【作者】 赵洪雯；

【导师】 吴雄飞；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 局灶节段性肾小球硬化（focal segmental glomerulosclerosis,FSGS）为不同病因和发病机理引起的一组临床病理综合征,其病理特征为局灶节段性肾小球固缩及疤痕化,主要临床表现为蛋白尿及肾功能损害。FSGS包括原发性和继发性,原发性FSGS病因尚不清楚,继发性FSGS的病因包括过敏性紫癜、系统性红斑狼疮,以及艾滋病、静脉药物滥用、肾小球肥大和肾单位减少等。FSGS的发病率高且有逐渐增加的趋势,治疗以免疫抑制和肾素血管紧张素系统（renin angiotensin system,RAS）阻断为主,缺乏对受损肾小球修复再生性治疗,常不能有效控制病情进展,肾功能进行性恶化,最终导致慢性肾衰竭（chronic renal failure,CRF）。因此如何从根本上延缓甚至逆转FSGS进展成为肾脏病治疗的热点与难点,探索新的治疗手段解决这一难题是当前国内外肾病界努力的方向。无论是病因不清楚的原发性FSGS还是继发性FSGS,其发病机制均尚不十分清楚。目前公认有以下三种可能:家族性基因遗传;体内存在特异性致FSGS的通透性因子;肾小球足细胞（podocyte）（上皮细胞）和其他固有细胞损伤包括内皮细胞（endothelial cell,EC）和系膜细胞（mesangial cell）。在三种可能机制中,足细胞和其他固有细胞的损伤在FSGS发病机制中的重要作用越来越受到重视,尤其是足细胞损伤及其与EC的相互作用。最近的研究认为,足细胞损伤是FSGS发病的始动因素,FSGS的本质是一种由不同病因引起的肾小球足细胞病,其证据主要包括:（1）肾小球足细胞的损伤和剥脱,使肾小球基底膜裸露,是FSGS的常见和早期病变,进而导致球囊粘连和硬化;（2）损伤和剥脱的肾小球足细胞可以转分化为巨噬细胞,并产生转化生长因子β（transforming growth factorβ,TGFβ）等多种促纤维化和硬化的细胞因子,促进并加速肾小球硬化、肾小管萎缩和间质纤维化;（3）足细胞损伤使其丧失其生理功能,如合成和分泌血管内皮生长因子（vascular endothelial growth factor,VEGF）明显减少。正常情况下VEGF在足细胞合成,以旁分泌的形式作用于临近EC表面的VEGF受体,是维持毛细血管袢完整和EC增殖的必要条件。VEGF的五种亚型中VEGF165的含量最多,作用最明显。足细胞损伤导致VEGF合成和分泌不足,肾小球毛细血管袢的正常结构和功能无法维持;同时由于VEGF在调整肾脏微血管变化中也起重要作用,VEGF不足也可导致肾间质和肾小管周微血管减少,形成肾脏局部缺血缺氧状态,引起并加速肾小管萎缩和肾间质的纤维化。局部的缺血缺氧状态以及肾小管萎缩和间质纤维化反之又引起和加重肾小球硬化,并形成恶性循环,最终导致CRF。由于肾小球足细胞损伤VEGF合成和分泌不足,可以导致肾小球其他固有细胞损伤如EC损伤的修复障碍,VEGF受体表达下调,使肾小球毛细血管袢结构维持更加困难,这种足细胞和EC损伤的相互作用形成瀑布效应,一旦启动,很难自行终止。所以,我们推测更新与修复足细胞、恢复VEGF合成与分泌、重建EC细胞的正常功能,可能是延缓或阻断FSGS进展的关键。国外研究显示,慢性肾功能不全时存在内皮祖细胞（endothelial progenitor cells,EPCs）功能不全,无法正常修复受损的肾小球EC和小管间质血管的EC,而肾动脉内给予体外培养的骨髓单个核细胞获得的EPCs,可以降低抗体诱导的EC、足细胞损伤并减轻系膜细胞活化。这些研究结果提示FSGS与EPCs、VEGF关系密切。EPCs最早由Asahara等于1997年发现并报道。它是一群具有游走特性,具有向受损伤EC“归巢”的特性,尚未表达成熟血管内皮细胞表型,体外培养后能增殖并分化为血管内皮细胞的前体细胞。它不仅参与人胚胎血管生成,而且在骨髓、脐带血及外周血中均存在,在出生后的血管新生过程中也具很强的促血管生成作用,并以血管发生方式形成新生血管的EPCs。这一发现,更新了传统意义上的出生后血管生成、血管损伤修复的理论,为缺血性疾病的治疗提供了新的思路。有关EPCs在成体血管形成中作用的研究主要集中在缺血心肌、缺血肢体、损伤角膜、损伤皮肤的修复等方面。由此我们设想,EPCs和VEGF可能对FSGS时足细胞和EC及其他肾小球固有细胞损伤有修复和更新作用,进而可能成为延缓FSGS进展的有效手段。随着日益成熟的分离培养、诱导和扩增技术,EPCs可以相对容易地从外周血或骨髓中获得,结合近年基因工程技术的迅速发展及在临床不同领域的应用,为证实我们这一设想提供了可实施的技术手段。本研究拟采用文献报道的改良阿霉素肾病诱导的FSGS模型,即单侧肾切除后分次注射阿霉素,如果不给予其他干预措施,动物将在肾切除和注射阿霉素后12-16周发展为FSGS。在本实验中阿霉素第二次注射后1周,即FSGS的进展过程中,将体外培养的EPCs移植,依赖EPCs的“归巢”特性使EPCs向受损的大鼠肾脏归巢,观察其对FSGS的进展是否有延缓作用。由于VEGF的半衰期仅30-40分钟,直接补充VEGF的作用时间非常短暂,而且全身使用有诸多问题包括增加肿瘤风险、浓度不能维持以及价格昂贵等,因而将VEGF165重组腺病毒在体外构建并转染培养获得的EPCs再移植给阿霉素诱导的进展期FSGS大鼠,EPCs在体内修复足细胞等细胞损伤的同时,持续合成分泌VEGF165,观察转染VEGF165的EPCs对FSGS进展是否比单用EPCs有更好的延缓作用,探讨EPCs移植和VEGF165转染EPCs移植在FSGS进展中的作用和可能机制,为临床采用EPCs移植和VEGF转染EPCs移植治疗FSGS的设想提供实验依据。主要实验方法及结果:1.大鼠骨髓来源的内皮祖细胞的分离、体外诱导培养及鉴定采用密度梯度离心法从雄性SD大鼠骨髓中得到单个核细胞,在特定的条件下进行分离纯化,通过贴壁法培养、体外扩增获取贴壁细胞,采用流式细胞表型检测、免疫细胞化学及免疫双荧光等方法进行鉴定。结果证实,通过此方法获得的细胞即是EPCs,为EPCs移植作好了准备。