本文選題：脈絡(luò)膜新生血管 + 年齡相關(guān)性黃斑變性��； 參考：《第四軍醫(yī)大學(xué)》2010年博士論文

【摘要】： 研究背景脈絡(luò)膜新生血管(choroidal neovascularization, CNV)是近40種眼病的共有體征,視網(wǎng)膜下病理性新生血管的生長不但損傷正常脈絡(luò)膜視網(wǎng)膜結(jié)構(gòu),而且常發(fā)生出血滲漏,往往造成嚴(yán)重視力損害。CNV的發(fā)生機(jī)制復(fù)雜,涉及多種細(xì)胞、因子和信號通路,確切機(jī)制尚未完全闡明。 成年個體新生血管的形成包含兩種機(jī)制:血管生成(angiogenesis)指原有的血管細(xì)胞發(fā)生增生、移行,形成新的血管;血管發(fā)生(vasculogenesis)指由干細(xì)胞分化而來的血管細(xì)胞構(gòu)成新血管。干細(xì)胞在一般情況下處于“休眠”狀態(tài),在外因刺激或病理狀態(tài)下會進(jìn)入血液循環(huán),移行至周邊組織,表現(xiàn)出不同程度的更新和分化能力,參與組織修復(fù)和新生物形成。近期的研究表明,CNV的生成兼具這兩種方式,既有原位組織細(xì)胞的增殖,又有骨髓來源細(xì)胞(bone marrow-derived cells, BMCs)的參與,但BMCs究竟以何種方式參與CNV生成、BMCs對CNV發(fā)展有何影響有待進(jìn)一步闡明。此外,BMCs是一個異質(zhì)性細(xì)胞群體,包含多種干/祖細(xì)胞,究竟是哪種干/祖細(xì)胞參與了CNV的生成、它們在CNV發(fā)生發(fā)展中發(fā)揮著怎樣的作用、是否可作為CNV治療的靶點等諸多問題有待研究。 目的和內(nèi)容探討B(tài)MCs在CNV發(fā)生發(fā)展中的作用,在危險因素影響CNV發(fā)展中的介導(dǎo)作用,及作為干細(xì)胞療法細(xì)胞載體的潛能。⑴建立C57-綠色熒光蛋白(green fluorescent protein, GFP)嵌合體小鼠,觀察在嵌合體CNV發(fā)生發(fā)展過程中,BMCs在CNV聚集、分化和表達(dá)蛋白情況;⑵探討尼古丁對參與CNV生成的BMCs的趨化、分化和蛋白表達(dá)能力的影響;⑶觀察骨髓來源基質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)向CNV的趨化特異性和時間窗,觀察MSCs參與CNV生成情況,探討MSCs是否具備作為藥物載體的潛能;⑷構(gòu)建表達(dá)人源性色素上皮衍生因子(pigment epithelium derived factor, PEDF)的腺病毒,觀察腺病毒轉(zhuǎn)導(dǎo)的MSCs對CNV的抑制作用,探討利用MSCs靶向治療CNV的新策略。 方法⑴通過GFP轉(zhuǎn)基因小鼠與野生型C57小鼠的骨髓移植建立嵌合體小鼠,利用532nm激光建立小鼠CNV模型,脈絡(luò)膜鋪片觀察GFP-BMCs向CNV趨化、參與血管結(jié)構(gòu)組成的情況,免疫熒光染色觀察CNV中BMCs分化的細(xì)胞類型(包括血管內(nèi)皮細(xì)胞、血管平滑肌細(xì)胞和巨噬細(xì)胞)及表達(dá)血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)和堿性成纖維細(xì)胞生長因子(basic fibroblast cell growth factor, bFGF)的情況;⑵激光光凝建立C57-GFP嵌合體小鼠CNV模型后,即日在其飲水中添加尼古丁(100μg/ml),對照組嵌合體小鼠建立CNV模型后普通飼養(yǎng),4周后眼球切片HE染色觀察CNV厚度與直徑,脈絡(luò)膜鋪片觀察CNV表面積、GFP-BMCs的趨化和參與構(gòu)成血管情況,免疫熒光染色觀察CNV中BMCs分化情況及各因子表達(dá)變化;⑶體外培養(yǎng)GFP轉(zhuǎn)基因小鼠骨髓來源MSCs,野生型小鼠激光建模后0.5-1小時尾靜脈注射4.0×106 GFP-BMCs或GFP-MSCs,激光后1、3、7天,心、肝、脾、肺、心臟切片熒光觀察比較兩種細(xì)胞在器官內(nèi)的滯留情況;野生型小鼠激光后0.5-1小時、3天及6天,分別注射4.0×106 GFP-MSCs,激光后7天觀察比較單次注射、雙次注射和三次注射后GFP-MSCs在體內(nèi)移行和向CNV趨化的情況;酶聯(lián)免疫吸附測定(enzyme linked immunosorbent assay, ELISA)和免疫熒光染色檢測激光后不同時間眼內(nèi)干細(xì)胞趨化因子基質(zhì)細(xì)胞衍生因子-1(stromal cell derived factor-1, SDF-1)的表達(dá);脈絡(luò)膜鋪片檢測參與構(gòu)成新血管的GFP-MSCs,免疫熒光染色分析CNV中GFP-MSCs分化的細(xì)胞類型;⑷構(gòu)建表達(dá)人源性PEDF、含報告基因GFP、E1 E3缺陷的5型腺病毒(adenovirus, Ad),轉(zhuǎn)導(dǎo)體外培養(yǎng)的野生型小鼠骨髓來源MSCs,倒置熒光顯微鏡觀察及流式細(xì)胞儀測定感染效率,ELISA檢測體外人源性PEDF表達(dá)情況;激光后0.5-1小時小鼠尾靜脈分別注射4.0×106 Ad-PEDF/MSCs、對照病毒Ad-GFP/MSCs、GFP-MSCs及0.4ml磷酸鹽緩沖液(Phosphate buffered saline, PBS),分別于激光后1、3、5、7天利用免疫熒光染色和ELISA檢測眼內(nèi)人源性PEDF表達(dá),激光后7天眼球切片組織學(xué)分析和脈絡(luò)膜鋪片分析比較各組CNV厚度、直徑及表面積。