【作者】 刘艳丽；

【导师】 刘执玉； 田铧； 毕玉顺； 丁兆习；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 淋巴水肿按发病因素可分为:原发性和继发性二大类,继发性淋巴水肿是由丝虫感染、外伤、手术创伤、大剂量放疗等诸多后天因素导致的淋巴回流障碍性疾病,以组织间隙的高蛋白水肿为特征,至今仍是医学领域的一个难题。长期以来,国内、外在临床上治疗淋巴水肿的方法主要是绷带捆扎、按摩、针灸、理疗等物理疗法或手术等,但这些方法只能起到短期缓解作用,都不能从根本上治愈淋巴水肿。从根本上解决问题的方法只有促进淋巴管的新生,新生的淋巴管才能促进淋巴回流,减轻淋巴水肿。血管内皮生长因子(vascular endothelial growth factor,VEGF)家族是特异性作用于脉管内皮细胞的生长因子,其结构、功能相似,基因结构与血小板源性生长因子(PDGF)高度同源,其参于形成链间和链内二硫键的8个半胱氨酸是保守的。该家族包括VEGF-A(即传统意义上的VEGF)、VEGF-B、VEGF-C、VEGF-D、VEGF-E以及胎盘源性生长因子(PIGF)等,他们的生物学效应是由三个表达于特定细胞表面的受体来介导的:VEGFR-1(Flt-1),VEGFR-2(KDR或Flk-1)和VEGFR-3(Flt-4);传统理论认为VEGF-A与细胞表面受体VEGFR-1,VEGFR-2结合,特异性促进血管新生,VEGF-C在不同蛋白酶解下与细胞表面受体VEGFR-2或VEGFR-3结合,在不同条件下可促进淋巴管新生或血管新生。根据VEGF-C可以选择性诱导淋巴管增生这一功能,我们不难推测,利用VEGF-C基因治疗淋巴管生成缺陷性疾病以及淋巴管阻塞导致的淋巴回流障碍性疾病应该是可行的。目前关于利用VEGF-C选择性诱导淋巴管增生的功能探讨淋巴水肿治疗的研究已有相关报道:Marika J等探讨了一种人类原发性淋巴水肿的基因治疗模型:通过病毒介导VEGF-C基因的治疗发现VEGF-C可以在模型小鼠皮肤中产生有功能的淋巴管,提示生长因子的基因治疗对人类的淋巴水肿治疗是可行的。因此我们设计推断VEGF-C同样可应用于基因治疗淋巴管生成缺陷性疾病如淋巴水肿,而用于基因治疗研究的关键性物质基础则是VEGF-C基因的真核表达载体。因此,自1999年起,本课题组针对VEGF-C在肿瘤淋巴转移中的生物学功能及其应用方面开始了一系列研究,运用分子生物学技术构建了VEGF-C基因的真核表达载体并发表了相关的论文,为了获得VEGF-C对于淋巴管内皮细胞的调节作用和在淋巴管生成作用中的有关线索,探索应用VEGF-C基因治疗某些淋巴管形成缺陷性疾病和抑制肿瘤的淋巴管转移的新方法,本实验利用大鼠后肢环形切除部分皮肤并清扫深部淋巴管和淋巴结构建大鼠继发性淋巴水肿模型,并在水肿区局部皮下注射编码有VEGF-C基因的裸露质粒(pcDNA3.1/VEGF-C),通过MRI,超声、HE染色、免疫细胞化学等技术研究pcDNA3.1/VEGF-C是否可以促进淋巴管增生并且从解剖,功能和病理上改善淋巴水肿。根据VEGFs家族的特性,近两年来分别有波兰,日本等学者分别用VEGF-A,VEGF-C等VEGF家族成员从胚胎干细胞中诱导出脉管母细胞表型的细胞,随后这些细胞分化并表达淋巴管内皮细胞特异性标记物(Prox-1,LYVE-1),并在基质明胶或者细胞外基质上,可形成管状结构,为淋巴水肿的基因治疗开启了一条新的思路。骨髓基质干细胞是一类可进行自我复制和更新的细胞,在不同的理化环境和细胞因子的诱导下能够跨胚层向骨、软骨、脂肪、神经、肌肉等多种谱系分化。与胚胎干细胞相比,这种干细胞有许多优点:易于从自身取材,无免疫排斥反应之忧,涉及较少伦理问题;体外易于扩增、易分离、以及体外操作简便,因此,在组织器官缺损性疾病,组织器官退行性疾病,遗传缺陷性疾病等多方面有重要的应用前景。但是,到目前为止,骨髓基质干细胞在继发性淋巴水肿的治疗方面,甚至在干细胞向淋巴管内皮细胞诱导方面尚处于起始阶段,为了获取相关线索,探索应用VEGFs家族体外诱导骨髓基质干细胞向淋巴管内皮细胞分化的方法研究,我们利用VEGF-A,VEGF-C和EGF-A/VEGF-C联合用药在内皮细胞条件培养基中诱导骨髓基质干细胞,观察其向淋巴管内皮细胞分化的可能性,寻找VEGFs基因诱导骨髓基质干细胞向淋巴管内皮细胞分化的方法研究,为骨髓基质干细胞治疗继发性淋巴水肿提供新思路和实验依据。目的:1、本课题旨在探讨VEGF-C基因真核表达载体对大鼠继发性淋巴水肿模型的治疗作用及对淋巴管新生的作用,2、寻找VEGFs基因诱导骨髓基质干细胞向淋巴管内皮细胞分化的方法研究,为骨髓基质干细胞治疗继发性淋巴水肿提供新的思路和实验依据。研究方法:1、继发性淋巴水肿模型的制作,将VEGF-C真核表达载体pcDNA3.1-VEGF-C注射到模型大鼠手术处的皮下组织,用核磁共振,B超,光镜以及免疫荧光等方法检测,观察VEGF-C基因对继发性淋巴水肿的治疗作用,及对淋巴管新生的作用。2、常规分离,纯化,培养并鉴定大鼠骨髓基质干细胞:3、取三代骨髓基质干细胞分别在VEGF-A,VEGF-C及VEGF-A/VEGF-C及条件培养基中诱导分化,于第10天,用光镜,免疫荧光,RT-PCR及Western Blot做血管内皮细胞特异性标记物CD3,CD34和淋巴管内皮细胞特异性标记物LYVE-1、Prox-1四种标志物鉴定,观察干细胞向淋巴管内皮细胞的诱导情况。4、制备鼠尾胶原凝胶,将骨髓基质干细胞来源的淋巴管内皮细胞种植于预先制备好的胶原凝胶基质表面,观察干细胞来源的淋巴管内皮细胞形成管状结构的能力。结果:1、成功构建大鼠继发性淋巴水肿模型,将VEGF-C真核表达载体pcDNA3.1-VEGF-C注射到模型大鼠手术处的皮下组织,核磁共振,B超,光镜以及免疫荧光等方法检测,发现VEGF-C基因治疗组继发性淋巴水肿消退较对照组明显(P＜0.05),并在模型鼠忠处皮下发现大量新生淋巴管。2、贴壁培养法成功培养SD雄性大鼠rMSC,通过流式细胞仪检测CD29,CD90阳性,CD34,CD45阴性证实;骨髓基质干细胞在条件培养基诱导培养10天后免疫荧光,PT-PCR及Western Blot检测发现:与对照组相比,VEGF-A,VEGF-C及VEGF-A/VEGF-C组淋巴管内皮细胞标志物LYVE-1及Prox-1均为阳性,且血管内皮细胞标志物CD31,CD34表达为阴性。3、各组骨髓基质干细胞来源的淋巴管内皮细胞在三维培养中均无明显的管状结构形成。结论:1、VEGF-C基因对模型大鼠继发性淋巴水肿有一定的治疗效果,并能促进模型鼠患处皮下组织大量淋巴管新生。2、VEGF-A,VEGF-C及VEGF-A/VEGF-C在一定的条件培养基中均能诱导骨髓基质干细胞向淋巴管内皮细胞分化,且不表达血管内皮细胞特异性标志物:但目前三维培养并无明显的管状结构形成。创新性:1、采用VEGF-C真核表达载体pcDNA3.1-VEGF-C探讨VEGF-C基因对模型大鼠继发性淋巴水肿的治疗作用及对淋巴管新生的作用;并结合B超,MRI等先进方法结合血管内皮细胞特异性标志物CD34及淋巴管内皮细胞特异性标志物LYVE-1对淋巴水肿的治疗效果进行检测;2、首次采用VEGF-A,VEGF-C及VEGF-A/VEGF-C基因在体外成功诱导大鼠骨更多还原

