本文選題：氧誘導(dǎo)視網(wǎng)膜病變 + 再血管化��； 參考：《復(fù)旦大學(xué)》2013年博士論文

【摘要】：第一部分：探討OIR模型視網(wǎng)膜小膠質(zhì)細(xì)胞和血管變化的關(guān)系 試驗(yàn)選用出生后7天(P7)小鼠,與同籠母鼠一起置入氧濃度為75±3%的密閉氧箱,5天后返回正常氧環(huán)境,制作OIR模型。分別取P12、P15、P17、P21小鼠視網(wǎng)膜,采用視網(wǎng)膜鋪片及切片免疫組化觀察血管改變。結(jié)果顯示：與正常視網(wǎng)膜血管相比,OIR模型P12時(shí)可見(jiàn)視網(wǎng)膜中央僅第一級(jí)大血管存在,第二級(jí)、第三級(jí)血管及毛細(xì)血管網(wǎng)消失,大片無(wú)灌注區(qū)形成,周邊部有血管網(wǎng)分布。P17時(shí)在視網(wǎng)膜周邊部和中央部無(wú)灌注區(qū)交界處大量新生血管簇形成,中央部大血管迂曲。P21時(shí)視網(wǎng)膜中央部基本血管化。視網(wǎng)膜垂直切片可見(jiàn)新生血管芽突破內(nèi)界膜,長(zhǎng)入玻璃體。視網(wǎng)膜鋪片及切片結(jié)果提示OIR造模成功。 成功建立OIR模型后,本研究進(jìn)一步觀察了OIR模型中視網(wǎng)膜小膠質(zhì)細(xì)胞動(dòng)態(tài)變化及其與視網(wǎng)膜血管的關(guān)系。采用免疫熒光染色鋪片和切片觀察OIR模型血管化過(guò)程中血管和小膠質(zhì)細(xì)胞的關(guān)系,應(yīng)用RT-PCR及western blot檢測(cè)方法對(duì)小膠質(zhì)細(xì)胞的特異性標(biāo)記物CDllb的mRNA和蛋白表達(dá)進(jìn)行定量分析,提示小膠質(zhì)細(xì)胞增減變化。 結(jié)果顯示：正常視網(wǎng)膜小膠質(zhì)細(xì)胞主要分布在視網(wǎng)膜內(nèi)層,胞體較小,突起細(xì)長(zhǎng)伸展,呈靜息態(tài),和血管關(guān)系密切。OIR模型中,小膠質(zhì)細(xì)胞胞體變大,突起變短粗,在血管化區(qū)域和新生血管區(qū)分布明顯多于無(wú)血管區(qū)。RT-PCR檢測(cè)提示P15時(shí),OIR視網(wǎng)膜小膠質(zhì)細(xì)胞特異性標(biāo)記物CD11bmRNA顯著低于正常組,隨時(shí)間增加,P21時(shí)超過(guò)正常水平。Western blot定量檢測(cè)CD11b蛋白結(jié)果與RT-PCR結(jié)果一致。體外低氧培養(yǎng)小膠質(zhì)細(xì)胞提示OIR模型中視網(wǎng)膜血管閉塞后的改變引起了小膠質(zhì)細(xì)胞改變。共聚焦圖片顯示小膠質(zhì)細(xì)胞聚集在新生血管芽上面及前方,包繞出芽的內(nèi)皮細(xì)胞尖端,提示小膠質(zhì)細(xì)胞吞噬基質(zhì)為新生血管形成開(kāi)辟空間,在內(nèi)皮細(xì)胞出芽形成血管腔的過(guò)程中起一定作用。 本部分研究表明OIR模型中視網(wǎng)膜小膠質(zhì)細(xì)胞被激活,開(kāi)始時(shí)減少,隨著時(shí)間增加,小膠質(zhì)細(xì)胞增加。在視網(wǎng)膜無(wú)灌注區(qū)血管化過(guò)程中,小膠質(zhì)細(xì)胞與新生血管芽密切相關(guān),提示其在視網(wǎng)膜無(wú)灌注區(qū)血管化過(guò)程中發(fā)揮重要作用。 第二部分：M-CSF對(duì)OIR模型中小膠質(zhì)細(xì)胞作用研究 OIR造模后,采用免疫組化觀察OIR模型視網(wǎng)膜M-CSF及CSF-1R分布變化,并且應(yīng)用RT-PCR及western blot檢測(cè)OIR模型視網(wǎng)膜M-CSF及CSF-1R mRNA和蛋白動(dòng)態(tài)表達(dá)變化。結(jié)果提示OIR模型M-CSF和CSF-1R主要分布于視網(wǎng)膜內(nèi)層,與正常視網(wǎng)膜組織對(duì)比,未見(jiàn)顯著變化。正常小鼠和OIR模型小鼠視網(wǎng)膜部分小膠質(zhì)細(xì)胞均可高表達(dá)CSF-1R。 將OIR造模成功的小鼠隨機(jī)分為兩組,注藥組腹腔注射M-CSF (40μg/kg),對(duì)照組注射等量注射用水。分別觀察小鼠行為、記錄各個(gè)時(shí)間點(diǎn)小鼠體重,視網(wǎng)膜鋪片免疫組化計(jì)數(shù)小膠質(zhì)細(xì)胞數(shù)目,應(yīng)用RT-PCR及western blot檢測(cè)OIR模型視網(wǎng)膜小膠質(zhì)細(xì)胞標(biāo)記性蛋白CD11b的mRNA和蛋白豐度動(dòng)態(tài)表達(dá)變化。 