采用雄性大鼠EPCs的目的是将雄性大鼠特有的Y染色体作为外源性EPCs的标记。2.腺病毒介导的VEGF165基因转染内皮祖细胞的实验研究用限制性内切酶XbaⅠ+HindⅢ从质粒载体中切出VEGF165基因片段,与KpnⅠ+HindⅢ双酶切的pAdTrack-CMV形成转移质粒pAdTrack-CMV-VEGF165,PmeⅠ酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183,并在293细胞中包装成Ad-VEGF165,PCR和Western Blot法鉴定目的蛋白表达。贴壁法体外培养获得大鼠骨髓来源的EPCs（同前）,Ad-VEGF165体外转染EPCs,检测转染后目的基因蛋白表达情况。结果显示:利用CsCl₂法由pAdTrack-VEGF165和pAdEasy-1共转化BJ5183感受态菌,可获得阳性重组体细菌克隆。PCR和Western Blot以及基因测序分析进行鉴定,表明重组腺病毒已含有目的基因,并能够高效表达目的蛋白,序列正确;病毒滴度为1.4×10¹⁰pfu/ml。Ad-VEGF165基因转染培养第6d的EPCs后24h开始培养上清中VEGF蛋白表达显著增加。证实体外构建VEGF165重组腺病毒成功并且可以成功转染EPCs,EPCs合成和分泌VEGF显著增加,为VEGF165转染EPCs移植到动物的在体实验提供了基础。3.内皮祖细胞移植或VEGF165基因转染内皮祖细胞移植对进展性FSGS的进展的影响雌性SD大鼠随机分为正常对照组（对照组）、阿霉素肾病组（肾病组）、EPC移植组（EPC组）和VEGF转染EPCs后移植组（VEGF组）。肾病组、EPC组和VEGF组行单肾切除,术后1w和2w分别尾静脉注射阿霉素制作阿霉素诱导的FSGS,对照组假手术并在相同时间点注射生理盐水。于第二次注射阿霉素后1w,5Gy的X线全身照射后,EPC组尾静脉注射移植1×10⁶的EPCs,VEGF组尾静脉注射移植1×10⁶转染了VEGF165重组腺病毒的EPCs,对照组和肾病组仅行同等剂量的X线照射和生理盐水注射。在切肾前（0w）和切肾后的第4（即EPCs移植后1w）、8、12、16w测大鼠体重、尿蛋白,处死大鼠取血测血清肌酐（SCr）、白蛋白（ALB）,原位杂交法观察肾组织中Y染色体阳性细胞掺入情况,以证实雄性供鼠来源的EPCs到达并整合入雌性受鼠的肾组织中;观察肾脏超微结构和组织学变化,图像分析计算肾小球硬化指数、肾小球毛细血管袢管腔开放程度及毛细血管袢截面积,同时免疫组化和Western Blot检测肾组织中VEGF的表达,并检测与细胞外基质（Extracellular matrix,ECM）代谢关系非常密切的转化生长因子β1（transforming growth factorβ1,TGFβ1）和多种肾脏固有细胞向肌成纤维细胞表型转化的标志性蛋白α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）在肾组织中的表达,作为各组肾组织慢性化转化程度的指标。结果显示:（1）原位杂交的结果证实雄性大鼠来源的EPCs到达并整合到雌性阿霉素肾病大鼠的肾脏组织中,而且这种整合从移植后1w直到实验终点,但有逐渐减弱趋势。整合的部位位于肾小球内和肾小管上皮细胞。（2）肾病组大鼠的一般情况最差,次第为EPC组、VEGF组,对照组一般情况较好,一般情况包括大鼠精神、进食和体重。（3）实验室检查:EPC组和VEGF组从第4w开始尿蛋白即显著低于肾病组,在第8w和第12w VEGF组尿蛋白显著低于EPC组（p＜0.05）;SCr测定显示,EPC组从第8w开始SCr显著低于肾病组,VEGF组SCr从第4w开始显著低于肾病组,至第16w时有所增高,但显著低于肾病组和EPC组;ALB结果显示,EPC组的ALB从第4w开始下降,各时间点均显著低于对照组,但第12、16w时显著高于肾病组;VEGF组的结果与EPC组的结果相似,但在12w和16w,VEGF组ALB显著高于EPC组（p＜0.05）。这些结果提示EPCs移植参与了EPC组和VEGF组尿蛋白下降、肾功能异常出现时间的延后以及血清ALB提高。（4）肾脏组织学和图像分析的结果显示,第16w时EPC组和VEGF组肾小球毛细血管管腔开放程度显著高于肾病组,且VEGF组显著高于EPC组,VEGF组的毛细血管腔开放最好;毛细血管袢截面积的结果与肾小球毛细血管腔开放程度的结果相似,系膜基质相对含量也显示EPC组和VEGF组较肾病组显著降低,VEGF组又显著低于EPC组;肾病组肾小球硬化指数显著高于EPC组和VEGF组,而VEGF组的肾小球硬化指数最低。（5）透射电镜观察肾组织超微结构的结果显示:EPC组和VEGF组肾小球足细胞损伤修复和肾小管上皮细胞损伤的减轻均早于肾病组,而VEGF组修复出现的时间更早。（6）免疫组化染色和Western Blot分析显示:EPC组和VEGF组的肾组织VEGF表达较对照组和肾病组明显增强,直到实验终点;VEGF组肾组织的VEGF表达较EPC组更强,VEGF蛋白表达也证实此结果;肾病组、EPC组和VEGF组肾组织TGF-β1、α-SMA的表达均随实验时间的延长逐渐增强,但EPC组和VEGF组较同时间点的肾病组明显减弱,而VEGF组的表达最弱;其蛋白表达也证实此结果。结论1.采用密度梯度离心法收集骨髓中的单个核细胞,在特定的诱导条件下通过贴壁培养可以获取足够数量的EPCs,在短时间内可在体外扩增,并最终在体外分化为有功能的具有内皮细胞特异性标志物的成熟血管内皮细胞。2.采用DNA重组技术成功构建了Ad-VEGF165重组腺病毒,且Ad-VEGF165能高效、稳定地表达VEGF165目的基因且具有良好的安全性;构建的Ad-VEGF165能够顺利转染体外培养的骨髓来源的EPCs,可以高效表达VEGF的目的蛋白。3.来源于异体骨髓的EPCs和体外构建的VEGF165基因重组腺病毒转染的EPCs,在阿霉素诱导的FSGS模型进展过程中经尾静脉移植后,EPCs掺入并整合入肾小球和肾小管上皮细胞,参与肾小球和肾小管的修复与细胞更新,并能够延缓FSGS的慢性化进展,为临床采用EPCs移植治疗FSGS的设想提供了理论依据。更多还原

