【摘要】：背景： 抗青光眼濾過性手術(shù)是治療青光眼的重要方法，但手術(shù)失敗率高達15%-30%，其主要原因是濾過泡瘢痕形成。雖然臨床上應(yīng)用5-氟脲嘧啶(5-fluorouracil,5-FU)、絲裂霉素C (mitomycin C, MMC)提高了濾過術(shù)的成功率，但仍然存在某些嚴(yán)重的并發(fā)癥，如濾過泡滲漏、術(shù)后淺前房以及角膜毒性等。因此，尋求一種更為有效、安全的抗瘢痕藥物仍是研究的重點。各種研究表明轉(zhuǎn)化生長因子-β (Transforming growth factior-β,TGF-β)與組織瘢痕形成的關(guān)系最為密切。TGF-β有5個亞型，哺乳動物只表達3個亞型，即TGF-β1、TGF-β2和TGF-β3，其中TGF-β2被認為是與眼部瘢痕形成最密切的亞型。TGF-βII型受體(Transforming growth factior-β receptor II, TβRII)是TGF-β信號傳遞的始動受體，TGF-β首先與TβRII結(jié)合激活募集到TGF-β I型受體(Transforminggrowth factior-β receptor I, TβRI)，并磷酸化TβRI，啟動胞內(nèi)信號轉(zhuǎn)導(dǎo)。TβRII磷酸化TβRI的GS區(qū)，使TβRI磷酸化是信號傳遞的關(guān)鍵步驟。因此，以TβRII作為封阻靶點，干擾TβRII與TGF-β結(jié)合，阻斷TGF-β信號轉(zhuǎn)導(dǎo)，以達到抗纖維化的作用，這將對抗纖維化研究提供新的途徑。本課題組前期已經(jīng)成功建立SELEX (SystematicEvolution of Ligands by Exponential Enrichment)篩選平臺，并從ssDNA隨機序列庫中篩選得到靶向TβRII胞外段蛋白的核酸適配子。然而適配子對TGF-β與其受體結(jié)合是否具有阻抑效應(yīng)，需實驗進一步驗證，從而篩選得到具有干擾效應(yīng)的適配子，為抗瘢痕治療的研究提供實驗依據(jù)。 目的： 本研究擬在體外實驗驗證靶向TβRII的核酸適配子對TGF-β2生物學(xué)效應(yīng)的干擾作用，并初步探討適配子干擾效應(yīng)的機制，進一步篩選得到具有封阻TGF-β2生物學(xué)作用的適配子。 方法： 1.取人眼Tenon囊組織貼壁法體外培養(yǎng)成纖維細胞(Human Tenon fibroblasts,HTFs)，第5代~第10代細胞應(yīng)用于實驗。 2.不同濃度TGF-β2(1、2、5、10、20ng/ml)誘導(dǎo)HTFs24h，以及TGF-β2(2、5ng/ml)誘導(dǎo)HTFs不同時間(6、24、48、72h)，應(yīng)用Western blot檢測細胞α-SMA、pSmad2蛋白表達；共聚焦細胞免疫熒光技術(shù)檢測α-SMA及F-actin定位表達。 3.凝膠收縮實驗檢測不同濃度TGF-β2(1、2、5、10ng/ml)作用下細胞收縮力的變化，以及適配子S58/68與TGF-β2(2ng/ml)共同作用下細胞收縮力的改變。 4.核酸適配子S58/68分別與TGF-β2共同作用HTFs24h，應(yīng)用Western blot檢測細胞α-SMA、pSmad2蛋白表達，共聚焦細胞免疫熒光技術(shù)檢測α-SMA及F-actin定位表達。 結(jié)果： 1. TGF-β2(1、2、5、10、20ng/ml)誘導(dǎo)HTFs24h后，，α-SMA表達較對照組明顯增加(P 0.05)，10ng/ml、20ng/ml濃度組較2ng/ml、5ng/ml濃度組蛋白表達下降；濃度為2ng/ml TGF-β2組α-SMA表達在24-48h達到高峰。TGF-β2促進細胞骨架蛋白F-actin表達，并能顯著提高HTFs的收縮力。 2.適配子S58具有明顯阻抑TGF-β2誘導(dǎo)HTFs表達α-SMA、細胞骨架蛋白F-actin，并顯著抑制TGF-β2介導(dǎo)HTFs的細胞收縮。不同濃度適配子組(20nM、100nM)作用之間無明顯差異。 3. TGF-β2(2ng/ml)誘導(dǎo)HTFs表達磷酸化Smad2，作用在30min~1h達高峰，2h開始出現(xiàn)pSmad2表達下降；適配子S58具有明顯阻抑TGF-β2誘導(dǎo)HTFs Smad2磷酸化水平以及pSmad2向細胞核內(nèi)轉(zhuǎn)位。 4.適配子S68對TGF-β2誘導(dǎo)HTFs的表型轉(zhuǎn)化無明顯的干擾作用。 結(jié)論： 1.在體外實驗中，TGF-β2可誘導(dǎo)HTFs明顯表達α-SMA，具有一定的濃度依賴性；并且2-5ng/ml TGF-β2濃度組對HTFs誘導(dǎo)α-SMA表達作用達到一個峰值；表明TGF-β2可誘導(dǎo)HTFs向肌成纖維細胞轉(zhuǎn)分化。 2.