【作者】 刘容；

【导师】 周建英；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 引言:肺癌在近三十年来国内肿瘤死亡构成位居首位,且死亡率增幅高达167%,一直是严重威胁人类生命的疾病,和临床治疗的难点。肿瘤组织的血管新生是肿瘤组织增殖和转移的重要条件之一,抗血管新生已经成为实体肿瘤(如NSCLC)治疗的一个重要途径,血管内皮生长因子(VEGF)是主要的血管新生因子,参与肿瘤血管新生的全过程,成为抗血管新生的重要靶标。小干扰RNA(siRNA)在哺乳动物细胞中能降解同源序列的靶基因的mRNA,RNA干扰(RNAi)技术以其高效性,特异性以及多功能性的优点成为基因研究的强有力工具。近年来在动物体内进行RNA干扰逐渐成为研究的热点,动物实验中选择合适的基因转染方式是实验技术的重要环节。非病毒载体具有低毒、低免疫反应、外源基因整合几率低、无基因插入片断大小限制,以及使用简单、制备方便、便于保存和检验等优势,因此非病毒性载体的研究日益受到重视。本实验将采用质粒作为载体,介导针对人VEGF的siRNA,转染人肿腺癌A549细胞,并采用不同的基因转染方式用于动物实验中,比较不同基因转染方式在A549细胞RNA干扰实验中的效率,观察VEGFsiRNA对动物移植肿瘤生长的影响,并观察VEGFsiRNA对A549细胞诱导的血管新生的影响。第一部分VEGFsiRNA抑制A549细胞裸鼠移植肿瘤的早期生长目的:利用siRNA质粒介导抑制A549细胞表达内源性VEGF,观察其在体内外实验中对A549细胞表达VEGF的作用,及其在A549细胞裸鼠移植肿瘤实验中对肿瘤生长的影响,并分别用几种不同的转染方法和试剂转染VEGFsiRNA质粒,比较其对VEGF的抑制效率及其对A549细胞移植肿瘤生长的影响效率。方法:1.设计并构建三个带有抗VEGF的siRNA序列的质粒载体,与无意义序列siRNA质粒载体分别用PEI与Lipofectamine 2000两种转染试剂瞬时转染A549细胞,用ELISA与实时荧光定量RT-PCR检测A549细胞VEGFmRNA与分泌VEGF含量是否受到抑制,选出效果最佳的VEGF siRNA质粒载体。G418筛选稳定表达各个VEGF siRNA质粒的A549细胞克隆。细胞毒性评价MTT法分别比较PEI,Lipofectamine 2000转染A549细胞48小时后存活分数。2.裸鼠皮下注射A549细胞,待形成肉眼可见的皮下肿瘤后,随机分为7组,分别于肿瘤组织周围进行注射。空白对照组注射生理盐水;阴性对照组注射含无意义序列siRNA质粒;VEGFsiRNA组注射含VEGF siRNA质粒;VEGFsiRNA-PEI组注射VEGF siRNA质粒与转染试剂PEI混悬液;VEGFsiRNA-LIPO组注射VEGF siRNA质粒与转染试剂Lipofectamine 2000混悬液;PEI组注射转染试剂PEI混悬液;LIPO组注射转染试剂Lipofectamine 2000混悬液。观察肿瘤的生长情况。3.将稳定表达VEGFsiRNA与表达无意义序列siRNA的A549细胞,与未转染A549细胞分别注射于裸鼠皮下,观察肿瘤成瘤率以及肿瘤生长情况。4.分别剥离各组移植肿瘤,肿瘤组织匀浆上清液测总蛋白含量及VEGF含量。结果:1.PEI瞬时转染最高转染效率约41%,Lipofectamine 2000瞬时转染最高转染效率约48%。转染后A549细胞存活分数,分别为(65.4±4.3)%,(68.3±3.0)%,无显著差异。实时荧光定量RT-PCR检测结果显示,瞬时转染不同序列VEGFsiRNA后的A549细胞VEGF mRNA/GAPDH mRNA之比有显著降低,VEGFsiRNA1组降低了28%,VEGFsiRNA2组降低了40%,VEGFsiRNA3降低了30%(P＜0.05)。无意义序列siRNA组与对照组无显著差异(P＞0.05)。稳定表达VEGFsiRNA的A549细胞VEGF mRNA/GAPDH mRNA之比降低更为显著,VEGFsiRNA1组降低了49%,VEGFsiRNA2组降低了65%,VEGFsiRNA3降低了49%(P＜0.05)。无意义序列siRNA组与对照组无显著差异(P＞0.05)。ELISA的检测结果显示,瞬时转染不同序列VEGFsiRNA的各组A549细胞分泌VEGF量均降低,VEGFsiRNA1组降低了16%,VEGFsiRNA2组降低了19%,VEGFsiRNA3降低了17%(P＜0.05)。空白对照组和无意义序列siRNA组A549细胞VEGF分泌量无明显差异(P＞0.05)。经过稳定筛选后,各组A549细胞分泌VEG量降低更为显著,VEGFsiRNA1组降低了32%,VEGFsiRNA2组降低了61%,VEGFsiRNA3降低了42%(P＜0.05)。空白对照组和无意义序列siRNA组A549细胞VEGF分泌量无明显差异(P＞0.05)。2.未转染的A549细胞裸鼠移植瘤成瘤率100%。比较各组裸鼠肿瘤平均体积,发现排除转染试剂对肿瘤细胞的毒性因素后,发现VEGFsiRNA质粒注射组的肿瘤平均体积增长无明显抑制作用。在用裸DNA注射的VEGF siRNA组同样没有观察到有统计学意义的肿瘤平均体积降低。比较各组肿瘤倍增时间,发现排除转染试剂对肿瘤细胞的损伤因素后,发现VEGFsiRNA质粒注射组的肿瘤倍增时间并无显著降低。在用裸DNA注射的VEGF siRNA组同样没有观察到有统计学意义的肿瘤倍增时间延长。剥取移植肿瘤做ELISA分析VEGF含量结果发现,各组肿瘤组织匀浆上清液VEGF含量均没有明显差异(P＞0.05)。3.裸鼠腋部皮下分别注射未转染,稳定表达VEGFsiRNA和无意义序列siRNA的A549细胞后,移植肿瘤成瘤率100%。接种A549细胞第45天起,三组裸鼠的肿瘤平均体积有显著差异,VEGFsiRNA组肿瘤体积低于无意义序列siRNA组与对照组,且无意义序列siRNA组肿瘤平均体积也低于对照组(P＜0.05)。接种第8周末时,VEGFsiRNA组肿瘤平均体积比对照组降低了76.3%。肿瘤生长到体积为50 mm~3的时间VEGFsiRNA组比无意义序列siRNA组延迟了5.4天。VEGFsiRNA组与无意义序列siRNA组肿瘤倍增时间无显著差异(P＞0.05)。肿瘤组织匀浆上清液VEGF含量,VEGFsiRNA组肿瘤平均体积比无意义序列siRNA组降低了57.5%。肿瘤体积的对数回归趋势线分析及对数回归方程分析,显示VEGFsiRNA组的肿瘤生长速率并没有比对照组降低。结论:1.VEGFsiRNA质粒瞬时转染及稳定转染在体外能抑制A549细胞VEGF mRNA及分泌VEGF水平。2.裸鼠皮下移植肿瘤注射VEGFsiRNA质粒(无论是否与转染试剂PEI或Lipofectamine 2000共转染)三周,肿瘤生长及分泌VEGF量均没有明显受到抑制。转染VEGFsiRNA质粒没有对移植肿瘤生长起到抑制效应的原因可能是在动物体内转染效率过低。