【Abstract】 Focal segmental glomerulosclerosis （FSGS） is a group of clinical pathologic syndrome caused by various pathogenesis and mechanisms, with a pathological feature of focal and segmental glomerular solidification and scarring, and the clinical feature of nephrotic or non-nephrotic proteinuria. FSGS can be of primary renal diseases, or correlate with some other secondum diseases such as systemic lupus erythematosus, anaphylactoid purpura, acquired immunodeficiency syndrome, veinous drug abuse, glomerular hypertrophy, nephron reduction, etc.FSGS has high morbility and an increasing tendency. It is mainly treated by immune suppression and renin angiotensin system （RAS） blockage but without reparative regeneration of damaged glomerular, which usually causes progressive aggravation of renal function and chronic renal failure （CRF）. Therefore, the exploration of new methods of delayed or ameliorated FSGS becomes the urgent demand and the focus of attention of the nephropathic field in the world.Studies in recent years consider that FSGS is a kind of podocyte diseases caused by some etiological factors that podocyte damage is the most important factor in the development of FSGS. （1） When podocytes are damaged and denudated, glomerular basement membrane will be exposed and this is commonly the earlier period of FSGS, which will cause glomerular capsular adhesion and sclerosis. （2） Damaged and exfoliated podocyte will transdifferentiate to macrophage, which will produce some fibrotic and sclerotic cytokine. （3） Damaged podocytes lose their physiologic functions, such as synthesis and secretion of vascular endothelial growth factor （VEGF）. VEGF is synthesized in podocytes and will paracrine to VEGF receptors on jacent ECs which is the essential condition of EC proliferation and the glomerular capillary loop maintenance. VEGF also plays an important role in modulating capillary vessels in interstitium and peri-tubules. VEGF insufficiency will fail to keep the glomerular capillary loops normal and cause renal ischemia and hypoxia, which results in glomerular sclerosis and tubuar-linterstitial fibrosis. Together with podocytes damage, some other glomerular inherent cell damages occur, such as ECs. ECs damage will cause down regulation of VEGF receptors which forms the vicious cycle. At last the damages lead to CRF. In the vicious cycle, the podocytes recruitment, the VEGF reablement and the ECs recruitment may be the key point of amelioration of FSGS.Some studies showed that chronic renal insufficiency had endothelial progenitor cells （EPCs） dysfunction and intrarenal injection of bone marrow-derived EPCs could reduce