【作者】 袁莉；

【导师】 梅长林； 刘厚奇； 叶朝阳； 赵学智； 王雪琦；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 研究背景和目的急性肾损伤(AKI)是临床的常见疾病,重症死亡率达50%-80%。虽然在急性肾衰竭的发病机制和治疗上进行了诸多研究,但是到目前为止治疗的手段仍然有限,唯一可选择的治疗方法为透析治疗和在等待肾功能恢复过程中的支持治疗,寻找更有效地治疗AKI的方法一直是努力的方向。干细胞的定义为具有自我更新和多向分化潜能的克隆细胞,干细胞在治疗急性肾损伤方面有广阔的前景,已有多项研究证实干细胞对于急性肾损伤有治疗作用。MSC是目前最常用于研究发育以及疾病治疗的干细胞,其中又以成体骨髓来源MSC最为多见,随着研究的深入,MSC的来源日益增多,比如成体或者胚胎,胎儿组织,脐血等。由于干细胞的来源不同,各自分化,增生能力亦有差别,到目前为止尚不知道何种干细胞作为治疗疾病或新药研究是最佳的。成体骨髓MSC来源广泛,但是使用这一细胞也有其局限性,MSC在成人骨髓内含量极低,约占有核细胞数的0.001-0.0001%,并且骨髓MSC的数量和分化能力随着所以年龄的增长而减少和减弱。与成体MSC相比,胚胎组织来源的MSC增殖和分化能力强,免疫原性弱,所以使用胚胎来源干细胞作为干细胞的研究有其独特的优越性。目前从人早期胚胎中分离MSC的研究国内外研究较少。关于干细胞修复急性肾损伤的机制以往认为是通过直接转化为小管上皮细胞,目前认为小管再生主要通过残存细胞的增生和转分化,仅有很少的外源干细胞转化为肾小管上皮细胞。MSC的肾保护作用主要通过旁分泌作用,分泌细胞因子抗调亡,促分裂,免疫调节和促血管生成。通过对干细胞的旁／自分泌作用的深入了解,将以干细胞分泌功能作为调节干细胞保护的新靶点提供依据。VEGF是血管内皮细胞特异性有丝分裂原,具有促进血管内皮细胞分裂增殖、促进新生血管形成和侧支循环开放等作用。VEGF对于维持肾小球功能起关键作用,可以刺激间质血管生成,小管增值并且直接保护肾脏上皮细胞。最近有研究MSC经过RNA干扰后VEGF表达下降,急性缺血再灌注小鼠给予干扰后MSC后,与给予干扰MSC的小鼠比,肾脏修复明显减低,生存率下降,这说明,VEGF是MSC肾脏保护作用中重要的因子。因此我们准备以调节干细胞分泌VEGF为切入点,提高干细胞对AKI的治疗效果。本实验采用体外顺铂损伤肾小管上皮细胞模型和体外顺铂诱导的裸鼠急性肾损伤模型观察VEGF基因转染对于hMSC修复急性肾损伤的作用,并对其机制进行探讨。研究方法、内容及结果一、VEGF基因转染人早胚间充质干细胞选择携带VEGF165基因的重组腺病毒(Ad.VEGF165病毒颗粒由张裕东博士馈赠)转染hMSC,并使用携带GFP基因的重组腺病毒转染hMSC确定最佳的转染滴度。实验结果显示：腺病毒介导的GFP基因对于hMSC具有较高的转染效率,转染效率与病毒滴度(MOI)具有量效关系。病毒MOI为50时,转染效率>70%,转染后hMSC的生长曲线与未转染细胞相似。转染VEGF后,hMSC可有效表达VEGF,4-6天时达峰,14天左右恢复正常。腺病毒介导的VEGF基因可以安全、有效的转染hMSC,其携带的VEGF基因可获得较高的表达水平。二、VEGF基因转染的hMSC对顺铂诱导的肾小管上皮细胞损伤的修复作用使用10 uM顺铂预处理TCMK-1后24小时,TCMK-1分为正常组、hMSC+顺铂+TCMK-1共培养组、VEGF转染hMSC+顺铂+TCMK-1共培养组、空载体转染hMSC+顺铂+TCMK-1共培养组和顺铂+TCMK-1组。结果显示：与正常组相比,TCMK-1经过顺铂预处理后3天增值明显减慢,凋亡增加,PCNA表达增强(p<0.01)。与未共培养组相比,顺铂预处理后TCMK-1与各干细胞共培养均能缓解顺铂对TCMK-1的生长抑制,减少凋亡,促进PCNA的表达(p<0.01)；尤其以VEGF转染hMSC共培养组最明显(p< 0.01 versus cisplatin+TCMK-1+AD-hMSC, cisplatin+TCMK-1+hMSC)。本部分实验证实：VEGF转染hMSC和以提高hMSC对顺铂损伤TCMK-1的修复作用,机制可能与增加hMSC促增殖和抗调亡有关。三、VEGF基因转染的hMSC对顺铂诱导的小鼠AKI的修复作用选取6-8周龄的BALB/C雄性裸鼠,按18mg/kg经小鼠腹腔注射顺铂,建立药物性急性肾损伤小鼠模型。动物分组：A组(正常对照组)；B组(生理盐水治疗组)：注射生理盐水；C组(VEGF-hMSC组)：注射体外转染Ad.CMV-VEGF165的hMSC;D组(AD-hMSC):注射体外转染Ad.CMV-con的hMSC;E组(hMSC组)：注射hMSC。药物注射后第4天,小鼠肾脏病理解剖结果肾小管上皮细胞呈空泡变性,部分细胞坏死脱落入管腔,基底膜暴露；小鼠血中BUN, SCr升高。C组、D组和E组HE染色坏死小管计数,管型计数以及血清BUN, SCr均低于B组,以C组的检测指标降低最明显。与正常肾脏相比急性肾损伤后各组中肾脏PANC和TUNEL染色计数增加,C组、D组和E组PCNA计数均高于B组而TUNEL计数低于B组,其中C组的PCNA计数最高,TUNEL计数最低。与正常肾脏相比急性肾损伤后各组中肾脏肾小管周围CD34染色血管面积减少,C组、D组和E组CD34染色血管面积均高于B组,其中C组染色面积最高。PKH-26标记的hMSC大多定位于肺、肝、脾,肾脏仅看到很少量的干细胞,并且在VEGF转染和非转染组没有区别。在本部分实验中,我们成功的建立了顺铂诱导的急性肾损伤裸鼠模型,VEGF基因转染可以提高hMSC对急性肾损伤的修复作用,具体机制与VEGF基因转染可以提高干细胞的促增值,抗凋亡和保护血管微环境的作用有关。结论综合三部分结果,我们可以得出结论,使用VEGF腺病毒基因转染可以有效上调hMSC的VEGF水平。上调hMSC的VEGF水平可以增加hMSC的对受损肾小管上皮的保护作用,其机制与增加干细胞的促增值,抗凋亡和保护血管内皮功能有关。本研究为临床使用新的干细胞来源提供了基础,验证了关于提高干细胞治疗急性肾损伤作用有效的干预靶点。更多还原

【Abstract】 Acute kidney injury (AKI) is a common clinical disease, whose mortality rate is 50-80%. Despite advancements in the research of pathogenesis of AKI, no effective treatment is available at present. Supportive therapy remains to be the major option for the treatment of AKI patients who are waiting for recovery of renal function. This calls for the development of new and more effective treatments for AKI.Stem cell is defined as a cell that has the potential of self-renewal and multilineage differentiation, and has broad prospects in the treatment of acute renal failure. Many studies have