【摘要】：以糖尿病性視網(wǎng)膜病變?yōu)榇淼?包括早產(chǎn)兒視網(wǎng)膜病變、視網(wǎng)膜中央靜脈阻塞等視網(wǎng)膜缺血性疾病,以新生血管形成為主要病理標(biāo)志。視網(wǎng)膜循環(huán)障礙使血管內(nèi)皮細(xì)胞與周細(xì)胞受損,從而導(dǎo)致毛細(xì)血管失去正常的屏障功能,滲透性增加,造成周圍組織水腫、滲出,繼而引起視網(wǎng)膜組織缺血缺氧,并代償性生成組織結(jié)構(gòu)不完整的新生血管,導(dǎo)致出血、增殖,甚至視網(wǎng)膜脫離,嚴(yán)重威脅患者的視力。目前臨床上尚無有效的阻止新生血管形成的治療方法,視網(wǎng)膜激光光凝、血管內(nèi)皮生長因子抑制劑、光動(dòng)力療法、類固醇激素以及手術(shù)治療能在一定程度上減少新生血管的形成,但不能從根本上消除新生血管形成因素,并且伴隨著一系列的副損傷及復(fù)發(fā)的可能性。大量研究表明,血管內(nèi)皮結(jié)構(gòu)破壞和功能障礙在視網(wǎng)膜缺血性疾病和病理性新生血管形成和發(fā)展過程中發(fā)揮重要的作用,因此,控制視網(wǎng)膜缺血性疾病新生血管形成的關(guān)鍵在于修復(fù)受損傷的血管內(nèi)皮,改善視網(wǎng)膜的缺血缺氧狀態(tài)。內(nèi)皮祖細(xì)胞(EPCs)是一類能增殖并分化為血管內(nèi)皮細(xì)胞的前體細(xì)胞,具有修復(fù)血管內(nèi)皮損傷和參與新生血管形成的功能。移植EPCs能夠改善心、腦和肢體等局部缺血的損傷,增加缺血部位的血流量和毛細(xì)血管密度,提高對(duì)缺血性疾病的療效。如果能將EPCs有效的移植到缺血缺氧的視網(wǎng)膜局部,就有可能修復(fù)受損的血管內(nèi)皮,改善視網(wǎng)膜的血供,避免病理性新生血管的生成,這項(xiàng)研究不僅需要合適的細(xì)胞移植手段,還需要一種穩(wěn)定、高效的示蹤方法為實(shí)驗(yàn)提供客觀的觀察依據(jù)。目的 通過羧基熒光素二醋酸鹽琥珀酰亞胺酯(CFSE)標(biāo)記EPCs、Dil標(biāo)記的乙�；兔芏戎鞍�(DiI-AcLDL)標(biāo)記EPCs以及慢病毒介導(dǎo)綠色熒光蛋白(GFP)轉(zhuǎn)導(dǎo)EPCs,比較三種標(biāo)記物在體外和體內(nèi)對(duì)EPCs的示蹤情況,并觀察移植EPCs對(duì)視網(wǎng)膜血管損傷的修復(fù),為今后細(xì)胞移植選擇合適的標(biāo)記物,更好的跟蹤EPCs移植的療效提供實(shí)驗(yàn)基礎(chǔ)。 方法 (1)人臍帶血EPCs的培養(yǎng)及鑒定：通過羥乙基淀粉沉降法和Percoll密度梯度離心法分離人臍帶血中單個(gè)核細(xì)胞,在體外誘導(dǎo)分化成為EPCs,并通過觀察細(xì)胞形態(tài)學(xué)、流式細(xì)胞術(shù)分析細(xì)胞表面標(biāo)志、免疫熒光染色以及電鏡等方法進(jìn)行鑒定。(2)三種方法體外標(biāo)記EPCs:分別用CFSE、DiI-AcLDL以及慢病毒介導(dǎo)GFP基因轉(zhuǎn)導(dǎo)標(biāo)記EPCs,通過倒置相差顯微鏡觀察標(biāo)記前后細(xì)胞形態(tài)的改變,臺(tái)盼藍(lán)拒染法和貼壁細(xì)胞計(jì)數(shù)法測定標(biāo)記后EPCs的生存能力和粘附能力的變化,熒光顯微鏡下觀察熒光強(qiáng)度以及標(biāo)記熒光隨培養(yǎng)時(shí)間的變化情況,流式細(xì)胞術(shù)測定標(biāo)記陽性率,并綜合比較三種方法體外標(biāo)記細(xì)胞的優(yōu)缺點(diǎn)。(3)標(biāo)記的EPCs眼內(nèi)移植示蹤：利用多波長氪激光選擇性損傷視網(wǎng)膜,建立C57BL/6N小鼠視網(wǎng)膜血管損傷模型。分別收集DiI-AcLDL、CFSE、慢病毒介導(dǎo)GFP基因轉(zhuǎn)導(dǎo)三種方法標(biāo)記的EPCs,手術(shù)顯微鏡下采用微量注射器移植入玻璃體腔內(nèi)。分時(shí)段觀察眼底照相、視網(wǎng)膜石蠟切片、冰凍切片和視網(wǎng)膜鋪片,觀察標(biāo)記細(xì)胞在視網(wǎng)膜縱向切面和橫向平面的分布、熒光強(qiáng)度和持續(xù)時(shí)間等情況,并比較EPCs移植前后視網(wǎng)膜血管損傷修復(fù)情況。 