體外建立視網(wǎng)膜色素上皮(retinal pigment epithelium, RPE)細(xì)胞與MSCs的共培養(yǎng)體系,分析MSCs分泌的PEDF對RPE細(xì)胞增生和移行的影響。 結(jié)果⑴激光后大量GFP-BMCs聚集于激光斑處并整合入CNV中的血管結(jié)構(gòu)內(nèi),由其組成的血管面積約占CNV表面積的16.22%。GFP-BMCs主要分布于CNV區(qū)域(包括CNV深層的脈絡(luò)膜),少量散布于CNV表層的視網(wǎng)膜、角鞏膜緣、睫狀體、視盤周圍及遠(yuǎn)離CNV的視網(wǎng)膜、脈絡(luò)膜和鞏膜。CD31或αSMA陽性的GFP+細(xì)胞僅出現(xiàn)于CNV區(qū)域,F4/80+/GFP+細(xì)胞不僅出現(xiàn)在CNV區(qū)域,還出現(xiàn)在CNV表層的視網(wǎng)膜、角鞏膜緣和睫狀體內(nèi)。CNV內(nèi)CD31/GFP、αSMA/GFP和F4/80/GFP雙陽性細(xì)胞在全部GFP+細(xì)胞中的構(gòu)成比隨CNV進(jìn)展而變化,CD31/GFP或F4/80/GFP雙陽性細(xì)胞構(gòu)成比的最高值出現(xiàn)于激光后第2周,而αSMA+/GFP+細(xì)胞構(gòu)成比在激光后4周仍處上升趨勢。CNV區(qū)域的一些GFP-BMCs可表達(dá)促血管形成因子VEGF和bFGF。⑵尼古丁處理組小鼠CNV長度和表面積增大,CNV內(nèi)GFP-BMCs的面積和密度增加,GFP-BMCs來源的血管細(xì)胞面積增加,F4/80+/GFP+細(xì)胞構(gòu)成比下降,CNV內(nèi)VEGF和bFGF的表達(dá)及CNV下脈絡(luò)膜內(nèi)VCAM-1的表達(dá)上調(diào)。⑶在整個觀察期間,肺、肝和脾內(nèi)可見GFP+BMCs滯留,但未觀測到GFP+MSCs。單次、雙次或三次MSCs注射后趨化至CNV的MSCs的數(shù)量沒有明顯差異,雙次和三次注射組小鼠的脾內(nèi)發(fā)現(xiàn)大量GFP-MSCs。SDF-1免疫熒光染色顯示,激光后初期,SDF-1在激光斑處RPE層表達(dá),隨著CNV生成,CNV內(nèi)出現(xiàn)陽性染色;ELISA結(jié)果顯示,SDF-1表達(dá)水平在最初24小時內(nèi)迅速增加,在第2天時達(dá)頂峰,然后迅速下降。MSCs移植后外周血和骨髓的流式分析顯示,MSCs僅于第1天內(nèi)在骨髓中作短暫停留。脈絡(luò)膜鋪片顯示,激光后第1天,GFP-MSCs散布于激光斑周圍;第3天時,GFP-MSCs組成的“細(xì)胞環(huán)”圍繞激光斑,顯示出向CNV靠近的定向移行趨勢;第7天,GFP-MSCs進(jìn)入CNV內(nèi)并加入血管組成中。眼球切片顯示絕大多數(shù)GFP-MSCs位于激光斑處脈絡(luò)膜與光感受器間的CNV內(nèi)。在這些MSCs中發(fā)現(xiàn)了血管內(nèi)皮細(xì)胞、血管平滑肌細(xì)胞、巨噬細(xì)胞、上皮細(xì)胞和成纖維細(xì)胞的標(biāo)記物(CD31,αSMA, F4/80, keratin,和vimentin)的陽性表達(dá)。⑷Ad轉(zhuǎn)導(dǎo)小鼠MSCs后24小時,倒置熒光顯微鏡檢測到報告基因GFP的表達(dá),流式分析顯示(73.6±5.3)%的細(xì)胞轉(zhuǎn)導(dǎo)成功,AdPEDF轉(zhuǎn)導(dǎo)的MSCs在體外表達(dá)PEDF可持續(xù)至少8天。AdPEDF組小鼠的眼球切片中,人PEDF的陽性染色出現(xiàn)于MSCs內(nèi)及其周圍的細(xì)胞外基質(zhì)中;ELISA結(jié)果顯示,在整個觀察期間,眼內(nèi)人PEDF呈穩(wěn)定表達(dá),表達(dá)量是抑制CNV閾值的4倍多;其CNV的厚度、長度和表面積明顯減小。與AdPEDF/MSCs共培養(yǎng)的RPE細(xì)胞較之其他共培養(yǎng)條件下的RPE細(xì)胞增生和移行更快。 結(jié)論⑴BMCs分化為多種細(xì)胞類型參與CNV細(xì)胞構(gòu)成,分泌促血管形成因子促進(jìn)CNV發(fā)展,在CNV生成過程中發(fā)揮著重要作用。