結(jié)果顯示：注藥組體重P15時(shí)高于對(duì)照組小鼠10%,P17和P21時(shí)同對(duì)照組比較無(wú)明顯統(tǒng)計(jì)學(xué)差異,兩組小鼠行為未見(jiàn)明顯異常,主要組織器官大體解剖觀察亦未見(jiàn)明顯異常改變。視網(wǎng)膜鋪片免疫組化顯示P15、P17、P21時(shí)注藥組小膠質(zhì)細(xì)胞高于對(duì)照組71%、46%、31%,RT-PCR結(jié)果表明注藥組CDllb的mRNA表達(dá)分布高于對(duì)照組2.61、2.24、1.05倍,Western blot檢測(cè)CD11b蛋白表達(dá)與其mRNA趨勢(shì)一致。 本試驗(yàn)結(jié)果表明,OIR模型小鼠腹腔注射M-CSF可促進(jìn)視網(wǎng)膜小膠質(zhì)細(xì)胞增加,對(duì)小鼠無(wú)明顯毒副反應(yīng)產(chǎn)生。 第三部分：M-CSF對(duì)OIR模型視網(wǎng)膜血管化進(jìn)程的影響 將OIR造模成功的小鼠隨機(jī)分為兩組,注藥組腹腔注射M-CSF (40μg/kg),對(duì)照組注射等量注射用水,分別取P15、P17兩個(gè)時(shí)間點(diǎn)視網(wǎng)膜鋪片,FITC-GS染色、照相、拼圖后采用Image Pro Plus軟件分別描記視網(wǎng)膜邊界、無(wú)灌注區(qū)邊界、新生血管簇面積,分別計(jì)算每一張視網(wǎng)膜拼圖無(wú)灌注區(qū)面積比、新生血管簇面積比、血管化面積比,并做統(tǒng)計(jì)比較。Western blot檢測(cè)視網(wǎng)膜血管屏障蛋白ZO-1和Occludin動(dòng)態(tài)變化,MMP-9動(dòng)態(tài)變化,以及VEGF動(dòng)態(tài)變化。結(jié)果表明系統(tǒng)性應(yīng)用M-CSF可促進(jìn)OIR視網(wǎng)膜無(wú)灌注區(qū)血管化進(jìn)程,加速新生血管簇退化,增加視網(wǎng)膜血管屏障蛋白ZO-1表達(dá),減少M(fèi)MP-9表達(dá),減少VEGF表達(dá)。 為了進(jìn)一步研究是否因?yàn)镸-CSF引起的小膠質(zhì)細(xì)胞增加而促進(jìn)OIR視網(wǎng)膜無(wú)灌注區(qū)血管化進(jìn)程,其中部分系統(tǒng)性M-CSF注藥組小鼠接受腹腔和玻璃體腔注射氯膦酸二鈉脂質(zhì)體減少系統(tǒng)性巨噬細(xì)胞及視網(wǎng)膜小膠質(zhì)細(xì)胞,視網(wǎng)膜鋪片免疫組化觀察視網(wǎng)膜小膠質(zhì)細(xì)胞和血管變化,分別計(jì)算P15和P17時(shí)視網(wǎng)膜無(wú)灌注面積比,進(jìn)行統(tǒng)計(jì)。體外試驗(yàn)通過(guò)小膠質(zhì)細(xì)胞和人臍靜脈內(nèi)皮細(xì)胞(HUVEC)共培養(yǎng),采用跨膜電阻儀測(cè)量HUVEC上下的跨內(nèi)皮電阻抗(TER)。 結(jié)果顯示：氯膦酸二鈉脂質(zhì)體減少了M-CSF注藥組視網(wǎng)膜小膠質(zhì)細(xì)胞,血管發(fā)育障礙,血管較細(xì),血管網(wǎng)稀疏。氯膦酸二鈉脂質(zhì)體處理的OIR視網(wǎng)膜無(wú)灌注區(qū)面積增加。體外小膠質(zhì)細(xì)胞和HUVEC共培養(yǎng)試驗(yàn)結(jié)果提示,小膠質(zhì)細(xì)胞增加可促進(jìn)HUVEC細(xì)胞間TER增加。 本試驗(yàn)結(jié)果表明,系統(tǒng)性應(yīng)用M-CSF可促進(jìn)OIR視網(wǎng)膜無(wú)灌注區(qū)血管化進(jìn)程及血管屏障成熟,還可促進(jìn)新生血管簇退化,減少VEGF表達(dá)。去除視網(wǎng)膜內(nèi)小膠質(zhì)細(xì)胞及其來(lái)源時(shí),OIR視網(wǎng)膜內(nèi)小膠質(zhì)細(xì)胞減少,血管發(fā)育不良；小膠質(zhì)細(xì)胞增加可促進(jìn)血管內(nèi)皮屏障功能形成。研究提示系統(tǒng)性應(yīng)用M-CSF引起視網(wǎng)膜小膠質(zhì)細(xì)胞增加,可促進(jìn)缺血性視網(wǎng)膜病變無(wú)灌注區(qū)血管化進(jìn)程。
[Abstract]:The first part : To investigate the relationship between retinal microglial cells and vascular changes in OIR model