3.VEGF siRNA稳定表达的A549细胞,在移植瘤模型中可见肿瘤形成的初期有抑制生长的作用,使得肿瘤细胞增殖至50mm~3的体积所需的时间延长。而对肿瘤成形以后的生长无明显的抑制作用。第二部分VEGFsiRNA抑制A549细胞培养上清液对鸡胚尿囊膜上诱导的血管新生目的:利用VEGF siRNA抑制A549细胞VEGF的表达,研究VEGFsiRNA对A549细胞的培养上清液诱导鸡胚尿囊膜血管新生的影响。方法:选择表面无污染,无破损的,孵育至第9天的白皮受精种蛋。在气室范围中央划定约1×1厘米的开窗位置。用镊子尖端在蛋的钝端气室处打一小孔,慢慢剥去蛋壳,用小刀切破壳膜,显露出尿囊膜,用透明胶带封口制成假气室。次日,将鸡胚随机分成4组,每组12只,用微量加样器分别在鸡胚尿囊膜表面离开尿囊膜血管的空白处加100μL的DMEM培养液(阴性对照组),100μL未转染的A549细胞培养上清液(阳性对照组),100μL转染VEGF siRNA后的A549细胞培养上清液(VEGF siRNA组),100μL转染无意义序列siRNA后的A549细胞培养上清液(无意义序列siRNA组)。加样液取自培养72小时后的A549细胞培养上清液,细胞计数5×10~7,经冷冻脱水后加DMEM100μL溶解后待用。用ELISA法检测分别加入鸡胚尿囊膜表面的细胞培养上清液的VEGF浓度。继续孵育48小时,小心揭开透明胶带,暴露加药区尿囊膜,滴加数滴1:1甲醛与丙酮配成的固定液,室温下固定约20分钟,待血管内血液凝固后。剪下鸡胚尿囊膜,平铺于滤纸上干燥后置于载玻片上,于光学显微镜相同物镜倍数(10倍)下观察血管分支并摄片。同一倍数物镜下,随机取六个视野,计算机图象软件辅助下,描记视野内的尿囊膜血管一级与二级分支,手工记数血管分支点,测量血管总长度的相对值,取六个视野的平均数。比较各组血管分支数和血管分支总长度,上述两项结果作为评价血管密度的指标。结果:A549细胞培养上清液作用CAM后,对鸡胚的存活率没有显著影响。加入含VEGF的A549细胞培养上清液后,VEGFsiRNA组与阴性对照组鸡胚尿囊膜血管图相比,鸡胚尿囊膜上可见稍增多的血管分支,而阳性对照组和无意义序列siRNA组的尿囊膜上还可以见血管分支增长、增粗,走形异常,造成血管网结构异常。鸡胚尿囊膜上的加样液VEGF浓度,阴性对照组为零,VEGFsiRNA组约占无意义序列siRNA组与阳性对照组40%-44%;鸡胚尿囊膜血管分支点,VEGFsiRNA组、无意义序列siRNA组与阳性对照组比阴性对照组增加55%-45%;血管分支总长度VEGFsiRNA组比阴性对照组增加约53%,低于无意义序列siRNA组与阳性对照组,后两组比阴性对照组增加约97%及99%。结论:1.不同浓度的VEGF诱导鸡胚尿囊膜血管新生的程度不同,较低浓度VEGF诱导血管分支增多为主,较高浓度的VEGF诱导鸡胚尿囊膜血管分支增长增粗,及血管分支的走形改变,导致毛细血管网的结构异常。2.VEGF siRNA能抑制A549细胞表达VEGF,从而抑制鸡胚尿囊膜血管新生。更多还原

【Abstract】 Background:Antiangiogenic therapy has emerged as an important concept in the treatment of solid tumors, including non-small cell lung cancer (NSCLC). Vascular endothelial growth factor (VEGF) represents an important therapeutic target, as it is the primary mediator of angiogenesis and is induced by multiple tumor-relevant stimuli. Although antivascular strategy that targets the endothelial cells of the host was shown to be very efficient at reducing tumor growth and was not expected to trigger tumor resistance, recent studies have demonstrated that sustained high-level secretion of angiogenic stimulator from the tumor cells such as VEGF, can enable tumors to bypass antiangiogenic treatments. RNA interference (RNAi) is now being exploited as a powerful tool for reverse genetics, and shows great promise for therapeutic applications. Some authors developed vectors or viruses to produce RNAi which could be used in vivo experiments. However, the diffieultyof gene delivering still limits in vivo study. The aim of the present work was to determine whether blocking the ability of A549 cells to express endogenous VEGF by siRNA in a mouse xenograft experiments could affect the tumor growth and A549 cell induced angiogensis. Part 1 Small interfering RNA (siRNA) targeting VEGF inhibits initial A549 tumor growth in a xenograft murine model.Objective:To investigate the effect of VEGF siRNA on A549 cells in vitro using two transfection reagents and if it could inhibit A549 cell tumour growth in a xenograft murine modle.Materials and methods:Construction of siRNA vector: Three pairs of siRNAs were designed to downregulate human VEGF(GenBank Accession