CNV微環(huán)境趨化BMCs,并支持和調(diào)控BMCs的分化方向。BMCs與CNV微環(huán)境可能存在相互作用。⑵尼古丁促進(jìn)BMCs向CNV趨化和參與CNV生成,并影響CNV內(nèi)BMCs的分化。尼古丁對BMCs的這種作用可能部分通過調(diào)節(jié)局部因子(如VEGF、VCAM-1)表達(dá)實現(xiàn)。⑶MSCs僅出現(xiàn)于CNV而不在其他器官停留(骨髓中一過性停留),提示其在CNV模型中具有比BMCs更為卓越的特異趨化能力;一次激光光凝引起的MSCs趨化具有有限的時間窗,其原因可能是激光后CNV區(qū)域趨化因子的表達(dá)規(guī)律。激光后MSCs逐步趨化至CNV內(nèi),并分化為CNV生成所需的多種細(xì)胞類型。MSCs具備作為細(xì)胞載體的潛能。⑷經(jīng)基因修飾的MSCs到達(dá)CNV部位并在局部表達(dá)抗新生血管形成因子,從而抑制了CNV的生長,該效應(yīng)可能部分通過在CNV發(fā)展中發(fā)揮重要作用的RPE細(xì)胞介導(dǎo)。MSCs有望作為抗新生血管藥物的釋放系統(tǒng)用于CNV相關(guān)疾病的治療。
[Abstract]:Background choroidal neovascularization (choroidal neovascularization, CNV) is a common sign of nearly 40 kinds of ophthalmopathy. The growth of subretinal pathological neovascularization not only damages the normal choroid retina structure, but also often causes bleeding and leakage, often causing serious visual impairment of.CNV, which involves a variety of cells, factors and factors. The signal pathway, the exact mechanism has not been fully elucidated.
The formation of neovascularization in adult individuals consists of two mechanisms: angiogenesis (angiogenesis) refers to the proliferation, migration and formation of new vessels in the original vascular cells; angiogenesis (vasculogenesis) refers to the formation of new vascular cells derived from stem cells. In general, stem cells are in a "dormant" state, stimulated by external causes or The pathological state will enter the blood circulation, move to the peripheral tissue, show different degrees of regeneration and differentiation, participate in tissue repair and new biological formation. Recent studies have shown that the formation of CNV has both these two ways, both in situ tissue cell proliferation, and the involvement of bone marrow-derived cells, BMCs. But what is the way BMCs participates in CNV generation, and what influence BMCs has on the development of CNV needs further clarification. In addition, BMCs is a heterogeneous cell population, including a variety of stem / progenitor cells, which stem / progenitor cells are involved in the formation of CNV, how they play a role in the development of CNV and whether they can be used as a target for CNV treatment. There are many problems to be studied.
