本文選題：SP1 + Robo4　； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：糖尿病視網(wǎng)膜病變(diabetic retinopathy,DR)為糖尿病嚴(yán)重的微血管并發(fā)癥之一,其發(fā)病機(jī)制極為復(fù)雜,目前研究表明,視網(wǎng)膜在持續(xù)高糖環(huán)境的刺激下,可發(fā)生氧化應(yīng)激、炎癥反應(yīng)、神經(jīng)退行性改變和表觀遺傳學(xué)修飾異常等變化,它們之間相互作用并啟動(dòng)各自相關(guān)的信號(hào)通路,共同導(dǎo)致視網(wǎng)膜微血管病變的發(fā)生。根據(jù)視網(wǎng)膜血管病理改變的不同,可將DR的病程分為非增殖期和增殖期兩個(gè)階段。在DR非增殖期,病變主要表現(xiàn)為視網(wǎng)膜微血管內(nèi)皮細(xì)胞結(jié)構(gòu)損傷甚至丟失、周細(xì)胞凋亡、微血管細(xì)胞間緊密連接松弛、通透性增加等,進(jìn)而引起血-視網(wǎng)膜屏障的破壞。在DR增殖期,大面積的毛細(xì)血管閉塞使視網(wǎng)膜局部嚴(yán)重缺氧,引起眾多生長(zhǎng)因子和炎癥介質(zhì)的上調(diào),進(jìn)而導(dǎo)致視網(wǎng)膜新生血管的形成。因此保護(hù)視網(wǎng)膜微血管的完整性、抑制視網(wǎng)膜新生血管形成是預(yù)防和治療DR的關(guān)鍵。Roundabout 4(Robo4)是2002年被發(fā)現(xiàn)的Robo家族新成員,可特異性表達(dá)于血管內(nèi)皮細(xì)胞的表面,具有調(diào)節(jié)內(nèi)皮細(xì)胞通透性、影響新生血管形成等功能。近期研究發(fā)現(xiàn),Robo4的表達(dá)變化與DR的發(fā)生發(fā)展密切相關(guān)。在體外低氧條件培養(yǎng)的人視網(wǎng)膜血管內(nèi)皮細(xì)胞(human retinal endothelial cells,HREC)中,隨著低氧作用時(shí)間的延長(zhǎng),細(xì)胞內(nèi)Robo4表達(dá)逐漸升高,而抑制Robo4的表達(dá)可有效改善低氧誘導(dǎo)的HREC增殖和遷移能力的異常,且在體內(nèi)糖尿病動(dòng)物模型中,發(fā)現(xiàn)伴隨DR的發(fā)生和病程延長(zhǎng),Robo4在視網(wǎng)膜內(nèi)的表達(dá)水平逐漸增加。由此提示,Robo4在DR發(fā)生發(fā)展過(guò)程中可能發(fā)揮重要的作用,但與其相關(guān)的具體作用機(jī)制尚不清楚。在有關(guān)Robo4表達(dá)調(diào)控機(jī)制的研究中,利用基因啟動(dòng)子預(yù)測(cè)軟件分析Robo4上游的啟動(dòng)子區(qū)域,發(fā)現(xiàn)其中存在多個(gè)specificity protein 1(SP1)的結(jié)合位點(diǎn),且研究表明SP1作為常見(jiàn)的轉(zhuǎn)錄因子,可調(diào)控下游多個(gè)靶基因的轉(zhuǎn)錄而影響DR的發(fā)生發(fā)展,由此推測(cè)Robo4可能為SP1的另一靶向因子,在DR發(fā)病中,SP1通過(guò)調(diào)控Robo4的轉(zhuǎn)錄活性而影響視網(wǎng)膜微血管病變的發(fā)生。本研究于體內(nèi)檢測(cè)增殖性糖尿病視網(wǎng)膜病變(proliferative diabetic retinopathy,PDR)患者纖維血管膜中SP1與Robo4的表達(dá)情況,并利用高糖條件培養(yǎng)HREC,以模擬DR的體內(nèi)環(huán)境,探討此時(shí)SP1與Robo4的表達(dá)水平及二者間的轉(zhuǎn)錄調(diào)控機(jī)制,并進(jìn)一步探討該機(jī)制在高糖條件下對(duì)HREC功能的影響。方法1.檢測(cè)SP1與Robo4在PDR患者纖維血管膜中的表達(dá)情況收集臨床中手術(shù)獲取的PDR患者的纖維血管膜,對(duì)其進(jìn)行HE染色,觀察膜組織的病理結(jié)構(gòu),采用免疫組織化學(xué)染色法檢測(cè)SP1與Robo4蛋白在纖維血管膜中的表達(dá)水平,并利用雙重免疫熒光標(biāo)記法檢測(cè)二者表達(dá)的共定位情況。2.檢測(cè)SP1與Robo4在體外高糖培養(yǎng)的HREC中的表達(dá)情況體外用高糖培養(yǎng)基培養(yǎng)HREC,利用RT-q PCR和Western blot法檢測(cè)伴隨高糖作用時(shí)間的延長(zhǎng),SP1與Robo4在HREC中m RNA轉(zhuǎn)錄和蛋白表達(dá)水平的變化。3.檢測(cè)向HREC中分別轉(zhuǎn)染SP1、Robo4小干擾RNA(small interfering RNA,si RNA)后SP1與Robo4的表達(dá)變化向體外高糖培養(yǎng)的HREC中轉(zhuǎn)染SP1 si RNA以抑制細(xì)胞內(nèi)SP1的表達(dá)水平,利用RT-q PCR法和Western blot法檢測(cè)Robo4 m RNA轉(zhuǎn)錄水平和蛋白表達(dá)量的變化,同樣方法轉(zhuǎn)染Robo4 si RNA,觀察其是否對(duì)SP1的表達(dá)水平亦產(chǎn)生影響。4.檢測(cè)SP1與Robo4在HREC中的轉(zhuǎn)錄調(diào)控機(jī)制采用染色質(zhì)免疫共沉淀技術(shù)(Ch IP)分別檢測(cè)HREC在正常和高糖兩種培養(yǎng)條件下SP1蛋白與Robo4啟動(dòng)子區(qū)的結(jié)合活性,并在Ch IP實(shí)驗(yàn)結(jié)果的基礎(chǔ)上,利用熒光素酶報(bào)告基因系統(tǒng)(Luciferase Assay System)檢測(cè)SP1蛋白與Robo4基因間的確切結(jié)合位點(diǎn)。5.檢測(cè)SP1調(diào)控Robo4對(duì)HREC功能的影響向高糖條件培養(yǎng)的HREC中轉(zhuǎn)染si RNA以抑制SP1與Robo4的表達(dá)水平,并通過(guò)Transwell法、細(xì)胞劃痕法、單層細(xì)胞通透性實(shí)驗(yàn)和細(xì)胞管腔形成實(shí)驗(yàn)分別觀察SP1/Robo4表達(dá)變化對(duì)HREC遷移、單層HREC通透性和管腔形成能力的影響。結(jié)果1.PDR患者纖維血管膜的病理組織結(jié)構(gòu)主要分為四種類型:以增殖細(xì)胞為主要成分的膜組織、以新生血管居多的膜組織、新生血管破裂后紅細(xì)胞大量溢出的血管膜與血管退化后以致密纖維組織為主要成分的纖維膜,SP1與Robo4在不同類型纖維血管膜中的表達(dá)均異常增高,且二者的表達(dá)共定位于纖維血管膜的微血管內(nèi)皮細(xì)胞中;2.在體外高糖培養(yǎng)的HREC中,SP1與Robo4的m RNA轉(zhuǎn)錄與蛋白表達(dá)水平隨高糖作用時(shí)間的延長(zhǎng)呈同步性穩(wěn)定增加;3.