The results showed that the central part of the retina was only the first order big blood vessel , the second stage , the third stage blood vessel and the capillary network disappeared , and the peripheral part had the distribution of vascular network .

After successfully establishing the OIR model , this study further observed the dynamic changes of retinal microglial cells in OIR model and their relationship with retinal vessels . The expression of CDllb mRNA and protein in microglial cells was quantitatively analyzed by RT - PCR and western blot .

The results showed that normal retinal microglial cells were mainly distributed in the inner layer of the retina , the cells were small , the protrusions were elongated and stretched , and the distribution of CD11bmRNA was significantly lower in the OIR model than in the normal group .

In this part , the retinal microglial cells in the OIR model were activated and decreased at the beginning . As the time increased , the microglial cells increased . During the vascularization of the retina without perfusion , the microglial cells were closely related to the neovascular shoots , suggesting that they play an important role in the vascularization of the retina without perfusion .

The Effect of M - CSF on Microglia in OIR Model

The expression of M - CSF and CSF - 1R in the retina of OIR model was observed by immunohistochemistry . The results suggested that the OIR model M - CSF and CSF - 1R were mainly distributed in the inner layer of the retina and compared with normal retinal tissues .

The mice were randomly divided into two groups : M - CSF ( 40 渭g / kg ) was injected intraperitoneally in the injection group , and the control group injected the same amount of water for injection . The mouse behavior was observed , the weight of mouse body weight was recorded at each time point , the number of microglial cells was counted by immunohistochemistry . RT - PCR and western blot were used to detect the changes of mRNA and protein abundance in the retinal microglial cell marker protein of OIR model .

The results showed that the expression of CDllb mRNA in the injection group was higher than that of the control group ( 71 % , 46 % , 31 % ) when compared with the control group ( 71 % , 46 % , 31 % ) when P17 and P21 were higher than that in the control group .

The results showed that the intraperitoneal injection of M - CSF by OIR model could promote the increase of retinal glial cells and no obvious toxic side effect to mice .

The third part : Effect of M - CSF on retinal vascularization in OIR model

The mice were randomly divided into two groups : M - CSF ( 40 渭g / kg ) was injected intraperitoneally in the injection group and the control group injected with the same amount of water for injection . The changes of the area ratio , the area ratio and the area ratio of the retinal vascular barrier protein ZO - 1 , and the area of the neovascularization were respectively calculated by Western blot . The results showed that the systemic application of M - CSF could accelerate the vascularization of the retinal blood vessel barrier protein ZO - 1 and the vascular endothelial cell , and to reduce the expression of MMP - 9 and reduce the expression of VEGF .

In order to further study whether the microglial cells induced by M - CSF were increased to promote the vascularization of the OIR retina , some systemic M - CSF injection group mice received intraperitoneal and vitreous cavity injection of diclofenac sodium liposome to reduce systemic macrophages and retinal microglial cells . The retinal microglial cells and vascular changes were observed by immunohistochemistry . The ratio of retinal free area ratio was calculated by immunohistochemistry . In vitro experiments were carried out by co - culture of microglial cells and human umbilical vein endothelial cells ( HUVEC ) , and the trans - endothelium electrical impedance ( TER ) of HUVEC was measured by a transmembrane resistance meter .

The results showed that disodium clodronate decreased the area of retinal microglial cells , vascular development disturbance , vessel thinner , and sparse vascular network in M - CSF injection group , and the area of OIR retinal non - irrigated area treated by diclofenac sodium liposome was increased .

The results showed that the systemic application of M - CSF could promote the vascularization and vascular barrier maturation of OIR retinal non - irrigated area , and also promote the degeneration of neovascularization and reduce the expression of VEGF . In the removal of microglial cells in the retina and their origin , the microglial cells in the retina of OIR decreased and the vascular dysplasia was poor ;
Conclusion Systemic application of M - CSF results in the increase of retinal glial cells , which can promote the vascularization of ischemic retinopathy .
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R774.1

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3 曾R，

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