結(jié)果 (1)從人臍帶血分離原代培養(yǎng)EPCs,在培養(yǎng)過程中表現(xiàn)為典型EPCs的形態(tài)變化特點(diǎn),不同程度的表達(dá)CD34、CD133和’VEGFR-2等細(xì)胞表面標(biāo)志,可吞噬DiI-AcLDL并同時(shí)結(jié)合FITC-UEA-I,電鏡檢查可見內(nèi)皮細(xì)胞特有的W-P小體,證明所培養(yǎng)的細(xì)胞群體中大部分是正在分化中的EPCs。(2)CFSE標(biāo)記后EPCs呈現(xiàn)綠色熒光,標(biāo)記陽性率可達(dá)95%以上；DiI-AcLDL標(biāo)記EPCs呈紅色熒光,標(biāo)記陽性率可達(dá)80%；CFSE和DiI-AcLDL標(biāo)記熒光可持續(xù)4周,熒光強(qiáng)度隨培養(yǎng)時(shí)間的延長逐漸下降。慢病毒介導(dǎo)GFP基因轉(zhuǎn)導(dǎo)EPCs后4天,激光共聚焦顯微鏡下可觀察到細(xì)胞發(fā)出綠色熒光,隨后綠色熒光陽性細(xì)胞逐漸增多,熒光強(qiáng)度逐漸增強(qiáng),轉(zhuǎn)導(dǎo)后4周轉(zhuǎn)染效率超過30%。三種標(biāo)記方法細(xì)胞形態(tài)無明顯改變,在標(biāo)記2天和7天后,檢測生存能力及粘附能力較未標(biāo)記的細(xì)胞無明顯變化。(3)體內(nèi)試驗(yàn)中,通過視網(wǎng)膜激光光凝成功建立小鼠視網(wǎng)膜血管損傷模型。將標(biāo)記的EPCs進(jìn)行玻璃體腔注射,眼內(nèi)移植4周后,眼底照相可見激光斑色素沉著及瘢痕形成較未移植者減輕。移植EPCs后4周眼球石蠟切片HE染色,可見神經(jīng)纖維層血管周圍有細(xì)胞聚集,視網(wǎng)膜各層結(jié)構(gòu)比較規(guī)整,形成瘢痕較小。移植后視網(wǎng)膜冰凍切片顯示,DiI-AcLDL和CFSE標(biāo)記EPCs移植后2天于視網(wǎng)膜表面可見熒光細(xì)胞,1周時(shí)可見熒光細(xì)胞聚集于損傷部位,4周可見熒光細(xì)胞分布于視網(wǎng)膜各層,以視網(wǎng)膜血管富集的神經(jīng)纖維層和內(nèi)核層為主。慢病毒介導(dǎo)GFP基因轉(zhuǎn)導(dǎo)EPCs移植后各時(shí)間點(diǎn)視網(wǎng)膜冰凍切片未見熒光細(xì)胞。伊文思藍(lán)灌注血管造影可清晰顯示視網(wǎng)膜毛細(xì)血管網(wǎng)的結(jié)構(gòu),視網(wǎng)膜血管損傷模型激光斑處可見熒光滲漏。移植CFSE標(biāo)記的EPCs后2天行視網(wǎng)膜鋪片,可見綠色熒光標(biāo)記細(xì)胞群聚分布于視網(wǎng)膜上；移植后1周,綠色熒光標(biāo)記細(xì)胞在激光損傷周圍聚集；移植后4周,綠色熒光標(biāo)記細(xì)胞形成類似管狀結(jié)構(gòu),證實(shí)EPCs參與視網(wǎng)膜血管修復(fù)。 結(jié)論 (1)CFSE和Dil-AcLDL適合短期示蹤EPCs。CFSE標(biāo)記EPCs效率最高,起始熒光最強(qiáng),費(fèi)用最低,操作最簡便,短期示蹤更有優(yōu)勢。CFSE標(biāo)記EPCs聯(lián)合視網(wǎng)膜冰凍切片與伊文思藍(lán)灌注視網(wǎng)膜鋪片為EPCs眼內(nèi)移植的示蹤建立了多角度的觀察方法。慢病毒介導(dǎo)GFP基因轉(zhuǎn)導(dǎo)EPCs在長期示蹤方面更有潛力。(2)利用視網(wǎng)膜激光光凝建立了小鼠視網(wǎng)膜血管損傷模型,并通過玻璃體腔注射進(jìn)行EPCs眼內(nèi)移植。通過體內(nèi)示蹤,證實(shí)ECPs具有向損傷視網(wǎng)膜定向歸巢的能力,并能夠參與視網(wǎng)膜血管損傷的修復(fù)。