在高糖培養(yǎng)的HREC中,轉(zhuǎn)染SP1 si RNA后,SP1的表達(dá)水平受到明顯抑制,同時(shí)Robo4的m RNA和蛋白水平也顯著降低,相反細(xì)胞內(nèi)轉(zhuǎn)染Robo4 si RNA僅能有效抑制Robo4的表達(dá)水平,對(duì)SP1的表達(dá)無(wú)顯著影響;4.Ch IP結(jié)果顯示在正常培養(yǎng)的HREC中,Robo4啟動(dòng)子-2400/-2700bp區(qū)域可與轉(zhuǎn)錄因子SP1相結(jié)合,而在高糖培養(yǎng)的HREC中,啟動(dòng)子-2400/-2700bp與-1800/-2100bp區(qū)域均與SP1有結(jié)合活性,Luciferase結(jié)果顯示,HREC在正常條件下,SP1可與Robo4啟動(dòng)子區(qū)-2452/-2448bp位點(diǎn)結(jié)合調(diào)控Robo4的轉(zhuǎn)錄水平,而在高糖條件下,SP1可作用于Robo4啟動(dòng)子區(qū)-2452/-2448bp和-1912/-1908bp兩個(gè)結(jié)合位點(diǎn),進(jìn)而增強(qiáng)Robo4的轉(zhuǎn)錄活性;5.高糖條件可引起HREC的遷移能力增強(qiáng)、單層HREC通透性增加和管腔形成能力下降,向細(xì)胞內(nèi)轉(zhuǎn)染SP1 si RNA或Robo4 si RNA后,HREC的遷移能力有所下降,單層HREC通透性顯著降低,且HREC的管腔形成能力得到明顯改善。結(jié)論本實(shí)驗(yàn)研究SP1與Robo4在DR發(fā)生發(fā)展中的作用及調(diào)控機(jī)制,發(fā)現(xiàn)SP1與Robo4在PDR患者的纖維血管膜內(nèi)表達(dá)異常增高,提示二者可能參與纖維血管膜的形成,同時(shí)在體外高糖培養(yǎng)的HREC中,SP1與Robo4表達(dá)呈同步性上升,且下調(diào)SP1的表達(dá)可明顯抑制高糖誘導(dǎo)的Robo4表達(dá)升高,說(shuō)明SP1對(duì)Robo4表達(dá)存在一定的調(diào)控作用。進(jìn)一步研究SP1與Robo4間的轉(zhuǎn)錄調(diào)控機(jī)制,發(fā)現(xiàn)高糖條件可誘導(dǎo)HREC中SP1過(guò)表達(dá),并通過(guò)與Robo4啟動(dòng)子區(qū)-2452/-2448bp和-1912/-1908bp兩個(gè)結(jié)合位點(diǎn)相互作用,從而增強(qiáng)Robo4的轉(zhuǎn)錄活性,提高Robo4的表達(dá)水平。且SP1/Robo4通路與HREC的功能密切相關(guān),抑制SP1/Robo4的表達(dá)水平可顯著改善高糖誘導(dǎo)的HREC功能異常,說(shuō)明SP1/Robo4通路在DR微血管病變的發(fā)生中可能發(fā)揮一定的促進(jìn)作用。
[Abstract]:Diabetic retinopathy (diabetic retinopathy, DR) is one of the severe microvascular complications of diabetes. Its pathogenesis is very complex. The current research shows that the retina can occur oxidative stress, inflammatory response, neurodegenerative changes and epigenetic modification, with the stimulation of continuous high glucose environment. According to the pathological changes of retinal vessels, the course of DR can be divided into two stages of non proliferative and proliferative stages. In the non proliferation stage of DR, the lesion is mainly caused by the damage or even loss of the retinal microvascular endothelial cell structure, and the pericytes withering. In DR proliferation period, large area of capillary occlusion causes severe hypoxia in the retina, causing a large number of growth factors and inflammatory mediators to rise, resulting in the formation of retinal neovascularization, thus protecting retinal microvessels. The integrity of the inhibition of retinal neovascularization is the key.Roundabout 4 (Robo4) for the prevention and treatment of DR (Robo4), a new member of the Robo family found in 2002, which is specifically expressed on the surface of vascular endothelial cells, regulating endothelial cell permeability and affecting neovascularization. Recent studies have found that the expression changes of Robo4 and DR In human retinal vascular endothelial cells (human retinal endothelial cells, HREC) in vitro hypoxia conditions, the expression of Robo4 in cells increases gradually with the prolongation of hypoxia, and the inhibition of the expression of Robo4 can effectively improve the proliferation and migration of hypoxia induced HREC, and in vivo In the animal model of diabetes, the expression level of Robo4 in the retina is gradually increased with the occurrence and duration of DR. Thus, it is suggested that Robo4 may play an important role in the development of