Myocilin Asn 450 Tyr突变促进人原代小梁网细胞中细胞外基质蛋白的表达及意义

Myocilin Asn 450 Tyr promotes the expression of extracellular matrix protein in human primary trabecular meshwork cells and the significance

目的研究小梁网糖皮质激素诱导反应蛋白基因Myocilin N450Y突变(c.A1348T: p.N450Y, Myoc-N450Y)对人原代小梁网细胞(HTMC)细胞外基质(ECM)蛋白相关基因表达的影响及其参与青光眼发生发展的机制。设计实验研究。研究对象人原代小梁网细胞。方法实验分为三组,共9个样本,分别为空载体组、野生型Myoc (Myoc-WT)组及Myoc-N450Y组,用慢病毒感染的方法将Myoc-WT基因及Myoc-N450Y突变基因在HTMC中过表达,用蛋白质印迹法及荧光定量PCR方法检测Myoc-WT组和Myoc-N450Y组ECM蛋白Ⅰ型胶原蛋白(COL Ⅰ)、Ⅳ型胶原蛋白(COL Ⅳ)、纤连蛋白(FN)及基质金属蛋白酶(MMP)相关基因基质金属蛋白酶2(Mmp2)、基质金属蛋白酶9(Mmp9)的表达差异;构建野生型Myoc基因(Myoc-WT)及Asn 450 Tyr突变的Myoc基因(Myoc-N450Y)慢病毒载体,包装并确定慢病毒感染HTMC的条件;应用Myoc-WT慢病毒颗粒及Myoc-N450Y慢病毒颗粒感染HTMC,采用蛋白印迹法及荧光定量PCR方法检测Myoc基因的过表达情况及N450Y突变后对Col Ⅰ、Col Ⅳ、Fn、Mmp2、Mmp9表达的影响。主要指标 Col Ⅰ、Col Ⅳ、Fn、Mmp2、Mmp9基因的表达量。结果酶切鉴定结果及测序结果显示成功构建过表达Myoc-WT基因及Myoc-N450Y基因的慢病毒载体,并且慢病毒颗粒感染HTMC的感染复数(MOI)为5。蛋白质印迹及荧光定量PCR结果显示Myoc-WT及Myoc-N450Y组Myoc基因的蛋白水平及mRNA水平均高效过表达,空载体组、Myoc-WT组及Myoc-N450Y组Myoc基因mRNA相对表达量分别为1.00±0.11、28.88±1.28、22.06±0.47(F=1020.02,P<0.001)。荧光定量PCR结果显示,Myoc-N450Y组Col Ⅰ、Col Ⅳ及Fn基因 mRNA相对表达量分别为2.08±0.07、1.76±0.08、2.63±0.06,显著高于Myoc-WT组的0.93±0.04、0.95±0.09、0.89±0.06(P均<0.001)。且与Myoc-WT组相比,Myoc-N450Y组COL Ⅰ及FN蛋白表达水平显著上调。Myoc-N450Y组Mmp2、Mmp9 mRNA相对表达量分别为0.32±0.02和0.33±0.15,分别显著低于Myoc-WT组的1.01±0.03和0.95±0.03(P均<0.001)。结论成功制备可在HTMC过表达Myoc基因的慢病毒。过表达Myoc-N450Y基因对HTMC中ECM蛋白相关基因Col Ⅰ、Col Ⅳ及FN的表达具有促进作用,对MMP蛋白相关基因Mmp2、Mmp9的表达具有抑制作用。推测Myoc-N450Y可能通过调节ECM蛋白相关基因的表达参与青光眼的发生发展。

Objective To investigate the effects of Myocilin point mutation(c.A1348T: p.N450Y, Myoc-N450Y) on the expression of extracellular matrix (ECM) protein in human primary trabecular meshwork cells (HTMC), so as to explore the mechanism of Myoc-N450Y causing glaucoma. Design Experimental study. Participants Human primary trabecular meshwork cells. Methods The experiment was divided into three groups: nine samples in total, empty vector group (Empty), wild type myoc (Myoc-WT) group and Myoc-N450Y group. Lentivirus mediated overexpression method was used to overexpress Myoc-WT gene or Myoc-N450Y gene in HTMC. Western Blot and qRT-PCR were used to quantify the expression of ECM protein collagenⅠ(COL Ⅰ), collagen Ⅳ(COL Ⅳ), fibronectin (FN) and matrix metalloproteinase (MMP) related gene matrix metalloproteinase 2 (Mmp2), matrix metalloproteinase 9 (Mmp9) in Myoc-WT or Myoc-N450Y group. Lentiviral vectors of Myoc-WT and Myoc-N450Y were constructed. Lentivirus expressing Myoc-WT or Myoc-N450Y was packaged and the multiplicity of infection (MOI) of HTMC was determined. HTMC was infected by Myoc-WT lentivirus particles or Myoc-N450Y lentivirus particles. Western Blot and qRT-PCR were used to detect the overexpression of Myoc gene and the effect of N450Y mutation on the expression of Col Ⅰ, Col Ⅳ, Fn, Mmp2 and Mmp9. Main Outcome Measures The expression of Col Ⅰ, Col Ⅳ, Fn, Mmp2 and Mmp9. Results The results of double enzyme digestion and sequencing showed that lentivirus vectors overexpressing Myoc-WT or Myoc-N450Y was successfully constructed, and the MOI in HTMC was 5. Western Blot and qRT-PCR results showed that protein level and mRNA level of Myoc gene were highly over-expressed in Myoc-WT and Myoc-N450Y groups. The relative expression level of Myoc gene in Empty group, Myoc-WT group and Myoc-N450Y group was 1.00±0.02, 28.88±1.28, 22.06±0.47 (F=1020.02,P<0.001). The relative mRNA expression level of Col Ⅰ, Col Ⅳ and Fn in Myoc-N450Y group was 2.08±0.07, 1.76±0.08,2.63±0.06, significantly higher than that of Myoc-WT group (0.93±0.04, 0.95±0.09, 0.89±0.06)(all P<0.001). Compared to the expression in Myoc-WT group, the protein level of COL Ⅰ and FN were significantly upregulated in Myoc-N450Y group. The relative mRNA levels of Mmp2 and Mmp9 in Myoc-N450Y group were 0.32±0.02 and 0.33±0.15, which was significantly lower than that of Myoc-WT group (1.01±0.03 and 0.95±0.03)(all P<0.001). Conclusion Lentiviruses overexpressing Myoc gene in HTMC were successfully prepared, and it was proved that overexpression of Myoc-N450Y promoted the expression of Col Ⅰ, Col Ⅳ, Fn and inhibit the expression of Mmp2 and Mmp9 in HTMC. Therefore, we speculate that Myoc-N450Y may be involved in the occurrence and development of glaucoma by regulating the expression of ECM protein-related genes.