本文選題：青光眼濾過術(shù)　切入點：瘢痕化　出處：《吉林大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：青光眼是全球第一位不可逆性致盲眼病,嚴重威脅人們的視功能和生活質(zhì)量。濾過性手術(shù)是目前治療青光眼的最主要手術(shù)方法,但是,其成功率一直受濾過泡瘢痕化的限制。目前,5-氟尿嘧啶(5-fluorouracil,5-Fu)和絲裂霉素C(Mitomycin C,MMC)等抗代謝藥物已經(jīng)被用于抑制濾過泡瘢痕形成,但是其對細胞的毒副作用引起濾過泡漏、眼內(nèi)炎、低眼壓、淺前房、角膜內(nèi)皮失代償?shù)葒乐氐牟l(fā)癥限制了其在抗瘢痕化中的進一步應(yīng)用。因此,探索新的副作用更少、更有效的抗瘢痕化藥物具有重要意義。結(jié)膜成纖維細胞(fibroblast,FB)是參與濾過泡瘢痕形成的主要細胞,濾過手術(shù)會刺激結(jié)膜成纖維細胞轉(zhuǎn)化為肌成纖維細胞,并促進結(jié)膜成纖維細胞增殖、遷移及合成和分泌細胞外基質(zhì)(extracellular matrix,ECM),從而促進創(chuàng)面修復(fù)及瘢痕形成。轉(zhuǎn)化生長因子β(transfer growth factorβ,TGF-β)是由巨噬細胞、淋巴細胞、成纖維細胞(FB)、血管內(nèi)皮細胞和某些基質(zhì)細胞分泌的一類生物活性多肽,是創(chuàng)口愈合和瘢痕形成過程中最主要的生長因子。TGF-β通過自分泌或旁分泌的方式調(diào)控細胞增殖、分化和ECM合成。這些功能使TGF-β成為一種重要的促纖維化生長因子,可以促進器官組織纖維化的發(fā)生,在青光眼濾過泡瘢痕形成過程中,TGF-β也是重要的致纖維化細胞因子。本研究應(yīng)用TGF-β1作用于人結(jié)膜成纖維細胞(human conjunctival fibroblast,Hcon F)后,用CCK-8實驗方法檢測Hcon F細胞增殖情況,結(jié)果顯示TGF-β1可以時間依賴性地促進Hcon F細胞增殖;然后用流式細胞儀檢測細胞周期進程,發(fā)現(xiàn)TGF-β1促使Hcon F細胞由G1期進入S期和G2/M期;再用Western blot和q RTPCR法分別在蛋白水平和m RNA水平上檢測結(jié)膜FB轉(zhuǎn)化為肌成纖維細胞(myofibroblast,MF)的標(biāo)志性α-肌動蛋白(α-smooth muscle actin,α-SMA)的表達變化,結(jié)果顯示TGF-β1作用后α-SMA表達增強,說明TGF-β1促進人結(jié)膜FB向MF轉(zhuǎn)化;我們還用細胞劃痕實驗和Transwell小室遷移實驗檢測了TGF-β1作用后Hcon F細胞遷移功能,結(jié)果發(fā)現(xiàn)TGF-β1可促進Hcon F細胞遷移;再用Western blot和q RT-PCR實驗方法檢測collagen-I和fibronectin這兩種細胞外基質(zhì)(extracellular matrix,ECM)的m RNA和蛋白表達變化,發(fā)現(xiàn)TGF-β1作用后,Hcon F細胞的collagen-I和fibronectin表達水平升高,說明TGF-β1促進Hcon F的ECM合成。以上結(jié)果表明TGF-β1可促進人結(jié)膜成纖維細胞的纖維化,可以在體外很好地模擬青光眼濾過術(shù)后創(chuàng)口的愈合過程。生物體內(nèi)存在最多的陰離子是Cl-,機體多種生物學(xué)功能的完成都離不開Cl-。氯離子通道是位于細胞膜或細胞器膜上的一類跨膜蛋白,主要負責(zé)轉(zhuǎn)運Cl-,哺乳動物的大多數(shù)組織器官中都存在氯離子通道。氯離子通道在機體內(nèi)主要維持細胞容積、膜電位和p H值的穩(wěn)定,同時也調(diào)節(jié)細胞的增殖、分化、凋亡、遷移等一系列生物學(xué)活動。氯離子通道阻滯劑5-硝基-2-(3苯丙氨基)苯甲酸(5-Nitro-2-(3-phenylpropylamino)benzoic acid,NPPB)可以非特異性地阻斷氯離子通道,目前常被用來研究氯離子通道的功能。在此基礎(chǔ)之上,我們用NPPB作用于TGF-β1誘導(dǎo)后的Hcon F細胞,再次用CCK-8實驗方法檢測細胞活力,發(fā)現(xiàn)NPPB抑制了TGF-β1誘導(dǎo)的Hcon F細胞增殖;用流式細胞儀檢測細胞周期進程,結(jié)果證實NPPB可抑制TGF-β1誘導(dǎo)的Hcon F細胞周期進展,將細胞停滯于細胞周期的G1期;我們通過Western blot和q RT-PCR方法檢測NPPB對TGF-β1誘導(dǎo)Hcon F細胞α-SMA、collagen-I和fibronectin表達的影響,發(fā)現(xiàn)NPPB可抑制TGF-β1誘導(dǎo)的α-SMA、collagen-I和fibronectin的合成;用細胞劃痕和Transwell小室遷移實驗測定Hcon F細胞的遷移功能,結(jié)果顯示NPPB可抑制TGF-β1誘導(dǎo)的細胞遷移。