[Abstract]:Diabetic retinopathy, including retinopathy of premature infants, central retinal vein occlusion and other retinal ischemic diseases, is characterized by neovascularization. Additionally, it causes edema and exudation of peripheral tissues, and then leads to ischemia and hypoxia of retinal tissues, and compensatory formation of new blood vessels with incomplete organizational structure, leading to bleeding, proliferation, and even retinal detachment, which seriously threatens the visual acuity of patients. Endothelial growth factor inhibitors, photodynamic therapy, steroid hormones and surgical treatment can reduce angiogenesis to a certain extent, but can not fundamentally eliminate angiogenesis factors, and accompanied by a series of side effects and the possibility of recurrence. Retinal ischemic diseases and pathological neovascularization play an important role in the formation and development of retinal ischemic diseases. Therefore, the key to control retinal neovascularization is to repair damaged vascular endothelium and improve retinal hypoxia and hypoxia. EPCs transplantation can improve the ischemic injury of heart, brain and limbs, increase the blood flow and capillary density in ischemic sites, and improve the therapeutic effect on ischemic diseases. If EPCs can be effectively transplanted to the ischemic and hypoxic retina region It is possible to repair damaged vascular endothelium, improve retinal blood supply, and avoid pathological neovascularization. This study requires not only appropriate cell transplantation, but also a stable and efficient tracing method to provide an objective basis for the experiment.
Carboxyfluorescein diacetate succinimide ester (CFSE) labeled EPCs, Dil labeled acetylated low density lipoprotein (DiI-AcLDL) labeled EPCs and lentivirus mediated green fluorescent protein (GFP) transduction of EPCs were used to compare the tracing of EPCs in vitro and in vivo, and to observe the repairing effect of transplanted EPCs on retinal vascular injury. It provides an experimental basis for selecting suitable markers for cell transplantation and better tracking the efficacy of EPCs transplantation.
