本文選題：Hox基因 + 增殖型糖尿病性視網(wǎng)膜病變��； 參考：《中南大學(xué)》2010年博士論文

【摘要】： 研究背景 隨著糖尿病發(fā)病率在全世界范圍內(nèi)不斷上升,糖尿病性視網(wǎng)膜病變(diabetic retinopathy,DR)已成為生后最常見的致盲原因之一。90%以上的糖尿病患者會(huì)導(dǎo)致視網(wǎng)膜病變,其中約60%1型糖尿病患者、20%2型糖尿病患者最終會(huì)進(jìn)展為增殖型糖尿病性視網(wǎng)膜病變[1](proliferative diabetic retinopathy,PDR), PDR已然成為我國(guó)目前以及未來防盲、治盲的重點(diǎn)。然而,鑒于PDR的病理機(jī)制尚未完全闡明,且目前的治療方法-光凝和玻璃體切除術(shù)對(duì)局部組織創(chuàng)傷較大、遠(yuǎn)期療效不明顯,在分子水平上探究其發(fā)病機(jī)制以期指導(dǎo)臨床顯得尤為重要。 PDR是一類以新生血管形成為主要病理特征的細(xì)胞增殖性疾病,其發(fā)生發(fā)展與細(xì)胞的激活、增殖和分化調(diào)控失常有關(guān),新生血管也是細(xì)胞異常增殖的結(jié)果。在決定細(xì)胞的定向分化與增殖的眾多基因中,Ⅰ型同源盒基因(Hox基因)是一類很重要的發(fā)育相關(guān)基因.一旦Hox基因或其轉(zhuǎn)錄調(diào)節(jié)蛋白功能異常,將直接影響細(xì)胞的增殖與分化過程。已有研究表明Hox基因參與血管生成的過程,特別是在抗腫瘤血管生成的靶向治療中起到重要作用；參與胚胎發(fā)育過程中肢體、器官等形態(tài)發(fā)生并且以種族特異性和階段特異性方式在造血增殖分化中起調(diào)控作用等。目前Hox基因與糖尿病關(guān)系罕有報(bào)道；其與PDR的關(guān)系,國(guó)內(nèi)外尚未見相關(guān)文獻(xiàn)報(bào)道。已有文獻(xiàn)證實(shí)高糖可使視網(wǎng)膜色素上皮細(xì)胞(retinal pigment epithelium, RPE)發(fā)生一些增殖分化和功能方面的變化,其中包括高糖致使RPE細(xì)胞分泌的細(xì)胞因子如血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)和色素上皮衍生因子(pigment epithelium derived factor,PEDF)表達(dá)量的改變以及促進(jìn)RPE細(xì)胞增生等。故我們?cè)O(shè)想在細(xì)胞增殖分化中起重要作用的Hox基因是否參與該變化過程?PDR是以新生血管為主要特征的病變,而作為主要血管刺激因子的VEGF和主要血管抑制因子PEDF之間的平衡對(duì)新生血管的形成是至關(guān)重要的。有不少文獻(xiàn)報(bào)道了hox基因參與血管生成過程,其中有部分文獻(xiàn)報(bào)道了某些Hox基因可分別參與VEGF和PEDF表達(dá)的調(diào)節(jié),我們又大膽設(shè)想Hox族基因是否能通過影響VEGF、PEDF的表達(dá),參與視網(wǎng)膜新生血管形成過程進(jìn)而與PDR的發(fā)生發(fā)展相關(guān)聯(lián)?為驗(yàn)證以上假設(shè),我們?cè)O(shè)計(jì)如下實(shí)驗(yàn),進(jìn)一步探討PDR的發(fā)病機(jī)理,以期在分子水平上為其防治帶來新的曙光。 目的 1觀察不同濃度葡萄糖培基對(duì)體外培養(yǎng)的ARPE-19細(xì)胞增殖分化以及VEGF、PEDF表達(dá)的影響。 2檢測(cè)不同濃度葡萄糖培基培養(yǎng)的ARPE-19細(xì)胞中Hox基因表達(dá),探討其與PDR發(fā)生、發(fā)展的關(guān)系。選擇各組間表達(dá)差異最明顯的某一Hox基因進(jìn)行下一步實(shí)驗(yàn)。 3觀察篩選的目的基因?qū)RPE-19細(xì)胞增殖分化和表達(dá)VEGF、PEDF的影響及探討其可能機(jī)制。 方法 1分別用高糖(18mM)和正常糖(5.5mM) DMEM培基傳代培養(yǎng)ARPE-19,比較傳代后兩組細(xì)胞每天(1-8天)增殖數(shù)量以及細(xì)胞形態(tài)；設(shè)高糖組和正常糖組(NG1：培養(yǎng)7天細(xì)胞組；NG2：培養(yǎng)14天細(xì)胞組；NG3：培養(yǎng)21天細(xì)胞組)細(xì)胞分別為對(duì)照組和實(shí)驗(yàn)組。RT-PCR和westernblot分別檢測(cè)各組細(xì)胞中VEGF、PEDF的表達(dá)。 2設(shè)高糖組和正常糖組細(xì)胞分別為實(shí)驗(yàn)組和對(duì)照組。應(yīng)用Hox族基因特異引物,采用RT-PCR結(jié)合圖像分析法檢測(cè)兩組細(xì)胞中39個(gè)Hox基因mRNA的表達(dá)水平。統(tǒng)計(jì)分析比較兩組細(xì)胞中Hox族基因表達(dá)水平,用基因／GAPDH灰度比值表示。選擇兩組細(xì)胞中表達(dá)差異最明顯的HoxB7基因進(jìn)行下一步實(shí)驗(yàn)。 3將ARPE-19細(xì)胞分為五組：ARPE-HOXB71、ARPE-HOXB72 ARPE-HOXB73、ARPE-Neg和未轉(zhuǎn)染組。