【作者】 曲华伟；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 研究背景原发性胆囊癌是胆道系统中占首位的恶性肿瘤,在消化道恶性肿瘤中占第5位。胆囊癌恶性程度极高,早期无特异表现,很多病人在诊断时已进展至晚期。胆囊癌治疗以手术治疗仅只有20%-30%的患者可以得到根治性切除,其对于化疗和放疗均不敏感。探索胆囊癌的基因治疗具有积极意义。实体肿瘤是血管依赖性病变,其生长、浸润和转移离不开血管生成。VEGF是目前已知肿瘤血管生成因子中直接刺激内皮细胞分裂增殖的血管内皮细胞有丝分裂原。VEGF的表达在胆囊癌大小、淋巴浸润各阶段有显著的不同。胆囊癌细胞分泌VEGF增加,除可以促进新生血管形成,还可以加速肿瘤演进。VEGF不但与胆囊癌的发生相关,还与胆囊癌浸润、转移及预后密切相关。针对VEGF抗胆囊癌血管形成的治疗研究具有重要意义。肿瘤治疗的关键是有效抑制VEGF基因表达。现在,可以下调VEGF基因表达的技术包括反义核酸技术、核酶技术以及RNA干扰技术。反义技术和核酶技术的基因抑制效果十分短暂、微弱,不适合长期研究基因功能。RNA干扰技术抑制效果强,持续时间长,技术流程简便,其表达非常稳定。本实验的基本思路是：通过构建针对人VEGF基因的shRNA干扰质粒载体,观察沉默VEGF基因对胆囊癌GBC-SD细胞生长和侵袭能力的影响；建立稳定转染shRNA-VEGF的细胞株,通过检测其VEGF受体表达变化,初步探讨VEGF与VEGF受体的相互作用；应用胆囊癌GBC-SD细胞株以及稳定转染细胞株分别建立胆囊癌的裸鼠移植瘤模型,观察各组细胞的成瘤能力。另一组动物实验中,向正常胆囊癌裸鼠移植瘤瘤内注射干扰质粒shRNA-VEGF2,观察其对裸鼠移植瘤VEGF基因表达的影响及对肿瘤的生长与血管生成的抑制作用,通过应用RNA干扰技术抑制胆囊癌VEGF基因表达从而抑制胆囊癌肿瘤生长与侵袭,为这一技术的临床应用提供实验和理论基础。目的：构建针对人胆囊癌VEGF基因的shRNA及其质粒表达载体。方法：设计针对人VEGF基因序列的4组寡核苷酸链模板,退火后与载体质粒pCY/U6/GFP/Neo连接,然后进行酶切鉴定和DNA序列测定。结果：酶切鉴定和DNA测序分析后,表明靶向VEGF的RNA干扰质粒载体构建成功。结论：成功构建针对人VEGF基因的shRNA表达载体pCY/U6/GFP/Neo-shRNA-VEGF。目的：将干扰质粒转染人胆囊癌细胞,观察其对VEGF基因沉默的效果,并采用荧光素酶报告基因验证RNA干扰位点的有效性。筛选出干扰效果最好的质粒并建立稳定转染细胞株。观察VEGF受体表达变化,初步探讨VEGF与VEGF受体之间的作用机制。方法：①干扰质粒转染胆囊癌GBC-SD细胞,分别采用半定量PCR、实时荧光定量PCR和Western印迹方法检测VEGF mRNA和蛋白表达情况,筛选出抑制靶基因表达效果最好的质粒；②因RNA干扰可能存在“脱靶效应”,为验证RNA干扰位点的准确性,我们设计含有VEGF靶位点的引物,将其与荧光素酶报告基因载体退火连接,再将其与第一部分的干扰质粒共转染人胆囊癌细胞,观察荧光素酶活性变化,明确RNA干扰位点的有效性；③将干扰效率最高的的表达质粒用来建立稳转细胞株。同时用NC表达质粒作为阴性对照稳转细胞株。经G418筛选,建立两个稳转细胞株,分别称为GBC-shV2和GBC-NC;④采用实时荧光定量PCR检测稳定转染细胞株的三种VEGF受体(Flt-1、KDR、NRP-1)的mRNA表达变化情况,并加入外源性VEGF因子到稳转细胞株中,分别检测24,48,72小时VEGF受体的表达变化情况。初步探讨VEGF与其受体之间的作用机制。结果：干扰质粒转染胆囊癌GBC-SD细胞后,半定量PCR检测显示,shVEGF1、shVEGF2、shVEGF3、shVEGF4干扰质粒对靶基因mRNA表达都有抑制作用,其中shVEGF2干扰质粒抑制作用最明显,抑制率达64%,与空白对照组相比,具有明显差异(P<0.05)；实时荧光定量PCR检测显示,shVEGF1、shVEGF2. shVEGF3干扰质粒对靶基因mRNA表达都有抑制作用,其中shVEGF2干扰质粒抑制作用最明显,抑制率达83%,与空白对照组相比,具有明显差异(P＜0.05); Western blot方法检测各组细胞中VEGF蛋白表达水平显示,shVEGF1、shVEGF2干扰质粒对靶基因蛋白表达有抑制作用,其中shVEGF2干扰质粒抑制作用最明显,抑制率达92%,与空白对照组相比,具有明显差异(P<0.05)。