Hox基因在高糖诱导人视网膜色素上皮细胞增殖分化中的作用研究

发布时间：2018-04-23 13:54

  本文选题：Hox基因 + 增殖型糖尿病性视网膜病变　； 参考：《中南大学》2010年博士论文

【摘要】： 研究背景 随着糖尿病发病率在全世界范围内不断上升,糖尿病性视网膜病变(diabetic retinopathy,DR)已成为生后最常见的致盲原因之一。90%以上的糖尿病患者会导致视网膜病变,其中约60%1型糖尿病患者、20%2型糖尿病患者最终会进展为增殖型糖尿病性视网膜病变[1](proliferative diabetic retinopathy,PDR), PDR已然成为我国目前以及未来防盲、治盲的重点。然而,鉴于PDR的病理机制尚未完全阐明,且目前的治疗方法-光凝和玻璃体切除术对局部组织创伤较大、远期疗效不明显,在分子水平上探究其发病机制以期指导临床显得尤为重要。 PDR是一类以新生血管形成为主要病理特征的细胞增殖性疾病,其发生发展与细胞的激活、增殖和分化调控失常有关,新生血管也是细胞异常增殖的结果。在决定细胞的定向分化与增殖的众多基因中,Ⅰ型同源盒基因(Hox基因)是一类很重要的发育相关基因.一旦Hox基因或其转录调节蛋白功能异常,将直接影响细胞的增殖与分化过程。已有研究表明Hox基因参与血管生成的过程,特别是在抗肿瘤血管生成的靶向治疗中起到重要作用；参与胚胎发育过程中肢体、器官等形态发生并且以种族特异性和阶段特异性方式在造血增殖分化中起调控作用等。目前Hox基因与糖尿病关系罕有报道；其与PDR的关系,国内外尚未见相关文献报道。已有文献证实高糖可使视网膜色素上皮细胞(retinal pigment epithelium, RPE)发生一些增殖分化和功能方面的变化,其中包括高糖致使RPE细胞分泌的细胞因子如血管内皮生长因子(vascular endothelial growth factor, VEGF)和色素上皮衍生因子(pigment epithelium derived factor,PEDF)表达量的改变以及促进RPE细胞增生等。故我们设想在细胞增殖分化中起重要作用的Hox基因是否参与该变化过程?PDR是以新生血管为主要特征的病变,而作为主要血管刺激因子的VEGF和主要血管抑制因子PEDF之间的平衡对新生血管的形成是至关重要的。有不少文献报道了hox基因参与血管生成过程,其中有部分文献报道了某些Hox基因可分别参与VEGF和PEDF表达的调节,我们又大胆设想Hox族基因是否能通过影响VEGF、PEDF的表达,参与视网膜新生血管形成过程进而与PDR的发生发展相关联?为验证以上假设,我们设计如下实验,进一步探讨PDR的发病机理,以期在分子水平上为其防治带来新的曙光。 目的 1观察不同浓度葡萄糖培基对体外培养的ARPE-19细胞增殖分化以及VEGF、PEDF表达的影响。 2检测不同浓度葡萄糖培基培养的ARPE-19细胞中Hox基因表达,探讨其与PDR发生、发展的关系。选择各组间表达差异最明显的某一Hox基因进行下一步实验。 3观察筛选的目的基因对ARPE-19细胞增殖分化和表达VEGF、PEDF的影响及探讨其可能机制。 方法 1分别用高糖(18mM)和正常糖(5.5mM) DMEM培基传代培养ARPE-19,比较传代后两组细胞每天(1-8天)增殖数量以及细胞形态；设高糖组和正常糖组(NG1：培养7天细胞组；NG2：培养14天细胞组；NG3：培养21天细胞组)细胞分别为对照组和实验组。RT-PCR和westernblot分别检测各组细胞中VEGF、PEDF的表达。 2设高糖组和正常糖组细胞分别为实验组和对照组。应用Hox族基因特异引物,采用RT-PCR结合图像分析法检测两组细胞中39个Hox基因mRNA的表达水平。统计分析比较两组细胞中Hox族基因表达水平,用基因／GAPDH灰度比值表示。选择两组细胞中表达差异最明显的HoxB7基因进行下一步实验。 3将ARPE-19细胞分为五组：ARPE-HOXB71、ARPE-HOXB72 ARPE-HOXB73、ARPE-Neg和未转染组。