靶向TGF-βⅡ型受体的核酸适配子对Tenon囊成纤维细胞转分化的作用及机制研究
发布时间：2018-08-04 21:57
【摘要】：背景： 抗青光眼滤过性手术是治疗青光眼的重要方法，但手术失败率高达15%-30%，其主要原因是滤过泡瘢痕形成。虽然临床上应用5-氟脲嘧啶(5-fluorouracil,5-FU)、丝裂霉素C (mitomycin C, MMC)提高了滤过术的成功率，但仍然存在某些严重的并发症，如滤过泡渗漏、术后浅前房以及角膜毒性等。因此，寻求一种更为有效、安全的抗瘢痕药物仍是研究的重点。各种研究表明转化生长因子-β (Transforming growth factior-β,TGF-β)与组织瘢痕形成的关系最为密切。TGF-β有5个亚型，哺乳动物只表达3个亚型，即TGF-β1、TGF-β2和TGF-β3，其中TGF-β2被认为是与眼部瘢痕形成最密切的亚型。TGF-βII型受体(Transforming growth factior-β receptor II, TβRII)是TGF-β信号传递的始动受体，TGF-β首先与TβRII结合激活募集到TGF-β I型受体(Transforminggrowth factior-β receptor I, TβRI)，并磷酸化TβRI，启动胞内信号转导。TβRII磷酸化TβRI的GS区，使TβRI磷酸化是信号传递的关键步骤。因此，以TβRII作为封阻靶点，干扰TβRII与TGF-β结合，阻断TGF-β信号转导，以达到抗纤维化的作用，这将对抗纤维化研究提供新的途径。本课题组前期已经成功建立SELEX (SystematicEvolution of Ligands by Exponential Enrichment)筛选平台，并从ssDNA随机序列库中筛选得到靶向TβRII胞外段蛋白的核酸适配子。然而适配子对TGF-β与其受体结合是否具有阻抑效应，需实验进一步验证，从而筛选得到具有干扰效应的适配子，为抗瘢痕治疗的研究提供实验依据。 目的： 本研究拟在体外实验验证靶向TβRII的核酸适配子对TGF-β2生物学效应的干扰作用，并初步探讨适配子干扰效应的机制，进一步筛选得到具有封阻TGF-β2生物学作用的适配子。 方法： 1.取人眼Tenon囊组织贴壁法体外培养成纤维细胞(Human Tenon fibroblasts,HTFs)，第5代~第10代细胞应用于实验。 2.不同浓度TGF-β2(1、2、5、10、20ng/ml)诱导HTFs24h，以及TGF-β2(2、5ng/ml)诱导HTFs不同时间(6、24、48、72h)，应用Western blot检测细胞α-SMA、pSmad2蛋白表达；共聚焦细胞免疫荧光技术检测α-SMA及F-actin定位表达。 3.凝胶收缩实验检测不同浓度TGF-β2(1、2、5、10ng/ml)作用下细胞收缩力的变化，以及适配子S58/68与TGF-β2(2ng/ml)共同作用下细胞收缩力的改变。 4.核酸适配子S58/68分别与TGF-β2共同作用HTFs24h，应用Western blot检测细胞α-SMA、pSmad2蛋白表达，共聚焦细胞免疫荧光技术检测α-SMA及F-actin定位表达。 结果： 1. TGF-β2(1、2、5、10、20ng/ml)诱导HTFs24h后，，α-SMA表达较对照组明显增加(P 0.05)，10ng/ml、20ng/ml浓度组较2ng/ml、5ng/ml浓度组蛋白表达下降；浓度为2ng/ml TGF-β2组α-SMA表达在24-48h达到高峰。TGF-β2促进细胞骨架蛋白F-actin表达，并能显著提高HTFs的收缩力。 2.适配子S58具有明显阻抑TGF-β2诱导HTFs表达α-SMA、细胞骨架蛋白F-actin，并显著抑制TGF-β2介导HTFs的细胞收缩。不同浓度适配子组(20nM、100nM)作用之间无明显差异。 3. TGF-β2(2ng/ml)诱导HTFs表达磷酸化Smad2，作用在30min~1h达高峰，2h开始出现pSmad2表达下降；适配子S58具有明显阻抑TGF-β2诱导HTFs Smad2磷酸化水平以及pSmad2向细胞核内转位。 4.适配子S68对TGF-β2诱导HTFs的表型转化无明显的干扰作用。 结论： 1.在体外实验中，TGF-β2可诱导HTFs明显表达α-SMA，具有一定的浓度依赖性；并且2-5ng/ml TGF-β2浓度组对HTFs诱导α-SMA表达作用达到一个峰值；表明TGF-β2可诱导HTFs向肌成纤维细胞转分化。 2.在体外实验中，靶向TGF-β II型受体的核酸适配子S58能明显阻抑TGF-β2诱导HTFs向肌成纤维细胞转分化。 3. Smad通路参与了TGF-β2诱导HTFs转分化的过程；核酸适配子S58明显阻抑TGF-β2介导HTFs表达pSmad2，以及pSmad2向细胞核内转位，因此推测适配子S58能够干扰TGF-β2与其受体结合，封阻受体激活而活化的下游信号通路，从而减弱TGF-β2介导的生物学效应；可能为青光眼滤过术后抗瘢痕治疗提供新的思路。 4.适配子S58与S68对TGF-β2在体外细胞干扰作用不同，可能与适配子的结构不同相关。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.6
本文编号：2165292
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.6
【参考文献】
相关期刊论文 前2条
1 谢琳;刘韧;朱旭东;贺翔鸽;陈彩宇;;人重组TGF-β RⅡ亲和核酸筛选方法的建立[J];第三军医大学学报;2008年23期
2 朱旭东,顾长国,邸雁飞;SELEX技术与适体[J];生命的化学;2002年03期
本文编号：2165292
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