CLC-2氯离子通道在TGF-β1诱导的HconF细胞纤维化中的作用研究

发布时间：2018-03-09 07:26

本文选题：青光眼滤过术　切入点：瘢痕化　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：青光眼是全球第一位不可逆性致盲眼病,严重威胁人们的视功能和生活质量。滤过性手术是目前治疗青光眼的最主要手术方法,但是,其成功率一直受滤过泡瘢痕化的限制。目前,5-氟尿嘧啶(5-fluorouracil,5-Fu)和丝裂霉素C(Mitomycin C,MMC)等抗代谢药物已经被用于抑制滤过泡瘢痕形成,但是其对细胞的毒副作用引起滤过泡漏、眼内炎、低眼压、浅前房、角膜内皮失代偿等严重的并发症限制了其在抗瘢痕化中的进一步应用。因此,探索新的副作用更少、更有效的抗瘢痕化药物具有重要意义。结膜成纤维细胞(fibroblast,FB)是参与滤过泡瘢痕形成的主要细胞,滤过手术会刺激结膜成纤维细胞转化为肌成纤维细胞,并促进结膜成纤维细胞增殖、迁移及合成和分泌细胞外基质(extracellular matrix,ECM),从而促进创面修复及瘢痕形成。转化生长因子β(transfer growth factorβ,TGF-β)是由巨噬细胞、淋巴细胞、成纤维细胞(FB)、血管内皮细胞和某些基质细胞分泌的一类生物活性多肽,是创口愈合和瘢痕形成过程中最主要的生长因子。TGF-β通过自分泌或旁分泌的方式调控细胞增殖、分化和ECM合成。这些功能使TGF-β成为一种重要的促纤维化生长因子,可以促进器官组织纤维化的发生,在青光眼滤过泡瘢痕形成过程中,TGF-β也是重要的致纤维化细胞因子。本研究应用TGF-β1作用于人结膜成纤维细胞(human conjunctival fibroblast,Hcon F)后,用CCK-8实验方法检测Hcon F细胞增殖情况,结果显示TGF-β1可以时间依赖性地促进Hcon F细胞增殖;然后用流式细胞仪检测细胞周期进程,发现TGF-β1促使Hcon F细胞由G1期进入S期和G2/M期;再用Western blot和q RTPCR法分别在蛋白水平和m RNA水平上检测结膜FB转化为肌成纤维细胞(myofibroblast,MF)的标志性α-肌动蛋白(α-smooth muscle actin,α-SMA)的表达变化,结果显示TGF-β1作用后α-SMA表达增强,说明TGF-β1促进人结膜FB向MF转化;我们还用细胞划痕实验和Transwell小室迁移实验检测了TGF-β1作用后Hcon F细胞迁移功能,结果发现TGF-β1可促进Hcon F细胞迁移;再用Western blot和q RT-PCR实验方法检测collagen-I和fibronectin这两种细胞外基质(extracellular matrix,ECM)的m RNA和蛋白表达变化,发现TGF-β1作用后,Hcon F细胞的collagen-I和fibronectin表达水平升高,说明TGF-β1促进Hcon F的ECM合成。以上结果表明TGF-β1可促进人结膜成纤维细胞的纤维化,可以在体外很好地模拟青光眼滤过术后创口的愈合过程。生物体内存在最多的阴离子是Cl-,机体多种生物学功能的完成都离不开Cl-。氯离子通道是位于细胞膜或细胞器膜上的一类跨膜蛋白,主要负责转运Cl-,哺乳动物的大多数组织器官中都存在氯离子通道。氯离子通道在机体内主要维持细胞容积、膜电位和p H值的稳定,同时也调节细胞的增殖、分化、凋亡、迁移等一系列生物学活动。氯离子通道阻滞剂5-硝基-2-(3苯丙氨基)苯甲酸(5-Nitro-2-(3-phenylpropylamino)benzoic acid,NPPB)可以非特异性地阻断氯离子通道,目前常被用来研究氯离子通道的功能。在此基础之上,我们用NPPB作用于TGF-β1诱导后的Hcon F细胞,再次用CCK-8实验方法检测细胞活力,发现NPPB抑制了TGF-β1诱导的Hcon F细胞增殖;用流式细胞仪检测细胞周期进程,结果证实NPPB可抑制TGF-β1诱导的Hcon F细胞周期进展,将细胞停滞于细胞周期的G1期;我们通过Western blot和q RT-PCR方法检测NPPB对TGF-β1诱导Hcon F细胞α-SMA、collagen-I和fibronectin表达的影响,发现NPPB可抑制TGF-β1诱导的α-SMA、collagen-I和fibronectin的合成;用细胞划痕和Transwell小室迁移实验测定Hcon F细胞的迁移功能,结果显示NPPB可抑制TGF-β1诱导的细胞迁移。由以上结果可以得出结论:NPPB可抑制TGF-β1诱导的Hcon F细胞纤维化。为进一步探索NPPB抑制Hcon F细胞纤维化过程的机制,我们在TGF-β1诱导的基础之上用NPPB作用于Hcon F细胞,然后用Western blot方法检测了Hcon F细胞PI3K和Akt磷酸化水平,发现TGF-β1可以促进Hcon F细胞PI3K和Akt的磷酸化,而NPPB可抑制TGF-β1诱导的PI3K和Akt磷酸化,由此结果可以推断,NPPB可能通过PI3K/Akt信号通路调控Hcon F的细胞纤维化过程。CLC-2作为氯离子通道家族中的一个亚型,是目前研究较为广泛和明确的一种氯离子通道类型。据报道NPPB不仅可以阻断CLC-2氯离子通道,对CLC-3氯离子通道也有阻断作用,鉴于NPPB是非特异性氯离子通道阻滞剂,我们用RNA干扰技术沉默Hcon F细胞中的CLC-2基因,然后再检测Hcon F细胞增殖、转化、迁移及ECM合成的变化,来明确CLC-2氯离子通道在Hcon F细胞纤维化过程中的作用。用CLC-2 si RNA和无义si RNA转染Hcon F细胞后,我们通过Western blot和q RT-PCR法检测CLC-2 si RNA的基因敲除效果,结果表明CLC-2 si RNA浓度依赖性地抑制CLC-2基因的m RNA和蛋白表达;之后,我们在TGF-β1诱导基础上,用CLC-2si RNA和si RNA-NC转染Hcon F细胞,再次用Western blot和q RTPCR法检测CLC-2表达情况,结果发现TGF-β1可促进Hcon F细胞CLC-2基因的m RNA和蛋白表达,而TGF-β1诱导的Hcon F细胞CLC-2表达可以被CLC-2 si RNA抑制;用TGF-β1诱导,再用CLC-2 si RNA和si RNA-NC转染之后,我们又通过CCK-8方法检测了Hcon F细胞增殖情况,结果发现CLC-2 si RNA转染会抑制TGF-β1诱导的Hcon F细胞增殖;我们还用Western blot和q RT-PCR法检测了α-SMA、collagen-I和fibronectin表达情况,结果表明CLC-2si RNA转染可以抑制TGF-β1诱导的α-SMA、collagen-I和fibronectin表达;之后我们又通过细胞划痕实验和Transwell小室迁移实验两种方法检测了CLC-2 si RNA转染对TGF-β1诱导的Hcon F细胞迁移的影响,结果也表明CLC-2 si RNA转染可以抑制TGF-β1诱导的Hcon F细胞迁移,这些结果说明CLC-2氯离子通道参与调控TGF-β1诱导的Hcon F细胞纤维化过程。为进一步探索CLC-2调控Hcon F细胞纤维化过程的机制,我们在TGF-β1诱导基础上,用CLC-2 si RNA转染Hcon F细胞后,然后通过Western blot方法测定了Hcon F细胞p-PI3K和p-Akt的表达水平,发现CLC-2 si RNA转染可抑制TGF-β1诱导的PI3K和Akt磷酸化,由此结果可以推测,CLC-2可能通过PI3K/Akt信号通路调控TGF-β1诱导的Hcon F细胞纤维化过程。本实验探讨了CLC-2氯离子通道在TGF-β1模拟的创口愈合过程中的调控作用,以及可能通过的信号通路,并得出结论,CLC-2氯离子通道通过PI3K/Akt信号通路调控TGF-β1诱导的HCon F细胞增殖、转化、迁移和ECM合成过程。提示CLC-2氯离子通道是滤过泡瘢痕形成过程的重要调控位点,为今后青光眼滤过术后抗瘢痕化提供了一个新的方向。
