组织型转谷氨酰胺酶在后囊膜混浊中的作用研究

发布时间：2018-03-24 06:33

  本文选题：组织型转谷氨酰胺酶　切入点：晶状体上皮细胞　出处：《华中科技大学》2010年博士论文

【摘要】： 第一部分tTG抑制剂对人晶状体上皮细胞表达FN、Col-Ⅳ的影响研究 目的观察组织型转谷氨酰胺酶(tissue transglutaminase, tTG)抑制剂单丹磺酰尸胺monodansyl-cadaverine (MDC)对转化生长因子β2 (transforming growth factor-β2, TGF-β2)诱导的人晶状体上皮细胞(Human lens epithelial cells, HLECs)表达纤维连接蛋白(Fibrone-ctin, FN)和Ⅳ型胶原(collagenⅣ, Col-Ⅳ)的影响。 方法将体外培养的人晶状体上皮细胞株HLE-B3分为5组：无血清培养基中培养的HLE-B3为正常对照组；加入10μg/L TGF-β2处理36h的HLE-B3为TGF-p2组;10μg／LTGF-p2与100、200和400μmol/L MDC共同处理36h的HLE-B3为不同浓度MDC组。用半定量RT-PCR检测tTG、FN、Col-Ⅳ在HLE-B3细胞中表达的变化。 结果正常体外培养的HLE-B3细胞中可表达一定量的tTG, FN及Col-Ⅳ。与正常对照组相比,TGF-β2组中tTG, FN和Col-Ⅳ的表达明显增强,差异有统计学意义(P0.01)。与TGF-β2组相比,100、200和400μmol/L MDC组中FN和Col-Ⅳ的表达含量明显减少(P0.01)。100、200和400μmol/L MDC组各组间FN和Col-Ⅳ的表达差异亦具有显著性意义(P0.01)。 结论MDC可剂量依赖性的抑制TGF-p2诱导的LECs中FN和Col-Ⅳ表达的增加。tTG可能通过促进HLECs表达FN和Col-Ⅳ等细胞外基质成分参与白内障术后后囊膜混浊(posterior capsule opacification, PCO)的形成。 第二部分RNA干扰对人晶状体上皮细胞tTG表达的影响研究 目的研究RNA干扰抑制组织型转谷氨酰胺酶(tissue transglutaminase, tTG)对人晶状体上皮细胞(Human lens epithelial cells, HLECs)中tTG表达的影响。 方法设计并合成特异性针对tTG的3对小干扰RNA(short interfering RNA, siRNA): siRNA-1、siRNA-2、siRNA-3,用Real-time RT-PCR和Western blot法检测转染了siRNA后人晶状体上皮细胞株(HLE-B3)中tTG基因在mRNA水平和蛋白水平的变化。 结果转染siRNA-1、siRNA-2、siRNA-348h后,tTG mRNA的表达分别下降为空白对照组的46.60%,12.84%,66.75%,与空白对照组相比差异具有统计学意义(P0.05)。转染siRNA-1、siRNA-2、siRNA-360h后,tTG蛋白的表达含量分别下降为空白对照组的60.49%,27.87%,55.91%(P0.01)。Real-time RT-PCR与Western blot检测结果一致显示了siRNA-2对tTG基因的抑制效果最佳。 结论我们设计并合成的靶向tTG的siRNA可以显著降低tTG的表达。 第三部分tTG-siRNA对人晶状体上皮细胞转分化和细胞外基质沉积的作用研究 目的探讨RNA干扰抑制组织型转谷氨酰胺酶(tissue transglutaminase, tTG)的表达对人转化生长因子β2(transforming growth factorβ2, TGF-β2)诱导的人晶状体上皮细胞(Human lens epithelial cells, HLECs)转分化和细胞外基质沉积的抑制作用。 方法将体外培养的人晶状体上皮细胞株HLE-B3分为正常对照组,TGF-β2组和TGF-β2+siRNA组。Western blot法检测各组细胞tTG、α平滑肌肌动蛋白(a-Smooth muscle actin, a-SMA)、纤维连接蛋白(Fibronectin, FN)和Ⅳ型胶原(collagen IV, Col-IV)的表达。 结果与正常对照组相比,TGF-β2组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显增加(P0.01)；与TGF-β2组相比,TGF-β2+siRNA组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显减少(P0.01)。 结论靶向tTG的siRNA可以明显抑制TGF-β2诱导的HLE-B3细胞合成a-SMA、FN、Col-Ⅳ。提示tTG是介导TGF-β2诱导人晶状体上皮细胞转分化和细胞外基质沉积的重要分子,tTG蛋白有望成为预防和治疗后囊膜混浊(posterior capsule opacification, PCO)的新靶点。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R776.1

【参考文献】

相关期刊论文 前3条

1 王辉;刘哲丽;郝旭红;徐丽;;增生性玻璃体视网膜病变中组织型转谷氨酰胺酶的表达[J];国际眼科杂志;2007年02期

2 陈春林;叶剑;;RNA干扰技术及其在眼科疾病中的应用[J];眼科新进展;2008年01期

3 王欣玲,张劲松,于韬;层黏连蛋白和纤维连接蛋白对人晶状体上皮细胞生长特性的影响[J];中华眼科杂志;2005年04期

，

本文编号：1657155