Number NM-003376) expression.Three pairs of VEGFsiRNA-plasmid and non-specific-plasmid were transfected into A549 cells through PEI or lipofectamine 2000, respectively. The most effective pair of VEGFsiRNA-plasmid was selected by ELISA and realtime RT-PCR. To generate A549 cell lines stably expressing VEGF siRNA by drug selection with G418. Transfection efficiency and toxicity were measured by fluorescence detection or MTT. A549 cells were injected in nude mice to gengerate xenograft than transfected with VEGF siRNA or co-transfected with PEI or lipo fectamine 2000, respectively. The stable A549 cell clone which expressesVEGFsiRNA-plasmid or non-specific-plasmid and A549 cell without transfection were injected in nude mice respectively, to evaluating their biologic effects.Results:The highest transfection efficiecy of PEI or . lipofectamine 2000 was 41% or48%. Cell survival rate with PEI or lipofectamine 2000 was similar as (65.4±4.3) %,(68.3±3.0) %. Transient-transfection of VEGFsiRNA-plasmid in A549 cell induced 28%,40%,30% reduction in the ratio of VEGF mRNA copies to hGAPDH mRNA copies, respectively.. Stable-transfectiong of VEGF siRNA plasmid induced 49%, 65%, 49%, reduction in the ratio of VEGF mRNA copies to hGAPDH mRNA copies, respectively. ELISA of VEGF revealed that tansient-transfection of VEGFsiRNA-plasmid in A549 cell induced slightly levels of reduction in VEGF secretion as compared with untransfected cells: VEGFsiRNAl induced 16% reduction in VEGF secretion, VEGFsiRNA2 induced 19%, VEGFsiRNA3 induced 17%. Stable-transfectiong of VEGF siRNA plasmid induce 32%,61%,42% reduction in VEGF secretion.After a certain period of injection with A549 cells into the axilla of nude mice, visible tumors had developed to be palpable at the injection sites. Transient-transfection of VEGFsiRNA-plasmid in tumour with or without PEI or lipofectamine 2000 showed on effect on tumour growth or VEGF in tumour content.Stable-transfection A549 cells of nude mice xenograft model showed tumour formation was 100%. Analysis of the growth curves show that VEGFsiRNA reduced tumor volume from day 45 after injection compared with non-silencing group or control group. The logarithmic regression analysis of the growth curves showed that VEGFsiRNA do not significantly reduce the growth rate of the tumors but induced a slight increased in VEGF siRNA group as compared with non-silencing siRNA group or control group. To control this reduction was not resulting from a non-silencing siRNA effect ,we compared VEGFsiRNA group with non-silencing siRNA group, showed that the mean tumor volume was reduced by 76.3%. The time for xenografts reached 50mm~3 after injection of the tumor cells in VEGFsiRNA group was delayed by 5.4 days compared with non-silencing siRNA group.The mean doubling time of tumors was not significantly modified by VEGFsiRNA treatments compared with non-silencing siRNA group. The mean doubling time of tumors was delayed by VEGFsiRNA