[Abstract]:Glaucoma is the first irreversible blindness worldwide, a serious threat to people's visual function and quality of life. Filtration surgery is the main method of surgical treatment of glaucoma, but the success rate has been affected by the bleb scarring restrictions. At present, 5- fluorouracil (5-fluorouracil, 5-Fu) and mitomycin C (Mitomycin C MMC) and anti metabolic drugs has been used to inhibit the bleb scarring, but its cell toxicity caused by bleb leak, endophthalmitis, low intraocular pressure, shallow anterior chamber, corneal endothelial decompensation and other serious complications limit its further application in anti scarring in fewer side effects. Therefore, exploring new the important significance of anti scarring drugs more effective. Conjunctival fibroblasts (fibroblast, FB) is mainly involved in cell formation of the bleb and filtration surgery will stimulate node fibroblast cells into muscle Fibroblasts, and promote conjunctival fibroblast proliferation, migration and synthesis and secretion of extracellular matrix (extracellular, matrix, ECM), so as to promote wound healing and scar formation. Transforming growth factor beta (transfer beta growth factor, TGF-) by macrophages, lymphocytes, fibroblasts (FB), a the class of bioactive peptides secretion of vascular endothelial cells and some stromal cells, wound healing and scar formation is the main growth factor beta.TGF- process by autocrine or paracrine regulation of cell proliferation, differentiation and ECM. These features make TGF- beta become an important profibrotic growth factor, can promote organ in the process of tissue fibrosis, the formation of filtering bleb scar, TGF- beta is also important fibrogenetic factors. The research and application of TGF- beta 1 in human conjunctiva fibroblast cells (human conjunc Tival fibroblast, Hcon F), using CCK-8 assay Hcon F cell proliferation, the results show that TGF- beta 1 can time dependently stimulated Hcon proliferation of F cells; then by flow cytometry cell cycle process, found that TGF- beta 1 prompted Hcon F cells into S phase and G2/M phase from G1 phase; with Western blot and Q RTPCR were used to detect the conjunctival FB into myofibroblasts in protein level and m level of RNA (myofibroblast, MF) marker alpha actin (-smooth muscle alpha actin, alpha -SMA) expression, the results show that TGF- beta 1 by alpha increased the expression of -SMA and TGF- beta 1 promotes human conjunctival FB transformation to MF; we also use cell scratch test and Transwell chamber migration assay of