针对HoxB7基因的mRNA序列设计了三条HoxB7特异性的小干扰RNA(small interfering RNA, siRNA)和一条阴性对照negtive-siRNA,并分别克隆入pRNAT质粒载体,将重组质粒转入ARPE-19细胞,荧光显微镜下检测转染效率,半定量RT-PCR和Werstern blot方法检测对HoxB7基因干扰效果以及各组细胞VEGF、PEDF的表达；利用MTT增生试验,观察HoxB7基因对各组细胞增生能力的影响；比较转染后各组细胞形态。 结果 1.HG组细胞长呈纺锤形或更狭长形状,而NG组细胞呈圆形或鹅卵石形。第4天开始HG组细胞较NG组细胞明显增殖,两组比较差异具有统计学意义(P0.05)；培养第8天时,HG组细胞数量由5.1±0.3E+04／ml增殖至31.2±2.1E+04／ml,较初始增殖约500%；而NG组细胞数量4.5±0.4E+04／ml增殖至14.1±1.2E+04／ml,较初始增殖不到200%。应用RT-PCR检测：NG组较HG组细胞PEDF表达水平增加, NG1与HG比较差异均有统计学意义(P0.05),NG2和NG3组分别与HG组比较差异具有显著性(P0.01)；NG组较HG组VEGF表达水平降低,NG2组与HG比较差异均有统计学意义(P0.05),NG3组与HG组比较差异具有显著性(P0.01)。应用Western blot检测：各实验组同对照组比较,随时间延长VEGF的蛋白表达逐渐降低,差异具有显著性(P0.01)；各实验组同对照组比较,随时间延长PEDF的蛋白表达逐渐增加,差异具有显著性(P0.01)。 2.Hox基因全部39个成员中有12个成员在两组细胞中都有表达,它们是HoxA5、HoxA6、HoxA13、HoxB3、HoxB5、HoxB7、HoxB13、HoxC6、HoxC13、HoxD1、HoxD3、HoxD10; HoxA5、HoxA13、HoxC13、HoxD1、HoxD3在两组细胞中的表达差异无统计学意义；HG组与NG组比较,HoxA6、HoxB3、HoxC6、HoxD10的表达较NG组细胞降低,差异有统计学意义(P0.05)；HG组中HoxB5、HoxB7、HoxB13基因的表达较NG组细胞增高,差异具有统计学意义(P0.05); HoxB7是两组细胞中表达差异最显著的基因之一。 3.与转染阴性质粒组和未转染组比较,干扰质粒pRNAT-HoxB7特异并有效的抑制了ARPE-19细胞中HoxB7基因的表达,稳定转染后,HoxB7-mRNA和蛋白抑制率依次为(50.15±2.31)%(55.41±2.99)%；且VEGF的表达较未转染组和阴性对照组降低(P0.01),同时PEDF的表达较未转染组和阴性对照组增高(P0.01)。干扰HoxB7基因后,MTT检测ARPE-19细胞增生能力下降,其在干扰48h和72h后的增殖抑制率依次为(16.18±2.53)%和(25.02±5.02)%。HoxB7基因干扰后,ARPE-19细胞形态未发生明显改变。 结论 1高糖可刺激hRPE细胞增殖分化；控制血糖可从转录及翻译水平上调hRPE细胞PEDF的表达,并抑制VEGF的表达。 2 Hox基因家族中有12个成员(HoxA5、HoxA6、HoxA13、HoxB3、HoxB5、HoxB7、HoxB13、HoxC6、HoxC13、HoxD1、HoxD3、HoxD10)可能参与了hRPE细胞的增殖分化过程；其中,HoxA6、HoxB3、HoxB5、HoxB7、HoxB13、HoxC6、HoxD10可能参与增殖型糖尿病视网膜病变的发生发展过程；HoxB7可能是与增殖型糖尿病视网膜病变的发生发展关系最密切的Hox基因成员之一。 3高糖可能通过上调HoxB7基因的表达促进hRPE细胞的增殖；HoxB7基因可能通过上调VEGF的表达,并抑制PEDF的表达参与PDR的发生发展过程。
[Abstract]:Background of the study