Purpose and content to explore the role of BMCs in the development of CNV, the mediating role of the risk factors in the development of CNV, and the potential of as a cell carrier for stem cell therapy. (1) the establishment of C57- green fluorescent protein (green fluorescent protein, GFP) chimerism mice, observed in the process of the development of chimerism CNV, BMCs in CNV aggregation, differentiation and development. To investigate the effect of nicotine on the chemotaxis, differentiation and protein expression of BMCs generated by CNV; (3) observe the chemotaxis and time window of mesenchymal stem cells (MSCs) to CNV, observe the participation of MSCs in the formation of CNV, and explore the potential of MSCs as a drug carrier; 4 To construct adenoviruses expressing human derived pigment epithelium derived factor (PEDF), observe the inhibitory effect of MSCs transduced on adenovirus on CNV, and explore a new strategy for the treatment of CNV by targeting MSCs.
Methods (1) the mice were built by bone marrow transplantation of GFP transgenic mice and wild type C57 mice. The mouse CNV model was established by 532nm laser. The chemotaxis of GFP-BMCs to CNV was observed and the structure of vascular structure was observed by GFP-BMCs. Immunofluorescence staining was used to observe the type of BMCs differentiated in CNV (including vascular endothelial cells and smooth blood vessel). Muscle cells and macrophages) and the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (basic fibroblast cell growth factor, bFGF). (2) after laser photocoagulation to establish a C57-GFP chimera model, nicotine was added to the drinking water day (100 mu), control After setting up CNV model in the group of chimerism mice, the thickness and diameter of CNV were observed by HE staining of the eyeball section 4 weeks later. The surface area of CNV was observed by the choroid spreading, the chemotaxis and participation of GFP-BMCs were observed. The differentiation of BMCs in CNV and the expression of various factors in CNV were observed by immunofluorescence staining, and (3) the bone marrow origin of GFP transgenic mice was cultured in vitro. S, after laser modeling in wild type mice, 4 x 106 GFP-BMCs or GFP-MSCs were injected into the tail vein in 0.5-1 hours. After 1,3,7 days, the retention of two cells in the organs was compared with the heart, liver, spleen, lung and heart. After the laser, 3 days and 6 days after the laser in wild type mice, 4 x 106 GFP-MSCs were injected respectively, and the comparison was observed 7 days after the laser. The transition of GFP-MSCs in the body and the chemotaxis to CNV after the second injection, double injection and three injection; the expression of enzyme linked immunosorbent assay, ELISA, and immunofluorescence staining for the stromal cell derivatization factor -1 (stromal cell derived factor-1) at different times after the laser Da; choroidal paving detection involved in the formation of GFP-MSCs of new blood vessels, and immunofluorescence staining analysis of GFP-MSCs differentiated cell types in CNV; (4) constructing 5 types of adenoviruses expressing human PEDF, GFP, E1 E3 deficiency (adenovirus, Ad), transduced in vitro cultured mouse bone marrow MSCs, inverted fluorescence microscope observation and flow pattern The infection efficiency was measured by the cytometer, and the expression of human PEDF in vitro was detected by ELISA. The tail veins of mice were injected 4 x 106 Ad-PEDF/MSCs respectively at 0.5-1 hours after the laser, compared with the virus Ad-GFP/MSCs, GFP-MSCs and 0.4ml phosphate buffer solution (Phosphate buffered saline, PBS). The expression of human PEDF in the eye, the histological analysis of the eyeball section and the choroidal spread 7 days after the laser, the thickness, diameter and surface area of CNV were compared. The co culture system of retinal pigment epithelium (RPE) cells and MSCs was established in vitro, and the effect of PEDF on the proliferation and migration of RPE cells was analyzed.