TGF- beta 1 after Hcon F cell migration, the results showed that TGF- beta 1 Hcon F can promote cell migration; then Western blot and Q RT-PCR experimental method of inspection Collagen-I and fibronectin these two kinds of extracellular matrix (extracellular matrix ECM) m RNA and protein expression of TGF- beta 1, found after a higher expression level of Hcon collagen-I and fibronectin F cells, indicating that TGF- beta 1 promotes ECM synthesis of Hcon F. The above results showed that TGF- beta 1 can promote the human conjunctiva fibroblast fibrosis, can well simulate the process of wound healing after glaucoma filtering surgery in vitro. The presence of organisms most anion is Cl-, the body of various biological functions are complete cannot do without Cl-. chloride channel is a transmembrane protein located on the cell membrane or organelle membrane of a class, mainly responsible for transport of Cl- chloride channel are the most mammalian tissues. Chloride channels in the body mainly maintain cell volume, membrane potential and P value of H is stable, but also regulate cell proliferation, differentiation, apoptosis, A series of biological activities. The migration of chloride channel blockers 5- nitro -2- (3 phenyl amino benzoic acid) (5-Nitro-2- (3-phenylpropylamino) benzoic acid, NPPB) can specifically block the chloride channel, the current is often used to study chlorine ion channel function. On this basis, we use the NPPB function in TGF- beta 1 Hcon after induction of F cells, again using CCK-8 assay cell viability, NPPB inhibited TGF- induced Hcon F 1 beta cell proliferation; cell cycle by flow cytometry, confirmed that NPPB can inhibit the progression of TGF- beta 1 induced Hcon F cell cycle arrest in cell cycle, cell G1 blot and Q Western; the RT-PCR NPPB method for the detection of TGF- beta 1 induced Hcon F cell alpha -SMA, expression of collagen-I and fibronectin, found that NPPB could inhibit TGF- alpha beta 1 induced by -SMA, collagen-I and fibronectin The transfer function synthesis; experimental determination of Hcon of F cells by cell scratch assay and Transwell chamber migration, the results showed that NPPB could inhibit TGF- cell migration induced by beta 1. From the above results, we can draw the conclusion: NPPB can inhibit TGF- beta 1 induced Hcon F cell fibrosis. To further explore the mechanism of NPPB inhibiting Hcon F fibrosis process we, on the basis of 1 TGF- induced by beta NPPB in Hcon F cells, then detected the Hcon F cell PI3K and Akt phosphorylation by Western blot method, TGF- beta 1 can promote Hcon F cells PI3K and Akt phosphorylation, while NPPB inhibited TGF- beta 1 and PI3K induced Akt phosphorylation thus, it could be concluded that NPPB may through PI3K/Akt signal transduction pathway of Hcon.CLC-2 cells in the fibrosis process of F as a subtype of chloride channel family, is currently studying a wide and clear chloride The channel type. It is reported that NPPB can not only block the CLC-2 chloride channel, also has a blocking