【Abstract】 Lymphedema is a problem of tissue fluid imbalance often due to defects in lymphatic uptake and/or transport.Primary lymphedema results from developmental defects of the lymphatic system with fewer vessels leading to insufficiencies in transport,whereas the more common secondary lymphedema arises as a consequence of surgical,malignant,inflammatory,or traumatic disruption of the lymphatics. Although secondary lymphedema is a common clinical condition,treatment for this disabling condition remains limited and largely ineffective.The rebuild of circulatory structures is a good choice to treat lymphedema,but the way how to rebuild lymphatic vessels effectively is still under study.Recent molecular studies have begun to elucidate the basis for lymphangiogenesis which has been shown to be stimulated by various cytokines, including those in the vascular endothelial growth factor(VEGF) family:VEGF, VEGF-B,VEGF-C,VEGF-D,Orfvirus-encoded VEGF-E,and the placenta growth factor.These ligands bind to VEGF receptors(VEGFR)-1,VEGFR-2,and VEGFR-3 with partially overlapping receptor specificities.In traditionally,the prototype VEGF binds VEGF receptor(VEGFR)-1 and VEGFR-2 and is angiogenic,whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3,is either angiogenic or lymphangiogenic in different assays,and the affinity of VEGF-C toward their receptors is regulated by proteolytic processing.The affinity of themature,proteolytically processed form of VEGF-C is 40 times higher for VEGFR-3 than for VEGFR-2.Furthermore,in adults VEGFR-3 is largely absent from blood vessel endothelia and remains predominantly expressed in the lymphatic endothelium.Targeted gene inactivation and transgene approaches in mice have also revealed critical roles for VEGF-C in lymphatic vessel function Adenoviral expression of VEGF-C can induce lymphangiogenesis in the skin of normal mice.In order to resolve the questions about lymphangiogenic specificity of VEGF family in lymphedema,the scientists in Japan and Poland found that Embryonic Stem Cells treating with VEGF-A and VEGF-C individually promote lymphatic endothelial cell formation in vitro and enhance formation of lymphatic vessel structures OP9 culture condition or 3-dimensional collagen matrix.It is powerful new in vitro tool to dissect the molecular mechanisms that treat lymphedema and control lymphatic