骨髓来源细胞在脉络膜新生血管发生发展中的作用及其治疗潜能

发布时间：2018-06-22 02:46

本文选题：脉络膜新生血管 + 年龄相关性黄斑变性　；参考：《第四军医大学》2010年博士论文

【摘要】： 研究背景脉络膜新生血管(choroidal neovascularization, CNV)是近40种眼病的共有体征,视网膜下病理性新生血管的生长不但损伤正常脉络膜视网膜结构,而且常发生出血渗漏,往往造成严重视力损害。CNV的发生机制复杂,涉及多种细胞、因子和信号通路,确切机制尚未完全阐明。成年个体新生血管的形成包含两种机制:血管生成(angiogenesis)指原有的血管细胞发生增生、移行,形成新的血管;血管发生(vasculogenesis)指由干细胞分化而来的血管细胞构成新血管。干细胞在一般情况下处于“休眠”状态,在外因刺激或病理状态下会进入血液循环,移行至周边组织,表现出不同程度的更新和分化能力,参与组织修复和新生物形成。近期的研究表明,CNV的生成兼具这两种方式,既有原位组织细胞的增殖,又有骨髓来源细胞(bone marrow-derived cells, BMCs)的参与,但BMCs究竟以何种方式参与CNV生成、BMCs对CNV发展有何影响有待进一步阐明。此外,BMCs是一个异质性细胞群体,包含多种干/祖细胞,究竟是哪种干/祖细胞参与了CNV的生成、它们在CNV发生发展中发挥着怎样的作用、是否可作为CNV治疗的靶点等诸多问题有待研究。目的和内容探讨BMCs在CNV发生发展中的作用,在危险因素影响CNV发展中的介导作用,及作为干细胞疗法细胞载体的潜能。⑴建立C57-绿色荧光蛋白(green fluorescent protein, GFP)嵌合体小鼠,观察在嵌合体CNV发生发展过程中,BMCs在CNV聚集、分化和表达蛋白情况;⑵探讨尼古丁对参与CNV生成的BMCs的趋化、分化和蛋白表达能力的影响;⑶观察骨髓来源基质干细胞(mesenchymal stem cells, MSCs)向CNV的趋化特异性和时间窗,观察MSCs参与CNV生成情况,探讨MSCs是否具备作为药物载体的潜能;⑷构建表达人源性色素上皮衍生因子(pigment epithelium derived factor, PEDF)的腺病毒,观察腺病毒转导的MSCs对CNV的抑制作用,探讨利用MSCs靶向治疗CNV的新策略。方法⑴通过GFP转基因小鼠与野生型C57小鼠的骨髓移植建立嵌合体小鼠,利用532nm激光建立小鼠CNV模型,脉络膜铺片观察GFP-BMCs向CNV趋化、参与血管结构组成的情况,免疫荧光染色观察CNV中BMCs分化的细胞类型(包括血管内皮细胞、血管平滑肌细胞和巨噬细胞)及表达血管内皮生长因子(vascular endothelial growth factor, VEGF)和碱性成纤维细胞生长因子(basic fibroblast cell growth factor, bFGF)的情况;⑵激光光凝建立C57-GFP嵌合体小鼠CNV模型后,即日在其饮水中添加尼古丁(100μg/ml),对照组嵌合体小鼠建立CNV模型后普通饲养,4周后眼球切片HE染色观察CNV厚度与直径,脉络膜铺片观察CNV表面积、GFP-BMCs的趋化和参与构成血管情况,免疫荧光染色观察CNV中BMCs分化情况及各因子表达变化;⑶体外培养GFP转基因小鼠骨髓来源MSCs,野生型小鼠激光建模后0.5-1小时尾静脉注射4.0×106 GFP-BMCs或GFP-MSCs,激光后1、3、7天,心、肝、脾、肺、心脏切片荧光观察比较两种细胞在器官内的滞留情况;野生型小鼠激光后0.5-1小时、3天及6天,分别注射4.0×106 GFP-MSCs,激光后7天观察比较单次注射、双次注射和三次注射后GFP-MSCs在体内移行和向CNV趋化的情况;酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)和免疫荧光染色检测激光后不同时间眼内干细胞趋化因子基质细胞衍生因子-1(stromal cell derived factor-1, SDF-1)的表达;脉络膜铺片检测参与构成新血管的GFP-MSCs,免疫荧光染色分析CNV中GFP-MSCs分化的细胞类型;⑷构建表达人源性PEDF、含报告基因GFP、E1 E3缺陷的5型腺病毒(adenovirus, Ad),转导体外培养的野生型小鼠骨髓来源MSCs,倒置荧光显微镜观察及流式细胞仪测定感染效率,ELISA检测体外人源性PEDF表达情况;激光后0.5-1小时小鼠尾静脉分别注射4.0×106 Ad-PEDF/MSCs、对照病毒Ad-GFP/MSCs、GFP-MSCs及0.4ml磷酸盐缓冲液(Phosphate buffered saline, PBS),分别于激光后1、3、5、7天利用免疫荧光染色和ELISA检测眼内人源性PEDF表达,激光后7天眼球切片组织学分析和脉络膜铺片分析比较各组CNV厚度、直径及表面积。