本文選題：氧誘導視網膜病變 + 再血管化��； 參考：《復旦大學》2013年博士論文

【摘要】：第一部分：探討OIR模型視網膜小膠質細胞和血管變化的關系 試驗選用出生后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小鼠視網膜,采用視網膜鋪片及切片免疫組化觀察血管改變。結果顯示：與正常視網膜血管相比,OIR模型P12時可見視網膜中央僅第一級大血管存在,第二級、第三級血管及毛細血管網消失,大片無灌注區(qū)形成,周邊部有血管網分布。P17時在視網膜周邊部和中央部無灌注區(qū)交界處大量新生血管簇形成,中央部大血管迂曲。P21時視網膜中央部基本血管化。視網膜垂直切片可見新生血管芽突破內界膜,長入玻璃體。視網膜鋪片及切片結果提示OIR造模成功。 成功建立OIR模型后,本研究進一步觀察了OIR模型中視網膜小膠質細胞動態(tài)變化及其與視網膜血管的關系。采用免疫熒光染色鋪片和切片觀察OIR模型血管化過程中血管和小膠質細胞的關系,應用RT-PCR及western blot檢測方法對小膠質細胞的特異性標記物CDllb的mRNA和蛋白表達進行定量分析,提示小膠質細胞增減變化。 結果顯示：正常視網膜小膠質細胞主要分布在視網膜內層,胞體較小,突起細長伸展,呈靜息態(tài),和血管關系密切。OIR模型中,小膠質細胞胞體變大,突起變短粗,在血管化區(qū)域和新生血管區(qū)分布明顯多于無血管區(qū)。RT-PCR檢測提示P15時,OIR視網膜小膠質細胞特異性標記物CD11bmRNA顯著低于正常組,隨時間增加,P21時超過正常水平。Western blot定量檢測CD11b蛋白結果與RT-PCR結果一致。體外低氧培養(yǎng)小膠質細胞提示OIR模型中視網膜血管閉塞后的改變引起了小膠質細胞改變。共聚焦圖片顯示小膠質細胞聚集在新生血管芽上面及前方,包繞出芽的內皮細胞尖端,提示小膠質細胞吞噬基質為新生血管形成開辟空間,在內皮細胞出芽形成血管腔的過程中起一定作用。 本部分研究表明OIR模型中視網膜小膠質細胞被激活,開始時減少,隨著時間增加,小膠質細胞增加。在視網膜無灌注區(qū)血管化過程中,小膠質細胞與新生血管芽密切相關,提示其在視網膜無灌注區(qū)血管化過程中發(fā)揮重要作用。 第二部分：M-CSF對OIR模型中小膠質細胞作用研究 OIR造模后,采用免疫組化觀察OIR模型視網膜M-CSF及CSF-1R分布變化,并且應用RT-PCR及western blot檢測OIR模型視網膜M-CSF及CSF-1R mRNA和蛋白動態(tài)表達變化。結果提示OIR模型M-CSF和CSF-1R主要分布于視網膜內層,與正常視網膜組織對比,未見顯著變化。正常小鼠和OIR模型小鼠視網膜部分小膠質細胞均可高表達CSF-1R。 將OIR造模成功的小鼠隨機分為兩組,注藥組腹腔注射M-CSF (40μg/kg),對照組注射等量注射用水。分別觀察小鼠行為、記錄各個時間點小鼠體重,視網膜鋪片免疫組化計數(shù)小膠質細胞數(shù)目,應用RT-PCR及western blot檢測OIR模型視網膜小膠質細胞標記性蛋白CD11b的mRNA和蛋白豐度動態(tài)表達變化。 結果顯示：注藥組體重P15時高于對照組小鼠10%,P17和P21時同對照組比較無明顯統(tǒng)計學差異,兩組小鼠行為未見明顯異常,主要組織器官大體解剖觀察亦未見明顯異常改變。視網膜鋪片免疫組化顯示P15、P17、P21時注藥組小膠質細胞高于對照組71%、46%、31%,RT-PCR結果表明注藥組CDllb的mRNA表達分布高于對照組2.61、2.24、1.05倍,Western blot檢測CD11b蛋白表達與其mRNA趨勢一致。 本試驗結果表明,OIR模型小鼠腹腔注射M-CSF可促進視網膜小膠質細胞增加,對小鼠無明顯毒副反應產生。 第三部分：M-CSF對OIR模型視網膜血管化進程的影響 將OIR造模成功的小鼠隨機分為兩組,注藥組腹腔注射M-CSF (40μg/kg),對照組注射等量注射用水,分別取P15、P17兩個時間點視網膜鋪片,FITC-GS染色、照相、拼圖后采用Image Pro Plus軟件分別描記視網膜邊界、無灌注區(qū)邊界、新生血管簇面積,分別計算每一張視網膜拼圖無灌注區(qū)面積比、新生血管簇面積比、血管化面積比,并做統(tǒng)計比較。Western blot檢測視網膜血管屏障蛋白ZO-1和Occludin動態(tài)變化,MMP-9動態(tài)變化,以及VEGF動態(tài)變化。結果表明系統(tǒng)性應用M-CSF可促進OIR視網膜無灌注區(qū)血管化進程,加速新生血管簇退化,增加視網膜血管屏障蛋白ZO-1表達,減少MMP-9表達,減少VEGF表達。 為了進一步研究是否因為M-CSF引起的小膠質細胞增加而促進OIR視網膜無灌注區(qū)血管化進程,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體減少系統(tǒng)性巨噬細胞及視網膜小膠質細胞,視網膜鋪片免疫組化觀察視網膜小膠質細胞和血管變化,分別計算P15和P17時視網膜無灌注面積比,進行統(tǒng)計。體外試驗通過小膠質細胞和人臍靜脈內皮細胞(HUVEC)共培養(yǎng),采用跨膜電阻儀測量HUVEC上下的跨內皮電阻抗(TER)。 結果顯示：氯膦酸二鈉脂質體減少了M-CSF注藥組視網膜小膠質細胞,血管發(fā)育障礙,血管較細,血管網稀疏。氯膦酸二鈉脂質體處理的OIR視網膜無灌注區(qū)面積增加。體外小膠質細胞和HUVEC共培養(yǎng)試驗結果提示,小膠質細胞增加可促進HUVEC細胞間TER增加。 本試驗結果表明,系統(tǒng)性應用M-CSF可促進OIR視網膜無灌注區(qū)血管化進程及血管屏障成熟,還可促進新生血管簇退化,減少VEGF表達。去除視網膜內小膠質細胞及其來源時,OIR視網膜內小膠質細胞減少,血管發(fā)育不良；小膠質細胞增加可促進血管內皮屏障功能形成。研究提示系統(tǒng)性應用M-CSF引起視網膜小膠質細胞增加,可促進缺血性視網膜病變無灌注區(qū)血管化進程。
[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 .
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R774.1

【共引文獻】

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