針對(duì)HoxB7基因的mRNA序列設(shè)計(jì)了三條HoxB7特異性的小干擾RNA(small interfering RNA, siRNA)和一條陰性對(duì)照negtive-siRNA,并分別克隆入pRNAT質(zhì)粒載體,將重組質(zhì)粒轉(zhuǎn)入ARPE-19細(xì)胞,熒光顯微鏡下檢測(cè)轉(zhuǎn)染效率,半定量RT-PCR和Werstern blot方法檢測(cè)對(duì)HoxB7基因干擾效果以及各組細(xì)胞VEGF、PEDF的表達(dá)；利用MTT增生試驗(yàn),觀察HoxB7基因?qū)Ω鹘M細(xì)胞增生能力的影響；比較轉(zhuǎn)染后各組細(xì)胞形態(tài)。 結(jié)果 1.HG組細(xì)胞長(zhǎng)呈紡錘形或更狹長(zhǎng)形狀,而NG組細(xì)胞呈圓形或鵝卵石形。第4天開始HG組細(xì)胞較NG組細(xì)胞明顯增殖,兩組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；培養(yǎng)第8天時(shí),HG組細(xì)胞數(shù)量由5.1±0.3E+04／ml增殖至31.2±2.1E+04／ml,較初始增殖約500%；而NG組細(xì)胞數(shù)量4.5±0.4E+04／ml增殖至14.1±1.2E+04／ml,較初始增殖不到200%。應(yīng)用RT-PCR檢測(cè)：NG組較HG組細(xì)胞PEDF表達(dá)水平增加, NG1與HG比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),NG2和NG3組分別與HG組比較差異具有顯著性(P0.01)；NG組較HG組VEGF表達(dá)水平降低,NG2組與HG比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),NG3組與HG組比較差異具有顯著性(P0.01)。應(yīng)用Western blot檢測(cè)：各實(shí)驗(yàn)組同對(duì)照組比較,隨時(shí)間延長(zhǎng)VEGF的蛋白表達(dá)逐漸降低,差異具有顯著性(P0.01)；各實(shí)驗(yàn)組同對(duì)照組比較,隨時(shí)間延長(zhǎng)PEDF的蛋白表達(dá)逐漸增加,差異具有顯著性(P0.01)。 2.Hox基因全部39個(gè)成員中有12個(gè)成員在兩組細(xì)胞中都有表達(dá),它們是HoxA5、HoxA6、HoxA13、HoxB3、HoxB5、HoxB7、HoxB13、HoxC6、HoxC13、HoxD1、HoxD3、HoxD10; HoxA5、HoxA13、HoxC13、HoxD1、HoxD3在兩組細(xì)胞中的表達(dá)差異無統(tǒng)計(jì)學(xué)意義；HG組與NG組比較,HoxA6、HoxB3、HoxC6、HoxD10的表達(dá)較NG組細(xì)胞降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；HG組中HoxB5、HoxB7、HoxB13基因的表達(dá)較NG組細(xì)胞增高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05); HoxB7是兩組細(xì)胞中表達(dá)差異最顯著的基因之一。 3.與轉(zhuǎn)染陰性質(zhì)粒組和未轉(zhuǎn)染組比較,干擾質(zhì)粒pRNAT-HoxB7特異并有效的抑制了ARPE-19細(xì)胞中HoxB7基因的表達(dá),穩(wěn)定轉(zhuǎn)染后,HoxB7-mRNA和蛋白抑制率依次為(50.15±2.31)%(55.41±2.99)%；且VEGF的表達(dá)較未轉(zhuǎn)染組和陰性對(duì)照組降低(P0.01),同時(shí)PEDF的表達(dá)較未轉(zhuǎn)染組和陰性對(duì)照組增高(P0.01)。干擾HoxB7基因后,MTT檢測(cè)ARPE-19細(xì)胞增生能力下降,其在干擾48h和72h后的增殖抑制率依次為(16.18±2.53)%和(25.02±5.02)%。HoxB7基因干擾后,ARPE-19細(xì)胞形態(tài)未發(fā)生明顯改變。 結(jié)論 1高糖可刺激hRPE細(xì)胞增殖分化；控制血糖可從轉(zhuǎn)錄及翻譯水平上調(diào)hRPE細(xì)胞PEDF的表達(dá),并抑制VEGF的表達(dá)。 2 Hox基因家族中有12個(gè)成員(HoxA5、HoxA6、HoxA13、HoxB3、HoxB5、HoxB7、HoxB13、HoxC6、HoxC13、HoxD1、HoxD3、HoxD10)可能參與了hRPE細(xì)胞的增殖分化過程；其中,HoxA6、HoxB3、HoxB5、HoxB7、HoxB13、HoxC6、HoxD10可能參與增殖型糖尿病視網(wǎng)膜病變的發(fā)生發(fā)展過程；HoxB7可能是與增殖型糖尿病視網(wǎng)膜病變的發(fā)生發(fā)展關(guān)系最密切的Hox基因成員之一。 3高糖可能通過上調(diào)HoxB7基因的表達(dá)促進(jìn)hRPE細(xì)胞的增殖；HoxB7基因可能通過上調(diào)VEGF的表達(dá),并抑制PEDF的表達(dá)參與PDR的發(fā)生發(fā)展過程。
[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 鹵 0.3E + 04 / ml to 31.2 鹵 2.1E + 04 / ml , and the initial proliferation was about 500 % .
鑰孨G緇勭粏鑳?yōu)鏁伴嚕?

本文編號(hào)：1792241