在體外實驗中，靶向TGF-β II型受體的核酸適配子S58能明顯阻抑TGF-β2誘導(dǎo)HTFs向肌成纖維細胞轉(zhuǎn)分化。 3. Smad通路參與了TGF-β2誘導(dǎo)HTFs轉(zhuǎn)分化的過程；核酸適配子S58明顯阻抑TGF-β2介導(dǎo)HTFs表達pSmad2，以及pSmad2向細胞核內(nèi)轉(zhuǎn)位，因此推測適配子S58能夠干擾TGF-β2與其受體結(jié)合，封阻受體激活而活化的下游信號通路，從而減弱TGF-β2介導(dǎo)的生物學(xué)效應(yīng)；可能為青光眼濾過術(shù)后抗瘢痕治療提供新的思路。 4.適配子S58與S68對TGF-β2在體外細胞干擾作用不同，可能與適配子的結(jié)構(gòu)不同相關(guān)。
[Abstract]:Background:
Glaucoma filtering surgery is an important method for the treatment of glaucoma, but the failure rate is up to 15%-30%, the main reason is the formation of filtration bleb scar. Although the clinical application of 5- fluorouracil (5-fluorouracil, 5-FU), mitomycin C (mitomycin C, MMC) improves the success rate of filtration, but there are still some serious complications, such as filtration. There are 5 subtypes of transforming growth factor - beta (Transforming growth factior- beta, TGF- beta), the most closely related to tissue scar formation, and 3 subtypes in mammals, and 3 subtypes in mammals. TGF- beta 1, TGF- beta 2 and TGF- beta 3, in which TGF- beta 2 is considered the most closely associated.TGF- beta II receptor of the eye scar (Transforming growth factior- beta receptor II, T beta) is the initiator of beta signaling. Tor I, T beta RI), and phosphorylation of T beta RI, starting the intracellular signal transduction of the GS region of.T beta RII phosphorylation T beta RI, making T beta phosphorylation a key step in signal transmission. Therefore, the beta binding is used as blocking target, interfering with beta binding and binding, blocking beta signal transduction, in order to achieve the effect of anti fibrinylation, which will provide a new way to resist fibrosis. We have successfully established the SELEX (SystematicEvolution of Ligands by Exponential Enrichment) screening platform and screened the nucleic acid aptamers targeting T beta RII extracellular segment protein from the random sequence library of ssDNA. However, it is necessary to further verify whether the aptamer has inhibition effect on the binding of TGF- beta to its receptor. The aptamers with interference effects were screened to provide experimental evidence for the study of anti scar therapy.
