本文選題：組織型轉(zhuǎn)谷氨酰胺酶　切入點：晶狀體上皮細(xì)胞　出處：《華中科技大學(xué)》2010年博士論文

【摘要】： 第一部分tTG抑制劑對人晶狀體上皮細(xì)胞表達(dá)FN、Col-Ⅳ的影響研究 目的觀察組織型轉(zhuǎn)谷氨酰胺酶(tissue transglutaminase, tTG)抑制劑單丹磺酰尸胺monodansyl-cadaverine (MDC)對轉(zhuǎn)化生長因子β2 (transforming growth factor-β2, TGF-β2)誘導(dǎo)的人晶狀體上皮細(xì)胞(Human lens epithelial cells, HLECs)表達(dá)纖維連接蛋白(Fibrone-ctin, FN)和Ⅳ型膠原(collagenⅣ, Col-Ⅳ)的影響。 方法將體外培養(yǎng)的人晶狀體上皮細(xì)胞株HLE-B3分為5組：無血清培養(yǎng)基中培養(yǎng)的HLE-B3為正常對照組；加入10μg/L TGF-β2處理36h的HLE-B3為TGF-p2組;10μg／LTGF-p2與100、200和400μmol/L MDC共同處理36h的HLE-B3為不同濃度MDC組。用半定量RT-PCR檢測tTG、FN、Col-Ⅳ在HLE-B3細(xì)胞中表達(dá)的變化。 結(jié)果正常體外培養(yǎng)的HLE-B3細(xì)胞中可表達(dá)一定量的tTG, FN及Col-Ⅳ。與正常對照組相比,TGF-β2組中tTG, FN和Col-Ⅳ的表達(dá)明顯增強,差異有統(tǒng)計學(xué)意義(P0.01)。與TGF-β2組相比,100、200和400μmol/L MDC組中FN和Col-Ⅳ的表達(dá)含量明顯減少(P0.01)。100、200和400μmol/L MDC組各組間FN和Col-Ⅳ的表達(dá)差異亦具有顯著性意義(P0.01)。 結(jié)論MDC可劑量依賴性的抑制TGF-p2誘導(dǎo)的LECs中FN和Col-Ⅳ表達(dá)的增加。tTG可能通過促進(jìn)HLECs表達(dá)FN和Col-Ⅳ等細(xì)胞外基質(zhì)成分參與白內(nèi)障術(shù)后后囊膜混濁(posterior capsule opacification, PCO)的形成。 第二部分RNA干擾對人晶狀體上皮細(xì)胞tTG表達(dá)的影響研究 目的研究RNA干擾抑制組織型轉(zhuǎn)谷氨酰胺酶(tissue transglutaminase, tTG)對人晶狀體上皮細(xì)胞(Human lens epithelial cells, HLECs)中tTG表達(dá)的影響。 方法設(shè)計并合成特異性針對tTG的3對小干擾RNA(short interfering RNA, siRNA): siRNA-1、siRNA-2、siRNA-3,用Real-time RT-PCR和Western blot法檢測轉(zhuǎn)染了siRNA后人晶狀體上皮細(xì)胞株(HLE-B3)中tTG基因在mRNA水平和蛋白水平的變化。 結(jié)果轉(zhuǎn)染siRNA-1、siRNA-2、siRNA-348h后,tTG mRNA的表達(dá)分別下降為空白對照組的46.60%,12.84%,66.75%,與空白對照組相比差異具有統(tǒng)計學(xué)意義(P0.05)。轉(zhuǎn)染siRNA-1、siRNA-2、siRNA-360h后,tTG蛋白的表達(dá)含量分別下降為空白對照組的60.49%,27.87%,55.91%(P0.01)。Real-time RT-PCR與Western blot檢測結(jié)果一致顯示了siRNA-2對tTG基因的抑制效果最佳。 結(jié)論我們設(shè)計并合成的靶向tTG的siRNA可以顯著降低tTG的表達(dá)。 第三部分tTG-siRNA對人晶狀體上皮細(xì)胞轉(zhuǎn)分化和細(xì)胞外基質(zhì)沉積的作用研究 目的探討RNA干擾抑制組織型轉(zhuǎn)谷氨酰胺酶(tissue transglutaminase, tTG)的表達(dá)對人轉(zhuǎn)化生長因子β2(transforming growth factorβ2, TGF-β2)誘導(dǎo)的人晶狀體上皮細(xì)胞(Human lens epithelial cells, HLECs)轉(zhuǎn)分化和細(xì)胞外基質(zhì)沉積的抑制作用。 方法將體外培養(yǎng)的人晶狀體上皮細(xì)胞株HLE-B3分為正常對照組,TGF-β2組和TGF-β2+siRNA組。Western blot法檢測各組細(xì)胞tTG、α平滑肌肌動蛋白(a-Smooth muscle actin, a-SMA)、纖維連接蛋白(Fibronectin, FN)和Ⅳ型膠原(collagen IV, Col-IV)的表達(dá)。 結(jié)果與正常對照組相比,TGF-β2組中tTG、α-SMA、FN、Col-Ⅳ的表達(dá)量均明顯增加(P0.01)；與TGF-β2組相比,TGF-β2+siRNA組中tTG、α-SMA、FN、Col-Ⅳ的表達(dá)量均明顯減少(P0.01)。 結(jié)論靶向tTG的siRNA可以明顯抑制TGF-β2誘導(dǎo)的HLE-B3細(xì)胞合成a-SMA、FN、Col-Ⅳ。提示tTG是介導(dǎo)TGF-β2誘導(dǎo)人晶狀體上皮細(xì)胞轉(zhuǎn)分化和細(xì)胞外基質(zhì)沉積的重要分子,tTG蛋白有望成為預(yù)防和治療后囊膜混濁(posterior capsule opacification, PCO)的新靶點。
[Abstract]:The effect of tTG inhibitor on the expression of FN and Col- IV in human lens epithelial cells
Objective To observe the expression of tissue transglutaminase (tissue transglutaminase tTG) inhibitors monodansylcadaverin (monodansyl-cadaverine MDC) on transforming growth factor beta 2 (transforming growth factor- TGF- beta 2, beta 2) in human lens epithelial cells induced by (Human lens epithelial cells, HLECs) expression of fibronectin (Fibrone-ctin, FN) and IV collagen (collagen IV, Col- IV) effect.