DR, but its specific mechanism is not clear. In the study of the regulatory mechanism of Robo4 expression, the gene promoter is used. The prediction software analyses the promoter region of the upstream of Robo4. It is found that there are multiple specificity protein 1 (SP1) binding sites, and the study shows that SP1 as a common transcription factor can regulate the transcription of multiple target genes in the downstream and affect the development of DR. Thus, it is presumed that Robo4 may be another target factor of SP1, and in DR pathogenesis, SP1 passes. Regulation of the transcriptional activity of Robo4 affects the occurrence of retinal microvascular lesions. In this study, the expression of SP1 and Robo4 in the fibrous vascular membrane of proliferative diabetic retinopathy (PDR) patients was detected in this study, and HREC was cultured with high glucose conditions to simulate the internal environment of DR, and to explore SP1 and Ro at this time. The expression level of bo4 and the transcriptional regulation mechanism between the two and the effect of the mechanism on the HREC function under high glucose. Method 1. to detect the expression of SP1 and Robo4 in the fibrous membrane of patients with PDR and to collect the fibrous membrane of the patients of PDR in the clinical operation, and to stain them by HE staining and to observe the pathological structure of the membrane tissue. The expression of the expression of SP1 and Robo4 protein in the fibrous vascular membrane was detected by immunohistochemical staining, and the co localization of the two were detected by double immunofluorescence..2. was used to detect the expression of SP1 and Robo4 in HREC in high glucose culture in vitro, and HREC was cultured with Gao Tangpei culture medium in vitro, and RT-q PCR and Western blot method were used. The change of M RNA transcriptional and protein expression levels between SP1 and Robo4 in HREC was detected by.3., and SP1 was transfected into HREC, and Robo4 small interference RNA (small interfering) was transfected to the high glucose culture in vitro. RT-q PCR and Western blot were used to detect the changes of Robo4 m RNA transcriptional level and protein expression. The same method transfected Robo4 Si RNA, and observed whether the expression level of SP1 was also affected by the transcription regulation mechanism of.4. detection and chromatin immunoprecipitation. The binding activity of SP1 protein and Robo4 promoter in two culture conditions, and on the basis of Ch IP experimental results, using the luciferase reporter gene system (Luciferase Assay System) to detect the exact binding site of SP1 protein and Robo4 gene,.5. SP1 regulation of Robo4 on the effect of Robo4 on the high sugar condition Si RNA was used to inhibit the expression of SP1 and Robo4, and the effects of SP1/Robo4 expression on HREC migration, monolayer HREC permeability and lumen formation ability were observed by Transwell, cell scratch, monolayer permeability and cell cavity formation. Results the pathological histological structure of the fibrous membrane of 1.PDR patients was mainly divided into two parts. The four types: the membrane tissue of the proliferating cell as the main component, the