Objective:
This study is to verify the interference of the nucleic acid aptamers of T beta RII to the biological effects of TGF- beta 2 in vitro, and to explore the mechanism of the aptamer interference effect, and to further screen the aptamers with the biological effect of blocking TGF- beta 2.
Method:
1. Human Tenon fibroblasts (HTFs) were cultured in vitro by tissue attachment method of human Tenon capsule. The cells of the fifth to tenth passages were used in the experiment.
2. different concentrations of TGF- beta 2 (1,2,5,10,20ng/ml) induced HTFs24h, and TGF- beta 2 (2,5ng/ml) induced HTFs at different time (6,24,48,72h), and Western blot was used to detect the expression of alpha -SMA, pSmad2 protein, and confocal cell immunofluorescence technique was used to detect alpha -SMA and localized expression.
3. gel contraction test was used to detect the changes in the contractile force of cells under the action of different concentrations of TGF- beta 2 (1,2,5,10ng/ml), and the changes of the contractile force of cells under the co action of aptamer S58/68 and TGF- beta 2 (2ng/ml).
4. aptamer S58/68 and TGF- beta 2 co acted on HTFs24h, and Western blot was used to detect the expression of alpha -SMA, pSmad2 protein, and confocal cell immunofluorescence technique was used to detect the expression of alpha -SMA and F-actin.
Result:
After 1. TGF- beta 2 (1,2,5,10,20ng/ml) induced HTFs24h, the expression of alpha -SMA was significantly higher than that of the control group (P 0.05), 10ng/ml, 20ng/ml concentration group was lower than 2ng/ml, 5ng/ml concentration histone expression decreased, and the concentration of 2ng/ml TGF- beta 2 was reached to the peak of beta 2 to promote cytoskeleton protein expression, and can significantly improve the contraction force.
2. aptamer S58 significantly inhibited TGF- beta 2 to induce HTFs expression of alpha -SMA, cytoskeleton protein F-actin, and significantly inhibited TGF- beta 2 mediated cell contraction in HTFs. There was no significant difference between the effects of different concentrations of the aptamer group (20nM, 100nM).
3. TGF- beta 2 (2ng/ml) induced HTFs to express phosphorylated Smad2, at the peak of 30min~1h, and 2H began to decline in pSmad2 expression, and the aptamer S58 had obvious inhibition of TGF- beta 2 induced HTFs Smad2 phosphorylation level and pSmad2 to the nucleus transposition.
4. aptamer S68 had no significant interference on the phenotype transformation of HTFs induced by TGF- beta 2.
Conclusion:
1. in vitro, TGF- beta 2 could induce HTFs to express obviously the expression of alpha -SMA, with a certain concentration dependence, and the concentration group of 2-5ng/ml TGF- beta 2 reached a peak in the expression of HTFs induced alpha -SMA, indicating that TGF- beta 2 could induce the differentiation of HTFs into myofibroblast.
2. In vitro, aptamer S58 targeting TGF-beta II receptor significantly inhibited the transdifferentiation of HTFs into myofibroblasts induced by TGF-beta 2.
3. Smad pathway participates in the process of TGF- beta 2 induced HTFs transdifferentiation; aptamer S58 obviously hinders TGF- beta 2 mediated HTFs expression pSmad2, and pSmad2 translocation into the nucleus. Therefore, it is speculated that the aptamer S58 can interfere with the binding of TGF- beta 2 to its receptor, the activation of blocking receptor activation and activation of the downstream signaling pathway, thus weakening TGF- beta 2 mediated organisms Learning effect may provide new ideas for anti scar treatment after glaucoma filtering surgery.
4. aptamers S58 and S68 have different interference effects on TGF- beta 2 in vitro, which may be related to the structure of aptamers.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R779.6

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