Method
(1) Culture and identification of EPCs from human umbilical cord blood: Mononuclear cells from human umbilical cord blood were isolated by hydroxyethyl starch sedimentation and Percoll density gradient centrifugation, and differentiated into EPCs in vitro. The cells were identified by morphology, flow cytometry, immunofluorescence staining and electron microscopy. Methods EPCs were labeled in vitro by CFSE, DiI-AcLDL and lentivirus-mediated GFP gene transduction. The morphological changes of the cells before and after labeling were observed by inverted phase contrast microscope. The viability and adhesion of the labeled EPCs were measured by Trypan blue staining and adherent cell counting. The fluorescence intensity was observed under fluorescence microscope. Flow cytometry was used to determine the positive rate of labeling and compare the advantages and disadvantages of the three methods. (3) Intraocular transplantation of labeled EPCs: C57BL/6N mice retinal vascular injury model was established by multi-wavelength krypton laser selective injury. EPCs labeled with LDL, CFSE and lentivirus mediated GFP gene transduction were transplanted into vitreous cavity by microinjector under operating microscope. Fundus photography, retinal paraffin section, frozen section and retinal paving were observed at different time intervals. The distribution, fluorescence intensity and persistence of labeled cells in longitudinal and transverse plane of retina were observed. Time and other conditions, and compare the repair of retinal vascular injury before and after EPCs transplantation.
Result
(1) The primary cultured EPCs were isolated from human umbilical cord blood and showed typical morphological changes during the culture process. The cells expressed CD34, CD133 and''VEGFR-2 in different degrees. They could phagocytose DiI-AcLDL and combine with FITC-UEA-I. The W-P bodies of endothelial cells were observed under electron microscope, which proved that most of the cultured cells were in large part of the population. The positive rate of EPCs labeled with DiI-AcLDL was up to 80%. The fluorescence intensity of CFSE and DiI-AcLDL labeled EPCs lasted for 4 weeks and gradually decreased with the time of culture. Four days after transduction of GFP gene by lentivirus, the positive rate of EPCs labeled with DiI-AcLDL was up to 95%. The green fluorescence was observed under confocal microscope, and then the green fluorescence positive cells increased gradually, the fluorescence intensity increased gradually, and the transfection efficiency exceeded 30% at 4 weeks after transduction. Changes. (3) In vivo, retinal vascular injury model was successfully established by laser photocoagulation in mice. After intravitreal injection of labeled EPCs, laser spot pigmentation and scar formation were observed in fundus photography at 4 weeks after transplantation, which were less severe than those in non-transplanted eyes. After transplantation, the frozen sections of the retina showed that fluorescent cells could be seen on the surface of the retina 2 days after transplantation of DiI-AcLDL and CFSE-labeled EPCs. At 1 week, fluorescent cells could be seen in the injured area. At 4 weeks, fluorescent cells could be seen in all layers of the retina. There were no fluorescent cells in the frozen sections of retina at each time point after lentivirus-mediated GFP gene transduction of EPCs transplantation. Evans blue perfusion angiography could clearly show the structure of retinal capillary network, and fluorescent leakage could be seen in the laser spot of retinal vascular injury model. Two days after transplantation, green fluorescent labeled cells clustered on the retina. One week after transplantation, green fluorescent labeled cells clustered around the laser injury. Four weeks after transplantation, green fluorescent labeled cells formed a similar tubular structure, confirming that EPCs participated in retinal vascular repair.
conclusion
(1) CFSE and Dil-AcLDL are suitable for short-term tracing of EPCs. CFSE has the highest efficiency, the strongest initial fluorescence, the lowest cost, the simplest operation and the advantages of short-term tracing. CFSE labeling EPCs combined with frozen section of retina and Evans blue perfusion retina paving have established a multi-angle observation method for the tracing of EPCs intraocular transplantation. Gene-transduced EPCs have more potential in long-term tracing. (2) Retinal vascular injury model was established by laser photocoagulation in mice, and intraocular transplantation of EPCs was performed by intravitreal injection.
【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】：2011
【分類號(hào)】：R774.1

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9 王玉環(huán);陳超;石文靜;;不同日齡新生小鼠的視網(wǎng)膜血管對(duì)氧療的敏感性[A];2006（第三屆）江浙滬兒科學(xué)術(shù)會(huì)議暨浙江省兒科學(xué)術(shù)年會(huì)論文匯編[C];2006年

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