confirmed that stem cell is effective for the treatment of acute renal failure.Mesenchymal stem cell (MSC) is now commonly used in research. Adult bone marrow-derived MSC is most prevalent, and with the in-depth study, more sources of MSC are available from adult or embryonic and fetal tissue and cord blood. But as differentiation and proliferation ability of stem cells from different sources are different, little is known about what kind of stem cell is best for disease treatment or new drug research. Although adult bone marrow MSC has a wide variety of sources, its use is limited, partly because the amount of MSC in adult bone marrow is very low, accounting for 0.001-0.0001% of the total number of nucleated cells. With the growth of donor age, the number of bone marrow MSC reduce and the differentiation capacity of MSC is iminished. Compared with adult-derived MSC, embryo-derived MSC have greater expansion and differentiation potentials. Immunologically, histocompatibility antigens are less intensively expressed in embryo-derived cells of early gestation terms than in adult cells.There are still controversies over the mechanism of how stem cells mediate the therapeutic actions. Although some early studies believed that this protective effect is attributed to the replacement of damaged cells by differentiated stem cells, recent studies suggest that only a small percentage of repaired tubular cells are stem cell-derived [1]. A more recent opinion is that stem cells exert their therapeutic effect in AKI by some paracrine/endocrine mechanism. Regulating the paracrine function would be a new method to enhance the therapeutic efficacy of MSC.Vascular endothelial growth factor (VEGF) can induce proliferation and anti-apoptotic response in renal tubular epithelial cells, and exert proangiogenic effects in AKI as well. Knockdown of VEGF by siRNA reduced the effectiveness of MSC in the treatment of ischemic AKI in a rat model, indicating that VEGF secreted by MSC plays an important renoprotective role in AKI when stem cell treatment is used.We therefore hypothesized that up-regulation of VEGF could strengthen the renal protective effect of MSC. The aim of the present study was to see whether the protective effect of stem cells could be enhanced by increasing VEGF secretion in a cisplatin-induced tubular cell damage model in vitro and a nude-mouse model of cisplatin-induced AKI in vivo.This study consists of 3 sections:Section 1:Adenoviral vector tansfection of hMSCAd.CMV-VEGF165 (provided by Dr. YuDong Zhang from the department of cardiothoracic surgery of the affiliated hospital of Nantong University, Nantong, JS, China) was used to infect hMSC. Ad.CMV-GFP was used to define the optimal transfection MOI. The result showed that the cell grown rate of hMSC infected with Ad-EGFP at MOI 50 was similar to that of regular hMSC without infection, and that hMSC remained to be a homogenous population of fibroblast-shaped cells. Therefore, the adenoviral vector was used to infect hMSC at MOI 50 in the subsequent experiments due to high efficiency and low toxicity. It was found that the peak level appeared at day 4-6 after transduction. At day 4 of transduction, VEGF level in the medium of