podocytes and endothelial cell （EC） injury, so as mesangial cell activation in experimental glomerulonephritis. Therefore, it is supposed that EPCs and VEGF may play an important role in the podocyte and EC recruitment, which might be an effective method in ameliorating FSGS.Endothelial progenitor cells （EPCs）, firstly reported by Asahara in 1997, are a group of precursor cells of ECs, which have not expressed mature EC phenotypes. EPCs could proliferate and differentiate into ECs. They take part in the vasculogenesis of human fetal vessels and exist in bone marrow, cord blood and peripheral blood with strong ability of promoting the angiogenesis after born. This conclusion updates the theory of traditional postnatal angiogenesis and vascular injury and repair, and offers some new thoughts for treatment of ischemic diseases. Recently, the studies on functions of EPCs on adult angiogenesis mainly focus on repairing of ischemic myocardium, ischemic limbs, injured corneal and skin, etc. With the development of isolation, cultivation, induction and amplification techniques, it is easy to obtain EPCs from peripheral blood or bone marrow. Thus in this experiment, EPCs derived from bone marrow were transplanted to progressive FSGS rats, or the recombinant adenovirus of VEGF165 were constructed in vitro and transfered to EPCs, then the gene tranfered EPCs were transplanted to progressive FSGS rats induced with Adriamycin injection. The progression of the FSGS were observed.Main Methods and Results1. Isolation, cultivation and identification of endothelial progenitor cells from rat bone marrowMononuclear cells were separated and purified from the male rats’ bone marrow by density gradient centrifugation. After short-time cultivation and amplification under special inductive conditions, enough quantity of EPCs could be obtained in vitro, and finally, EPCs could differentiate into functional mature vascular endothelial cells with endothelial cell specific markers, which offered possibility for clinical applications.2. Construction of vascular endothelial growth factor 165 gene recombinant adenovirus and transfection of endothelial progenitor cellsVEGF165 gene was liberated from the vector of pcDNA3.1/VEGF165 via Xba I and Hind III digestion, and subcloned into shuttle vector of pAdTrack-CMV plasmid digested by Kpn I +HindIII, forming transfer vector of pAdTrack-CMV-VEGF165. Then pAdTrack-CMV-VEGF165 was linearized with Pme I and cotransformed into BJ5183 cells with adenovirus genomic plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. The polymerase chain reaction （PCR）, sequencing and Western Blot