体外建立视网膜色素上皮(retinal pigment epithelium, RPE)细胞与MSCs的共培养体系,分析MSCs分泌的PEDF对RPE细胞增生和移行的影响。结果⑴激光后大量GFP-BMCs聚集于激光斑处并整合入CNV中的血管结构内,由其组成的血管面积约占CNV表面积的16.22%。GFP-BMCs主要分布于CNV区域(包括CNV深层的脉络膜),少量散布于CNV表层的视网膜、角巩膜缘、睫状体、视盘周围及远离CNV的视网膜、脉络膜和巩膜。CD31或αSMA阳性的GFP+细胞仅出现于CNV区域,F4/80+/GFP+细胞不仅出现在CNV区域,还出现在CNV表层的视网膜、角巩膜缘和睫状体内。CNV内CD31/GFP、αSMA/GFP和F4/80/GFP双阳性细胞在全部GFP+细胞中的构成比随CNV进展而变化,CD31/GFP或F4/80/GFP双阳性细胞构成比的最高值出现于激光后第2周,而αSMA+/GFP+细胞构成比在激光后4周仍处上升趋势。CNV区域的一些GFP-BMCs可表达促血管形成因子VEGF和bFGF。⑵尼古丁处理组小鼠CNV长度和表面积增大,CNV内GFP-BMCs的面积和密度增加,GFP-BMCs来源的血管细胞面积增加,F4/80+/GFP+细胞构成比下降,CNV内VEGF和bFGF的表达及CNV下脉络膜内VCAM-1的表达上调。⑶在整个观察期间,肺、肝和脾内可见GFP+BMCs滞留,但未观测到GFP+MSCs。单次、双次或三次MSCs注射后趋化至CNV的MSCs的数量没有明显差异,双次和三次注射组小鼠的脾内发现大量GFP-MSCs。SDF-1免疫荧光染色显示,激光后初期,SDF-1在激光斑处RPE层表达,随着CNV生成,CNV内出现阳性染色;ELISA结果显示,SDF-1表达水平在最初24小时内迅速增加,在第2天时达顶峰,然后迅速下降。MSCs移植后外周血和骨髓的流式分析显示,MSCs仅于第1天内在骨髓中作短暂停留。脉络膜铺片显示,激光后第1天,GFP-MSCs散布于激光斑周围;第3天时,GFP-MSCs组成的“细胞环”围绕激光斑,显示出向CNV靠近的定向移行趋势;第7天,GFP-MSCs进入CNV内并加入血管组成中。眼球切片显示绝大多数GFP-MSCs位于激光斑处脉络膜与光感受器间的CNV内。在这些MSCs中发现了血管内皮细胞、血管平滑肌细胞、巨噬细胞、上皮细胞和成纤维细胞的标记物(CD31,αSMA, F4/80, keratin,和vimentin)的阳性表达。⑷Ad转导小鼠MSCs后24小时,倒置荧光显微镜检测到报告基因GFP的表达,流式分析显示(73.6±5.3)%的细胞转导成功,AdPEDF转导的MSCs在体外表达PEDF可持续至少8天。AdPEDF组小鼠的眼球切片中,人PEDF的阳性染色出现于MSCs内及其周围的细胞外基质中;ELISA结果显示,在整个观察期间,眼内人PEDF呈稳定表达,表达量是抑制CNV阈值的4倍多;其CNV的厚度、长度和表面积明显减小。与AdPEDF/MSCs共培养的RPE细胞较之其他共培养条件下的RPE细胞增生和移行更快。结论⑴BMCs分化为多种细胞类型参与CNV细胞构成,分泌促血管形成因子促进CNV发展,在CNV生成过程中发挥着重要作用。CNV微环境趋化BMCs,并支持和调控BMCs的分化方向。BMCs与CNV微环境可能存在相互作用。⑵尼古丁促进BMCs向CNV趋化和参与CNV生成,并影响CNV内BMCs的分化。尼古丁对BMCs的这种作用可能部分通过调节局部因子(如VEGF、VCAM-1)表达实现。⑶MSCs仅出现于CNV而不在其他器官停留(骨髓中一过性停留),提示其在CNV模型中具有比BMCs更为卓越的特异趋化能力;一次激光光凝引起的MSCs趋化具有有限的时间窗,其原因可能是激光后CNV区域趋化因子的表达规律。激光后MSCs逐步趋化至CNV内,并分化为CNV生成所需的多种细胞类型。MSCs具备作为细胞载体的潜能。⑷经基因修饰的MSCs到达CNV部位并在局部表达抗新生血管形成因子,从而抑制了CNV的生长,该效应可能部分通过在CNV发展中发挥重要作用的RPE细胞介导。MSCs有望作为抗新生血管药物的释放系统用于CNV相关疾病的治疗。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R773.4

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