由以上結(jié)果可以得出結(jié)論:NPPB可抑制TGF-β1誘導(dǎo)的Hcon F細胞纖維化。為進一步探索NPPB抑制Hcon F細胞纖維化過程的機制,我們在TGF-β1誘導(dǎo)的基礎(chǔ)之上用NPPB作用于Hcon F細胞,然后用Western blot方法檢測了Hcon F細胞PI3K和Akt磷酸化水平,發(fā)現(xiàn)TGF-β1可以促進Hcon F細胞PI3K和Akt的磷酸化,而NPPB可抑制TGF-β1誘導(dǎo)的PI3K和Akt磷酸化,由此結(jié)果可以推斷,NPPB可能通過PI3K/Akt信號通路調(diào)控Hcon F的細胞纖維化過程。CLC-2作為氯離子通道家族中的一個亞型,是目前研究較為廣泛和明確的一種氯離子通道類型。據(jù)報道NPPB不僅可以阻斷CLC-2氯離子通道,對CLC-3氯離子通道也有阻斷作用,鑒于NPPB是非特異性氯離子通道阻滯劑,我們用RNA干擾技術(shù)沉默Hcon F細胞中的CLC-2基因,然后再檢測Hcon F細胞增殖、轉(zhuǎn)化、遷移及ECM合成的變化,來明確CLC-2氯離子通道在Hcon F細胞纖維化過程中的作用。用CLC-2 si RNA和無義si RNA轉(zhuǎn)染Hcon F細胞后,我們通過Western blot和q RT-PCR法檢測CLC-2 si RNA的基因敲除效果,結(jié)果表明CLC-2 si RNA濃度依賴性地抑制CLC-2基因的m RNA和蛋白表達;之后,我們在TGF-β1誘導(dǎo)基礎(chǔ)上,用CLC-2si RNA和si RNA-NC轉(zhuǎn)染Hcon F細胞,再次用Western blot和q RTPCR法檢測CLC-2表達情況,結(jié)果發(fā)現(xiàn)TGF-β1可促進Hcon F細胞CLC-2基因的m RNA和蛋白表達,而TGF-β1誘導(dǎo)的Hcon F細胞CLC-2表達可以被CLC-2 si RNA抑制;用TGF-β1誘導(dǎo),再用CLC-2 si RNA和si RNA-NC轉(zhuǎn)染之后,我們又通過CCK-8方法檢測了Hcon F細胞增殖情況,結(jié)果發(fā)現(xiàn)CLC-2 si RNA轉(zhuǎn)染會抑制TGF-β1誘導(dǎo)的Hcon F細胞增殖;我們還用Western blot和q RT-PCR法檢測了α-SMA、collagen-I和fibronectin表達情況,結(jié)果表明CLC-2si RNA轉(zhuǎn)染可以抑制TGF-β1誘導(dǎo)的α-SMA、collagen-I和fibronectin表達;之后我們又通過細胞劃痕實驗和Transwell小室遷移實驗兩種方法檢測了CLC-2 si RNA轉(zhuǎn)染對TGF-β1誘導(dǎo)的Hcon F細胞遷移的影響,結(jié)果也表明CLC-2 si RNA轉(zhuǎn)染可以抑制TGF-β1誘導(dǎo)的Hcon F細胞遷移,這些結(jié)果說明CLC-2氯離子通道參與調(diào)控TGF-β1誘導(dǎo)的Hcon F細胞纖維化過程。為進一步探索CLC-2調(diào)控Hcon F細胞纖維化過程的機制,我們在TGF-β1誘導(dǎo)基礎(chǔ)上,用CLC-2 si RNA轉(zhuǎn)染Hcon F細胞后,然后通過Western blot方法測定了Hcon F細胞p-PI3K和p-Akt的表達水平,發(fā)現(xiàn)CLC-2 si RNA轉(zhuǎn)染可抑制TGF-β1誘導(dǎo)的PI3K和Akt磷酸化,由此結(jié)果可以推測,CLC-2可能通過PI3K/Akt信號通路調(diào)控TGF-β1誘導(dǎo)的Hcon F細胞纖維化過程。本實驗探討了CLC-2氯離子通道在TGF-β1模擬的創(chuàng)口愈合過程中的調(diào)控作用,以及可能通過的信號通路,并得出結(jié)論,CLC-2氯離子通道通過PI3K/Akt信號通路調(diào)控TGF-β1誘導(dǎo)的HCon F細胞增殖、轉(zhuǎn)化、遷移和ECM合成過程。提示CLC-2氯離子通道是濾過泡瘢痕形成過程的重要調(diào)控位點,為今后青光眼濾過術(shù)后抗瘢痕化提供了一個新的方向。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R775

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