analysis were used to detect target gene DNA and protein. The titre of Ad-VEGF165 was measured with the aid of green fluorescent protein （GFP） expression. EPCs derived from rat bone marrow were obtained by adherent culture for 6 days. After transfected by Ad-VEGF165 gene for 24h, the expression of VEGF protein was assessed using Western blot. Results: Positive recombinant bacterial clones were obtained after cotransformation of BJ5183 bacterial cells with pAdTrack-CMV-VEGF165 and pAdEasy-1 by method of CsCl₂. PCR test, Western Blot and sequencing analysis indicated that the recombinant adenovirus contained the inserted VEGF165. The titre of purified recombinant adenovirus was 1.4×10¹⁰ pfu/ml. After transfection, the expression of VEGF protein was significantly higher than that of untransfected EPCs group.3. Endothelial progenitor cells transplantation or VEGF 165 gene transfected endothelial progenitor cells transplantation ameliorated the progressive of focal segmental glomerular sclerosisFemale SD rats were divided randomly into normal control group （control group）, adriamycin renal disease group （ADR group）, EPC transplantation group （EPC group） and VEGF165 gene transfected EPCs transplantation group （VEGF group）. ADR group, EPC group and VEGF group were carried out unilateral nephrectomy and injected with 5mg/kg and 3mg/kg of adriamycin via tail vein lweek（w） and 2w after operation, while the control group rats were carried out sham operation and then were injected with 0.9% Sodium Chloride solution of equal volume. After 5Gy X ray whole body irradiation were done 1w after the 2nd injection of Adriamycin, EPC group rats were transplanted with 1×10⁶ EPCs, and the VEGF group rats were transplanted with 1×10⁶ VEGF165 gene transfected EPCs via tail vein immediately, while the rats in control group and ADR group were only injected with 0.9% Sodium Chloride solution after whole body irradiation. The body weight and urine protein were measured before operation （0w） and 4w （1w after EPCs transplantation）, 8w, 12w and 16w after nephrectomy. Y chromatosome incorporation was detected with in situ hybridization. The renal histological and ultrastructural changes were evaluated. VEGF, transforming growth factorβ1 （TGFβ1）,α-smooth muscle actin （α-SMA） immunohistochemistry and Western Blot were detected. Results: （1） In situ hybridization showed that Y chromatosome positive cells incorporated in glomcrulus cells and tubular epithelial cells since 4^th week, and the incorporated positive cells could be found until the end of the experiment in EPC group and VEGF group. （2） Since the 4^th week, the body weight in