Results (1) a large number of GFP-BMCs gathered at the laser spot and integrated into the vascular structure of the CNV. The 16.22%.GFP-BMCs of the blood vessel area of the CNV surface area was mainly distributed in the CNV region (including the choroid of the deep CNV), and a small amount scattered in the retina, the corneo sclera, the ciliary body, the perioptic disc, and the view far from CNV on the surface of the CNV. The omentum, choroidal and scleral.CD31 or alpha SMA positive GFP+ cells only appeared in the CNV region. F4/80+/GFP+ cells not only appeared in the CNV region, but also appeared in the retina, the corneo sclera and the ciliary body.CNV in the CNV surface, and the constituent ratio of the alpha SMA/GFP and F4/80/GFP double positive cells in all GFP+ cells varied with the progression of the progression. The highest value of the constituent ratio of F4/80/GFP double positive cells appeared at second weeks after the laser, while the formation of alpha SMA+/GFP+ cells still rose at 4 weeks after the laser. Some of the GFP-BMCs expressed in the.CNV region could increase the length and surface of the CNV in the group of VEGF and bFGF. (2) nicotine treatment group, and the area and density of GFP-BMCs increased in CNV. The area of GFP-BMCs derived vascular cells increased, the proportion of F4/80+/GFP+ cells decreased, the expression of VEGF and bFGF in CNV and the expression of VCAM-1 in the choroid under CNV were up-regulated. (3) GFP+BMCs retention in the lungs, liver and spleen was observed throughout the observation period, but the GFP+MSCs. single, double or three MSCs injected into CNV MSCs were not observed. There were obvious differences. A large number of GFP-MSCs.SDF-1 immunofluorescence staining in the spleen of the mice with double and three injections showed that SDF-1 was expressed in the RPE layer at the laser spot in the early stage after the laser. With the formation of CNV, the positive staining was found in CNV. The results of ELISA showed that the expression level of SDF-1 increased rapidly within the first 24 hours and reached the peak at second days and then quickly. The flow analysis of peripheral blood and bone marrow after.MSCs transplantation showed that MSCs only stayed in the bone marrow in first days. The choroidal spread showed that the laser was scattered around the laser spot at first days after the laser; at third days, the "cell ring" of the GFP-MSCs was around the laser spot, showing the direction shift toward CNV; seventh days, GFP-MSCs Entry into the CNV and added to the vascular composition. The eyeball section shows that most of the GFP-MSCs is located in the CNV between the choroid and the photoreceptor at the laser spot. In these MSCs, the markers of vascular endothelial cells, vascular smooth muscle cells, macrophages, epithelial cells and fibroblasts (CD31, alpha SMA, F4/80, keratin, and vimentin) are found. 24 hours after MSCs transduction in Ad, the expression of the reporter gene GFP was detected by inverted fluorescence microscopy. Flow analysis showed that (73.6 + 5.3)% of the cell transduction was successful. The AdPEDF transduced MSCs expressed PEDF in the eyeball section of the.AdPEDF group of the.AdPEDF group at least 8 days in vitro, and the positive staining of human PEDF appeared in MSCs and its surrounding fine. In the extracellular matrix, ELISA results showed that the expression of PEDF in the eye was stable during the entire observation period, and the expression amount was 4 times more than the CNV threshold, and the thickness, length and surface area of CNV were significantly reduced. The RPE cells co cultured with AdPEDF/MSCs were more proliferating and migrating more RPE cells under other co culture conditions.
Conclusion (1) BMCs differentiates into a variety of cell types and participates in the composition of CNV cells, secreting the angiogenesis factor to promote the development of CNV, and plays an important role in the development of CNV, and plays an important role in the formation of CNV..CNV microenvironment chemotactic BMCs, and the support and regulation of the differentiation direction of BMCs may exist in the CNV microenvironment. (2) nicotine promotes BMCs to CNV and participates in CNV. It is produced and affects the differentiation of BMCs in CNV. This effect of nicotine on BMCs may be partly realized by regulating local factors (such as VEGF, VCAM-1). (3) MSCs appears only in CNV but not in other organs (an overstay in the bone marrow), suggesting that it has a more excellent specific chemotaxis than BMCs in the CNV model; a laser photocoagulation. The cause of MSCs chemotaxis has a limited time window, which may be due to the expression of chemokine in the CNV region after the laser. After the laser, MSCs gradually converge to CNV, and differentiate into a variety of cell types required for CNV generation,.MSCs has the potential as a cell carrier. (4) the gene modified MSCs arrives at the CNV site and is locally expressed in the expression of anti newborn. Angiogenic factors, which inhibit the growth of CNV, may be partly mediated by RPE cells, which play an important role in the development of CNV, and are expected to be used as a release system for anti neovascularization drugs for the treatment of CNV related diseases.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R773.4

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 詹文捷;明睛顆粒對骨髓來源細(xì)胞參與脈絡(luò)膜新生血管形成的干預(yù)作用研究[D];廣州中醫(yī)藥大學(xué);2013年

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