Diabetic retinopathy ( DR ) has become one of the most common causes of diabetic retinopathy . Diabetic retinopathy ( DR ) has become one of the most common causes of blindness in the world .



PDR is a kind of cell proliferative disease characterized by neovascularization . Its development is related to the activation , proliferation and differentiation of cells . In many genes determining the directional differentiation and proliferation of cells , the type I homeobox gene ( Hox gene ) is a very important development - related gene . Once the Hox gene or its transcriptional regulatory protein function is abnormal , it will directly affect the proliferation and differentiation of cells .
In the process of embryo development , the morphology of limbs , organs , etc . takes place and plays an important role in the differentiation of hematopoietic proliferation in the way of race - specific and stage - specific .
It has been reported that the Hox gene plays an important role in the development of vascular endothelial growth factor ( VEGF ) and pigment epithelium derived factor ( PEDF ) and promotes RPE cell proliferation .



Purpose



1 To observe the effects of different concentrations of glucose on proliferation and differentiation of ARPE - 19 cells and the expression of VEGF and PEDF in vitro .



The Hox gene expression in ARPE - 19 cells cultured in different concentrations of glucose was examined . The relationship between the expression of Hox gene and the occurrence and development of PDR was discussed . One Hox gene with the most obvious difference was selected for the next step .



3 To investigate the effects of the screened gene on proliferation and differentiation of ARPE - 19 cells and the expression of VEGF and PEDF and explore its possible mechanism .



method



ARPE - 19 was cultured in DMEM culture medium with high glucose ( 18 mM ) and normal sugar ( 5.5 mM ) , respectively , and the proliferation and cell morphology of two groups ( 1 - 8 days ) after passage were compared .
setting high - sugar group and normal sugar group ( NG1 : culturing the 7 - day cell group ;
NG2 : culturing the 14 - day cell group ;
The expression of VEGF and PEDF in each group was detected by RT - PCR and Western blot .



The expression level of Hox gene mRNA in two groups of cells was determined by RT - PCR and image analysis . The HoxB7 gene was selected to express the most significant difference in the expression of Hox gene in the two groups .



3 . ARPE - 19 cells were divided into five groups : ARPE - HOXB71 , ARPE - HOXB72 ARPE - HOXB73 , ARPE - Neg and untransfected group . Three HoxB7 - specific small interfering RNAs ( siRNA ) and a negative control group tive - siRNA were designed for the mRNA sequence of HoxB7 gene , and the recombinant plasmids were transferred to ARPE - 19 cells . The transfection efficiency , semi - quantitative RT - PCR and Werstern blot methods were used to detect the interference effect on HoxB7 gene and the expression of VEGF and PEDF in each group .
The effects of HoxB7 gene on proliferation of each group were observed by MTT assay .
The morphology of each group after transfection was compared .



Results



1 . The cells of HG group were spindle - shaped or more elongated , while NG - group cells were round or cobblestone - shaped . On the 4th day , the cells of HG - group were significantly higher than those in NG group , and the difference was statistically significant ( P0.05 ) .
At the 8th day of culture , the number of cells in HG group was increased from 5.1 卤 0.3E + 04 / ml to 31.2 卤 2.1E + 04 / ml , and the initial proliferation was about 500 % .
鑰孨G缁勭粏鑳炴暟閲，

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