ADR group, EPC group and VEGF group became lower than that in control group significantly; The rat weight in ADR group became lower than that in EPC group since 8w （p<0.05）, but the VEGF group rats had the heveaiest body weight among the three groups. The proteinuria and SCr reached peak and were more severe in ADR group than those in EPC group, VEGF group and control group since the 4^th week, then decreased gradually, but from the 8^th week, the proteinuria and SCr were lower in VEGF group than that in EPC group. （3） Histological results showed that in ADR group, mesangial proliferation and the tubular-interstitial fibrosis were more remarkable than that in EPC group, VEGF group and control group. The image analysis showed that the index of glomerular sclerosis in ADR group was significantly higher than that in EPC group and VEGF group, the opening degree of glomerular capillary cavity in EPC group and VEGF group was significantly higher than that in ADR group. The situation was the same in capillary sectional area, but it was better in VEGF group than that in EPC group. The observation by electron microscope showed that the podocytes and tubular epithelial cells leisions in ADR group were more severe than that in EPC group and VEGF group since the 8^th week, and in the latter two groups, there was early reparation of podocytes and tubular epithelial cells. （4） Immunohistochemistry and Western Blot analysis showed that there were more VEGF but less TGF-β1 andα-SMA expression in VEGF group and EPC group than that in ADR group, meanwhile, there were the most expression of VEGF and lowest TGF-β1 andα-SMA expression in VEGF group.Conclusion1. After short-time cultivation and amplification under special inductive conditions, enough quantity of EPCs can be obtained in vitro, and will finally differentiate into functional mature vascular endothelial cells with endothelial cell specific markers. No immunologic rejection arises after autogenous implantation, which offers possibility for clinical applications.2. The method of homologous recombination in bacteria is a convenient and efficient method to conduct recombinant adenovirus and the conducted Ad-VEGF165 can effectively mediate target gene expression in cultured EPCs, which builds a sound foundation for further study.3. EPCs transplantation can ameliorate renal damages and may delay the progression of glomerular sclerosis and tubular-interstial fibrosis induced by adriamycin. VEGF165 gene transfected EPCs transplantation has a stronger effect of delaying the progression of glomerular sclerosis and tubular-interstial fibrosis induced by adriamycin.更多还原