membrane tissue of the neovascularization, the large spillover of the red blood cells after the rupture of the neovascularization and the fibrous membrane of the dense fibrous tissue after the degeneration of the blood vessels. The expression of SP1 and Robo4 in the different types of fibrous vascular membrane are all abnormally increased and the expression of the two 2. in the microvascular endothelial cells in the fibrous vascular membrane; 2. in the high sugar cultured HREC in vitro, the m RNA transcription and protein expression level of SP1 and Robo4 increased synchronously with the prolongation of the time of high glucose; 3. in the high sugar culture HREC, the expression level of SP1 was obviously inhibited after transfecting SP1 Si RNA, and Robo4 M The protein level also decreased significantly. On the contrary, the transfection of Robo4 Si RNA only effectively inhibited the expression of Robo4, and had no significant effect on the expression of SP1. The 4.Ch IP results showed that the Robo4 promoter -2400/-2700bp region could be combined with the SP1 of the transcription factor in the normal cultured HREC. Both /-2100bp region and SP1 have binding activity. Luciferase results show that under normal conditions, SP1 can regulate the transcriptional level of Robo4 with the -2452/-2448bp locus of Robo4 promoter region in normal conditions, but in high glucose conditions, SP1 can act on the two binding sites of -2452/-2448bp and -1912/ Robo4 in the promoter region of Robo4, and then increase the transcriptional activity of Robo4; 5 High glucose conditions could increase the migration ability of HREC, increase the permeability of single layer HREC and decrease the formation ability of the lumen. After transfecting SP1 Si RNA or Robo4 Si RNA into the cells, the migration ability of HREC decreased, the permeability of single-layer HREC decreased significantly, and the capacity of the HREC lumen formation was obviously improved. The role of SP1 and Robo4 in the fibrous vascular membrane of PDR patients was found to be abnormal, suggesting that the two may participate in the formation of fibrous vascular membrane, while the expression of SP1 and Robo4 increased synchronously in HREC in high glucose culture in vitro, and the expression of SP1 could inhibit the elevation of Robo4 expression induced by high glucose. It is suggested that SP1 has a certain regulatory effect on the expression of Robo4, and further studies the transcriptional regulation mechanism between SP1 and Robo4. It is found that high sugar conditions can induce SP1 overexpression in HREC and interact with the two binding sites of -2452/-2448bp and -1912/-1908bp in the promoter region of Robo4, thus strengthening the transcriptional activity of Robo4 and improving the Robo4 expression level. And the SP1/Robo4 pathway is closely related to the function of HREC. Inhibition of the expression level of SP1/Robo4 can significantly improve the abnormal HREC function induced by high glucose, which indicates that the SP1/Robo4 pathway may play a certain role in the pathogenesis of DR microvascular lesions.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R774.1

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