Methods cultured human lens epithelial HLE-B3 cells were divided into 5 groups: serum free medium HLE-B3 as normal control group; adding 10 g/L TGF- beta 2 36h HLE-B3 treatment group TGF-p2; 10 g / LTGF-p2 with 100200 and 400 mol/L MDC treatment 36h HLE-B3 of different concentration MDC group. TTG was detected by semi quantitative RT-PCR, FN, expression of Col- IV in HLE-B3 cells.
Results HLE-B3 cells cultured in vitro can express a certain amount of tTG, FN and Col- IV. Compared with the normal control group, TGF- beta 2 in the group of tTG, the expression of FN and Col- IV increased significantly, the difference was statistically significant (P0.01). And TGF- beta 2 expression group, the contents of FN and Col- IV 100200 and 400 mol/L in the MDC group decreased significantly (P0.01) expression of.100200 and 400 mol/L between groups MDC FN and Col- IV was also of significance (P0.01).
Conclusion MDC can inhibit TGF-p2 dose dependently induced LECs expression in FN and Col- IV increased.TTG may promote the expression of HLECs FN and Col- IV, extracellular matrix components involved in posterior capsule opacification after cataract surgery (posterior capsule, opacification, PCO) is formed.
The study of the effect of the second part RNA interference on the expression of tTG in human lens epithelial cells
Objective to study the effect of RNA interference on the expression of tissue transglutaminase (tTG) in the expression of tTG in human lens epithelial cells (Human lens epithelial cells, HLECs).
The design and synthesis of specific methods in 3 pairs of small interfering RNA (siRNA tTG short interfering RNA, siRNA-1, siRNA-2, siRNA-3): siRNA, transfected human lens epithelial cell line by Real-time RT-PCR and Western blot (HLE-B3) changes in the tTG gene at mRNA and protein level.
The results of transfection of siRNA-1 siRNA-2, siRNA-348h, tTG mRNA expression were decreased to the control group 46.60%, 12.84%, 66.75%, and the control group with statistical significance difference (P0.05). SiRNA-1 siRNA-2, siRNA-360h after transfection, and the expression of tTG protein content were decreased to the control group 60.49%, 27.87%, 55.91% (P0.01).Real-time RT-PCR and Western blot results showed the inhibitory effect of siRNA-2 on the tTG gene.
Conclusion the siRNA target of the target tTG which we designed and synthesized can significantly reduce the expression of tTG.
The effect of third part tTG-siRNA on the transdifferentiation and extracellular matrix deposition of human lens epithelial cells
Objective to investigate the inhibitory effect of RNA interference of tissue transglutaminase (tissue, transglutaminase, tTG) on the expression of transforming growth factor beta 2 (transforming growth factor TGF- beta 2, beta 2) in human lens epithelial cells induced by (Human lens epithelial cells, HLECs) to inhibit differentiation and extracellular matrix deposition.
Methods cultured human lens epithelial HLE-B3 cells were divided into normal control group, to detect the expression of tTG beta 2 TGF- group and TGF- group 2+siRNA beta.Western blot, alpha smooth muscle actin (a-Smooth muscle, actin, a-SMA), fibronectin (Fibronectin, FN) and collagen type IV (collagen IV, Col-IV) expression.
Results compared with the normal control group, the expressions of tTG, alpha -SMA, FN and Col- IV in the TGF- beta 2 group increased significantly (P0.01), and the expressions of tTG, alpha, -SMA, and -SMA in TGF- beta 2+siRNA group were significantly reduced compared with those in the TGF- group (P0.01).
HLE-B3 synthesis of a-SMA cells, conclusion tTG targeting siRNA can inhibit TGF- induced FN beta 2, Col- IV. It suggests that tTG is an important molecule mediated TGF- beta 2 induced human lens epithelial cell transdifferentiation and extracellular matrix deposition, tTG protein is expected to become the prevention and treatment of posterior capsule opacification (posterior capsule, opacification, PCO) the new target.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R776.1

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本文編號：1657155