TAMRA
TGFβRIIF：5>CCC TAC TCT GTC TGT GGA TGA<3
TGFβRIIR ：5>GAC GTC ATT TCC CAG AGT AC<3
TGFβRIITM：FAM 5>CAG GTG GGA ACA GCG AGA TAC ATG G <3TAMRA
18SF: 5>GTA ACC CGT TGA ACC CCA TT <3
18SR: 5>CCA TCC AAT CGG TAG TAG CG <3
18SFTMP：FAM 5>ATG GGG ATC GGG GAT TGC AAT <3TAMRA
Quantitative RTPCR Reactions Take out 5μg RNA, using Superscript II retroviral to reverse transcriptase cDNA. The reaction system and the operation were carried out according to specification. Using Pterygium and normal bulbar conjunctival tissues cDNA as a template, TGFβ1, TGFβ2 primer, TGFβI and II receptor primer were respectively added, corresponding quantitative PCR reaction with add in the TaqMan probe, threehole repeated. PCR reactions happen in 96well plates. Reaction System consisit of the 50μL: 10×PCR buffer 5μL, MgCl2 1.5mmol/L, upstream and downstream primers 0.1μmol/L, 0.1μmol/L TaqMan probe, 5 U Taq enzyme. Set over in each cycle of degeneration, and the process automatically record the final 10 percent the average fluorescence value of cycle time in the last cycle at the end of the PCR. Fluorescence types choose FAM490, in accordance with the procedures set excitation and emission spectra selection filters which were 490nm and 530nm.
Quantitative PCR Analysis
All specimens Point curve were recorded, the default analysis mode was the background of the clawback (Background Substrated), choose baseline PCR deduction (PCR Base Line Substrated) mode for data analysis and correction. In the adjusted baseline cycle (Baseline Cycles) and the computational domain values (Threshold Value), drawn Ct values (Threshold Cycle). The ratio of TGFβ and 18S gene of the initial template was 2Ct18sCt TGFβ,to represent expression of TGFβ gene in the normal conjunctival tissues and pterygium relative to level of 18S. Ct β1 on behalf of TGFβ1 Ct value, Ct β2 on behalf of TGFβ2 Ct value. TGFβ receptor gene and 18S ratio of the initial template for the 2Ct18S CtTGFβR, on behalf of pterygium and normal conjunctival of TGFβ receptor gene relative expression level of 18S. CtTGFβR1 represent TGFβI receptor Ct value, Ct TGFβR2 represent TGFβ II receptor Ct value. All Ct value was got from the median.
Statistical Analysis Three holes of each specimen were determined, and their average value ±s was calculated. Each group was compared using t test.
Figure 1 Gel electropherogram of RNA extraction（略）
RESULTS
Extraction of RNA in Pterygium and Normal Conjunctival Tissue Using 4g/L formaldehyde denaturing gel electrophoresis to identify of 18 S and 28 S strip which was clear. No significant degradation of RNA (Figure 1). UV spectrophotometry measured OD260/280, the ratio was 1.82.0. RNA extraction was high purified and can be used for the experimental study.
Quantitative RTPCR Analysis the Expression of TGFβ1 and TGFβ2 in the Normal Conjunctival Tissue and Pterygium The results showed that the average expression levels of TGFβ1 in the normal bulbar conjunctival tissues and pterygium were 4.26×107±1.45×107and 10.67×107±7.47×107. TGFβ2 were 1.08×1010±0.68×1010and 8.23×1011±6.63×1011. TGFβ1 expression level in pterygium increase 2.94±2.81 times than in normal conjunctiva. There was significant difference (P<0.01）. TGFβ2 expression level in pterygium increase 7.5±1.4 times than in normal conjunctiva. A statistically significant difference can also be found(P<0.01）. It can be presumed that the average expression level of TGFβ1 and TGFβ2 in pterygium was far higher than in normal bulbar conjunctival tissues. TGFβ1 expression in normal conjunctiva tissue was higher than TGFβ2, but the change extent of TGFβ2 was larger than TGFβ1. TGFβ may play an important role in the development of pterygium lesions.
Quantitative RTPCR Analysis with the Normal Conjunctival Pterygium of TGFβRI, TGFβRII Expression
The results showed that the average expression of TGFβRI in normal bulbar conjunctiva tissue and pterygium was 0.003015±0.0036 and 0.000379±0.000281, TGFβRII was 5.33×105±5.05×105 and 1.002×105±9.04×106. The difference of TGFβRI was significant (P<0.05). Expression of TGFβRII level in pterygium was significantly lower than that in normal conjunctiva (P<0.01). It can be supposed that expressions of TGFβRI and TGFβRII were decreased in pterygium. Besides, there were more TGFβRI than TGFβRII both in pterygium and normal bulbar conjunctival tissues, suggesting that the decreased TGFβRI and TGFβRII may lead to the increasion of TGFβ secretion, resulting in the development of pterygium lesions. TGFβR was closely related with pterygium.
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