effect on the CLC-3 chloride channel, whereas NPPB is non-specific chloride channel blocker, we use RNA interference CLC-2 gene silencing Hcon in F cells, then detected Hcon F cell proliferation, migration and transformation, change ECM synthesis, to clear the CLC-2 chloride channel in Hcon F cell fibrosis. CLC-2 Si RNA and Si RNA nonsense transfected Hcon F cells, we detected by CLC-2 Si RNA Western blot and Q RT-PCR gene knockout effect. The results show that the CLC-2 Si RNA concentration dependent inhibition of m the RNA and protein expression of CLC-2 gene; later, we in TGF- beta 1 induced on the basis of CLC-2si RNA and Si RNA-NC transfected Hcon F cells detected by CLC-2 Western blot again and Q RTPCR expression, the results showed that TGF- can promote Hcon F beta 1 The expression of M RNA and protein of CLC-2 gene, and TGF- beta 1 induced Hcon F cells expression of CLC-2 can be CLC-2 Si RNA by TGF- inhibition; beta 1 induced by CLC-2, Si and Si after RNA RNA-NC transfection, we detected Hcon F cell proliferation by CCK-8 method, the results showed that CLC-2 Si RNA expression inhibition of TGF- beta 1 induced Hcon F cell proliferation; we also used Western blot and Q RT-PCR method to detect the alpha -SMA, collagen-I and fibronectin expression, the results showed that CLC-2si RNA transfection can inhibit TGF- alpha -SMA beta 1 induced the expression of collagen-I and fibronectin; then we by cell scratch assay and Transwell migration assay two methods to detect the effects of CLC-2 Si RNA 1 Hcon F induced migration of transfected cells to TGF- beta, Si results indicated that CLC-2 RNA transfection can inhibit TGF- beta 1 induced Hcon F cell migration, these results indicate that CLC-2 Chloride channels are involved in the regulation of TGF- beta 1 induced Hcon F cell fibrosis. To further explore the mechanism of CLC-2 regulation of Hcon F cells in fibrosis, we TGF- beta 1 induced by CLC-2 based on Si Hcon RNA was transfected into F cells, and then through the Western blot method was used to determine the expression level of Hcon p-PI3K and p-Akt F cells CLC-2 Si, found that RNA transfection can inhibit TGF- beta 1 and PI3K induced Akt phosphorylation, thus it suggests that CLC-2 may be through the process of PI3K/Akt signal transduction pathway of TGF- beta 1 induced Hcon F cell fibrosis. The experiment studied CLC-2 chloride ion channel in TGF- beta 1 simulated regulation in the process of wound healing well, probably through the signal pathway, and concluded that the CLC-2 chloride channel through PI3K/Akt signaling pathway TGF- beta 1 induced HCon F cell proliferation, migration and transformation, ECM suggested that CLC-2 chloride synthesis process. The subchannel is an important regulatory site for the formation of filter bleb scar formation, which provides a new direction for the anti scarring in the future after glaucoma filtering surgery.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R775

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