treatments or non-silencing siRNA compared with control group. ELISA of VEGF in tumor contents showed 57.5%reduction in the VEGF expression of tumors from VEGFsiRNA group as compared with non-silencing siRNA group.Conclusion:1 Transient-transfection or stable-transfection of VEGF siRNA plasmid could downregulate the expression of VEGF in human A549 cells in vitro.2 Transient-transfection of VEGFsiRNA-plasmid in vivo with or without PEI or lipofectamine 2000 showed on effect on tumour growth or VEGF in tumour content. Possible reason was low efficiency of transfection in vivo.3 Stable- transfection A549 cell of VEGF siRNA in a xenograft murine modle showed time for xenografts reached 50mm~3 was delayed but tumour growth rate was not inhibited. Part 2 Small interfering RNA (siRNA) targeting VEGF inhibits angiogenesis induced by A549 cell on CAM assaysObjective:To investigate whether blocking the ability of A549 cells to express endogenous VEGF by siRNA could affect VEGF induced angiogenesis in CAM assays.Materials and methods:Fertilized eggs on day 9 were exposed CAM and established CAM model. Chick embryos were randomly divided into four groups(n=12-14): negative control group , positive control group , VEGFsiRNA group , and nonsilencing siRNA group. On day 10, l00μl DMEM or cell culture supernatant were placed on CAM of each group for another 48 hours incubation.. The CAMs were harvested on day12, then fixed and placed on slide under the microscope.Results:The chick chorioallantoic membrane assays have been widely accepted as models for evaluating effects on angiogenesis or anti-angiogenesis.The vascular system of CAM is well developed and directly accessible to observation and experimentation. Compared with negative control group, the proliferation of microvessels was obviously increased when cell culture supernatant with VEGF was added in positive control group , non-silencing siRNA group or VEGFsiRNA group. Positive control group and non-silencing siRNA showed an extremely significant proliferated vessels compared with negative group, as part of the vascular net’s morphosis losing the tree type arrangement of vessels. VEGF content of VEGFsiRNA group were only 40%-44% of non-silencing siRNA group or positive control group. Vessels branch points of the chorioallantoic membrane were increased in positive control group by 99% , non-silencing siRNA group by 97% , VEGFsiRNA group by 53% compared with negative group.. Total vessel length of CAM were increased in positive group by55%, non-silencing siRNA group by 49% and VEGFsiRNA group by 45% compared with negative control group.Conclusion:.1. Low concentration of VEGF could induce angiogenensis around the chick chorioallantoic membrane as well as high level VEGF. However ,high VEGF also induced increased bulky and longer of vessel branch and change of vascular network.2. VEGFsiRNA partially inhibited A549 cells induced angiogenesis around the chick chorioallantoic membrane.更多还原