vessel formation.Bone marrow stromal cells(MSCs),also known as mesenchymal stem cells or colony-forming units(CFU) fibroblastic,are nonhematopoietic multipotent stem-like cells that adhere to culture dishes.They are capable of clonal expansion in culture, MSCs share characteristics with other multipotent stem cells such as neural stem cells, hematopoietic stem cells because they possess the ability to self-renew and can differentiate not only into osteoblasts,chondrocytes,neurons,and skeletal muscle cells but also into vascular endothelial cells and cardiomyocytes,it has been became a kinds of ideal cell in tissue and cell repair for its merits as followed:1.Bone marrow is an altenrative source of stem cells which are well suited for clinical application because they are easily obtained from patients and because autologous transplantation,which obviates immunologicin compatibilities is possible.2.Of the various p rogenitorc eUsth ate xistw ithinb onem arrow,m esenchymal stem cells(MSC) are particularly attractive for clinical use because they are easily isolated,can be expanded in culture,and can be genetically manipulated using currently available molecular techniques.3.MSCs are adult stem cells,has no ethics questions. Objectives:1.In order to resolve the questions about lymphangiogenic specificity of VEGF-C in lymphedema,We investigated the efficacy of local transfer of naked plasmid DNA encoding human VEGF-C(pcDNA3.1-VEGF-C) on a rat model of hindlimb lymphedema by following changes in lymphedema volume using water displacement volumetry,magnetic resonance imaging (MRI) and B ultrasound techniques.We additionally examined lymphangiogenesis using histological and immunohistochemical examinations.2.Although many researches have demonstrated that bone marrow-derived mesenchymal stern cells(MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes,chondroeytes,and adipocytes in vivo and in vitro,little information is available regarding its potential to differentiate into lymphatic endothelial cell.Thus,we investigated the differentiation of MSCs into cells of the lymphatic endothelial lineage,and investigated its bionomics by culture bone marrow stem cells on matrige with the presence of endothelial growth supplements,Methods:1、We produced a surgical model of secondary lymphedema in the rat hindlimb and treated with local intradermal VEGF-C(pcDNA3.1-VEGF-C) transfection to investigate the efficacy of gene transfer.Magnetic resonance imaging(MRI),B ultrasound,and water displacement volumetry demonstrated a change of lymphedema in therapy group as compared to controls.Histological and immunofluorescent studies demonstrated the numbers of newly formed lymphatic vessels in therapy group.Our results