VEGF-hMSC increased significantly, about 4 times that of uninfected hMSC. Adenovirus-mediated VEGF gene transfer was safe and effective, and transfected hMSC exhibited a high level of VEGF expression of.Section 2:Protection of VEGF-hMSC on cisplatin-impaired TCMK-1 in a co-culture systemTCMK-1 were pretreated with cisplatin for 24 h and cocultured with hMSC, VEGF-hMSC and AD-hMSC. Three days after cisplatin pretreatment, TCMK-1 slowed decreased regeneration, increased apoptosis and PCNA expression as compared with normal TCMK-1 (p< 0.01).Compared with TCMK-1 pretreatment with cisplatin alone, co-culturing with every kind of hMSC ceased the inhibitory effect of cisplatin on TCMK-1 growth, reduced apoptosis, and increased PCNA expression. These effects were most pronounced in VEGF-transfected hMSC. VEGF transfecton improved the ability of hMSC to impair cisplatin induce TCMK-1 injury. The repair mechanism may be related to promoting the proliferation and anti-apoptosis of hMSC. Section 3:Protection of VEGF-hMSC on cisplatin-induced renal injuryAKI was induced by subcutaneous injection of cisplatin (18 mg/kg).24 h after cisplatin administration, nude mice were divided into five groups:group A, normal group; group B, saline group; group C, Ad.CMV-VEGF165 transfected hMSC (VEGF-hMSC) group; group D, Ad.CMV-con transfected hMSC (AD-hMSC) group; and group E, hMSC group.Renal function and the tubular structure were impaired in group B 4 days after cisplatin administration.. The HE staining tubular necrosis count, tube count and the level of serum BUN were lower in VEGF-hMSC, AD-hMSC and hMSC groups than those in saline group.The PANC and TUNEL staining was increased in the kidney of all groups after AKI compared with that in normal kidney. PCNA count was higher in VEGF-hMSC, AD-hMSC and hMSC groups than that in saline group, while TUNEL count was lower. PCNA count in VEGF-hMSC group was the highest and TUNEL count was the lowest of the three groups. All hMSC treatment groups had a higher area percentage of peritubular capillary than the saline group (p<0.05). VEGF-hMSC therapy significantly restored the area percentage of peritubular capillary (p<0.01, versus AD-hMSC and hMSC).This study demonstrated that hMSC transfection with VEGF gene improved their therapeutic benefits in healing cisplatin-induced renal epithelial injury. VEGF-modified hMSC enhanced the renoprotective activity in the AKI nude-mice model compared to regular hMSC and vacant advirus transfected hMSC.In this part of the experiment, we successfully established a cisplatin-induced acute renal injury model in nude mice. VEGF gene transfer increased the ability of hMSC in repairing AKI. The specific mechanism may be related to the fact that VEGF up-regulation can improve stem cells to promote mitosis, anti-apoptosis and protection of the vascular micro-environment effects.This study indicates that implantation of VEGF-modified hMSC could provide advanced benefits in protection against renal tubular injury by anti apoptosis, improving microcirculation and proliferation in vitro and in vivo, thus providing a new source of stem cells for clinical use and a new method of improving the therapeutic effect of stem cells in AKI.更多还原