整合素连接激酶调节人Tenon's囊成纤维细胞生物学行为的信号转导机制

Fibroblast proliferation and cell matrix synthesis leading to fibrosis and scar formation is the major cause of filtration surgery failure. TGF-beta is the most potent proliferation-induced factor, while integrin is the bridge of the extracellular matrix-induced cell proliferation. ILK is the intersection point of integrin and TGF-beta signaling pathway. It was confirmed that α-SMA, COL-I, FN expression were significantly inhibited by ILK silence in Human Tenon's fibroblasts (HTFs)in our preliminary work,suggesting that ILK has a regulatory role of the migration, differentiation, collagen synthesis and other biological behavior in HTFs.This study intends to build the ILK-specific small interfering RNA lentiviral recombinants, using the specific blockers, to explore the possible signal pathway of ILK regulating the biological behavior of HTFs by detecting the expression of the kinase phosphorylation level and relative effect factors. Then, whether the ILK specific lentivirus-mediated siRNA could reduce ILK protein expression and filtration bleb scarring will be tested on rats filtration surgery model. In this study, extracellular stimulation, intracellular signal transduction, changes in cell function, and in vivo models observation correspond to clarifying the important regulatory role of ILK, helps to find new gene therapy approaches and drug targets for glaucoma anti-scarring therapy.

成纤维细胞增殖和细胞外间质合成是导致组织纤维化和瘢痕形成是滤过性手术失败的主要原因。TGF-β是最强力的增殖诱导因子，整合素是细胞外基质诱导细胞的桥梁，而ILK是整合素和TGF-β信号通路的交汇点。前期工作中已经证实ILK沉默后人Tenon's囊成纤维细胞（HTFs）中α-SMA、COL-I、FN的表达受到显著抑制，提示ILK对HTFs细胞迁移、分化、胶原合成等生物学行为有重要调控作用。本研究拟构建ILK特异性小干扰RNA慢病毒重组体，联合相关信号通路特异性阻断剂，检测相应的激酶磷酸化水平及效应因子的表达，探寻ILK调控HTFs可能的信号机制，并在大鼠滤过术模型中验证慢病毒-ILK-siRNA是否能够防治滤过道瘢痕化。本研究从细胞外刺激，细胞内信号转导，细胞功能变化，活体模型观察等多个层次对应阐明ILK的重要调控作用，希望为青光眼抗瘢痕化提供新的基因治疗方法和药物作用靶点。

在为期三年的研究中，本项目成功体外分离、培养HTFs细胞，在TGF-β2对HTFs细胞作用浓度和时间关系实验中，WB显示TGF-β2对ILK 表达的呈浓度及时间依赖性。尝试建立稳定表达ILK-的HTFs细胞株，但未成功，遂采用靶向基因SiRNA转染HTFs细胞株沉默ILK表达，确定其沉默效应及对HTFs增殖、分化、迁移、胶原分泌等生物学行为的调节作用。在下游通路的实验中我们发现， TGF-β2诱导下，p-AKT(ser473)、p-ERK (Thr202/Tyr204)、p-38MAPK(Thr180/ Tyr182)、p-JNK(Thr183/Tyr185)的磷酸化水平均受到抑制，而p-GSK-3β（Ser9）的磷酸化受到促进，提示ILK可以通过调节AKT、ERK1/2、JNK和p38 MAPK信号转导通路进一步对HTFs的增殖、转分化、细胞外基质合成及凋亡起调节作用。目前实验正在进一步使用上述各通路抑制剂进行干预，明确各条信号通路的调控作用及其之间的关系，从而探寻ILK调节人Tenon’s囊成纤维细胞生物学行为的可能机制。