indicate that VEGF-C gene therapy has produced new lymphatic vessels.2、Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described,and flow cytometry to detect the surface markers: CD29,CD90,CD34 and CD45.3、Purified MSCs were then plated onto dishes at cell density of 1 to 1.5×10~4 cells/cm~2 in the presence of VEGF(20 ng/mL) and / or VEGF-C(50 ng/mL) cultured in differentiation medium for 4 or 7 days to media containing 2% FBS.Thenexamined CD34,CD31,Prox-1 and LYVE-1 by immunocytochemistry,RT-PCR,Western Blot..4、Prepares collagen gel on the ice box and then put into37℃incubator. Inoculate the cells with good growth condition on the surface of the collagen gel.Then observe the growth of cultured cells by inverted phase contrast microscope.Results:1、The rat model got typical secondary lymphedema after operation, and one week after therapy,magnetic resonance imaging(MRI)(P<0.05),B ultrasound(P<0.05),and water displacement volumetry(P<0.05) demonstrated a reduction of lymphedema in therapy group as compared to controls.Histological and immunofluorescent studies demonstrated numerous newly formed lymphatic vessels in therapy group.2、Rat MSCs were isolated from bone marrow aspirate of Sprague-Dawley rats as previously described,and examination demonstrated be positive for CD29 and CD90 surface markers and negative for CD34 and CD45 by f low cytometry.5、Purified MSCs were then plated onto dishes in the presence of VEGF-A and /or VEGF-C cultured in differentiation medium for 10 days to media containing 2%FBS.The immunocytochemistry,RT-PCR,Western Blot demonstrated that compared to control group,the groups of VEGF-A, VEGF-C and VEGF-A +VEGF-C express Prox-1 and LYVE-1 but not CD34 or CD31.6、Lymphatic endothelial cells induced from MSCs can not grow with tubiform structure in collagen gel.Conclusions:1、Our results indicate that VEGF-C gene therapy has produced new lymphatic vessels which may have improved functional lymphatic drainage to reduce lymphedema volume in our model.2、In differentiation medium,MSCs treating with VEGF-A,VEGF-C and VEGF-A + VEGF-C individually promote lymphatic endothelial cell formation in vitro,and express the marks of Prox-1 and LYVE-1,but cannot grow with tubiform structure in collagen gelBring New Ideas:1、We investigated the efficacy of local transfer of naked plasmid DNA encoding human VEGF-C(pcDNA3.1-VEGF-C) on a rat model of hindlimb lymphedema by following changes in lymphedema volume using water displacement volumetry,magnetic resonance imaging(MRI) and B ultrasound techniques.2、Induced marrow stromal stem cells to lymphatic endotheliai cell with with VEGF-A,VEGF-C and VEGF-A + VEGF-C individually and detected by Immunocytochemistry,PT-PCR,Western Blot et al.更多还原