可见shVEGF2干扰质粒对VEGF基因表达抑制最明显；②通过荧光素酶报告基因验证实验,我们发现含有VEGF2靶位点的荧光素酶报告基因载体被shRNA-VEGF2成功干扰,与对照组比较,荧光素酶活性下降75%,有统计学意义(P<0.05)；③成功建立两个分别表达shRNA-VEGF2和NC质粒的稳转细胞株,分别命名为GBC-shV2和GBC-NC细胞。④实时荧光定量PCR检测显示,相对于阴性稳转细胞株,GBC-shV2细胞株中的Flt-1与KDR受体表达分别下降28%和18%,而NRP-1受体表达升高47%。我们将外源性VEGF因子加入稳转细胞株中,检测Flt-1受体表达变化：相对于阴性稳转细胞株,24小时表达75%,48小时表达183%,72小时表达411%。可见,外源性VEGF因子加入后,Flt-1受体mNRA的表达呈明显上升的趋势。结论：①shRNA-VEGF2干扰质粒可以明显抑制人胆囊癌细胞VEGF基因mRNA和蛋白的表达；②经荧光素酶报告基因实验证明RNA干扰的VEGF基因序列的靶位点是准确有效的；③成功建立稳定表达shRNA-VEGF2质粒的细胞株；④人胆囊癌GBC-SD细胞存在VEGF的受体(Flt-1、KDR、NRP-1)；⑤胆囊癌细胞VEGF蛋白可能存在自分泌机制；⑥VEGF可能对其Flt-1受体的表达有诱导作用。目的：通过对前面试验得到的稳定转染细胞株进行相关的生物学实验,探讨沉默VEGF基因对人胆囊癌GBC-SD细胞生长、黏附、迁移运动及侵袭能力的影响。方法：本实验采用的细胞株分别是正常胆囊癌细胞株GBC-SD以及前面试验得到的稳定转染细胞株GBC-shV2和GBC-NC。在本部分实验中拟采用MTT法绘制胆囊癌细胞的生长曲线；通过肿瘤细胞与细胞基质黏附实验以及肿瘤细胞与内皮细胞黏附实验,测定细胞不同吸光度值,评估胆囊癌细胞黏附能力变化；通过肿瘤细胞迁移运动实验,对比肿瘤细胞不同的迁移距离,评估胆囊癌细胞迁移运动能力变化；通过Transwell小室侵袭实验法,计算穿透膜的细胞数,评估胆囊癌细胞侵袭能力变化。结果：①从生长曲线看,GBC-shV2细胞与GBC-SD细胞相比,增殖生长明显变慢,细胞活力下降明显,达36%,差异有统计学意义(p<0.05)。而GBC-NC细胞的生长速度和GBC-SD没有区别(p>0.05)；②与GBC-SD细胞相比,GBC-shV2细胞与细胞外基质黏附率明显下降,达55%(p<0.05),与内皮细胞黏附率明显下降,达60%(p<0.05),而GBC-NC细胞与细胞外基质和内皮细胞的黏附率与GBC-SD细胞相比没有明显区别(p>0.05)；③与GBC-SD细胞相比,GBC-shV2细胞迁移运动距离明显减少,达61%(p<0.05),而GBC-NC细胞与与GBC-SD细胞相比没有明显区别(p>0.05)；④与GBC-SD细胞相比,GBC-shV2细胞穿透膜的细胞数明显减少,减少达51%(p<0.05),而GBC-NC细胞与与GBC-SD细胞相比没有明显区别(p>0.05)。结论：shRNA-VEGF干扰质粒能抑制胆囊癌细胞生长,可能使胆囊癌细胞的黏附,迁移运动以及侵袭能力降低。目的：应用胆囊癌GBC-SD细胞及稳转细胞株分别建立裸鼠移植瘤模型,观察其成瘤能力变化；瘤内注射重组质粒shRNA-VEGF2,观察对其裸鼠移植瘤VEGF基因表达的影响以及对肿瘤的生长与血管生成的抑制作用。方法：本实验分为裸鼠成瘤实验与荷瘤裸鼠治疗实验两部分。裸鼠成瘤实验：分别将GBC-SD, GBC-shV2, GBC-NC细胞建立裸鼠移植瘤模型,观察其成瘤时间,成瘤率,肿瘤体积变化,绘制生长曲线。荷瘤裸鼠治疗实验：首先建立正常胆囊癌GBC-SD细胞裸鼠异位移植瘤。分别将不同试剂(干扰质粒,空白质粒,培养液,转染试剂)多点多次注射入瘤内,观察肿瘤体积变化。动物实验结束后,进行肿瘤组织免疫组化检测。分别测定VEGF蛋白的积分光密度(IOD)与组织微血管密度(MVD)。结果：①裸鼠成瘤实验：GBC-shV2细胞成瘤时间在12.7天,成瘤率67%,肿瘤体积抑制率70%。GBC-shV2细胞组肿瘤组织无明显出血及坏死,肿瘤与周围组织粘连较松,易剥离；相对于GBC-SD细胞组,GBC-shV2组肿瘤组织VEGF蛋白的IOD值下降59%(p<0.05),MVD值下降64%(p<0.05)。②荷瘤裸鼠治疗实验：与注射培养液组(A组)相比较,注射shRNA-VEGF2质粒组(D组)的肿瘤体积明显减小,体积抑制率为45%；与A组比较,D组肿瘤组织VEGF的IOD值下降50%(p<0.05),MVD值下降59%(p<0.05)。结论：针对人胆囊癌GBC-SD细胞VEGF基因的RNA干扰技术可以显著抑制胆囊癌移植瘤中VEGF基因的表达,可以明显抑制胆囊癌移植瘤中的新生血管生成,从而间接抑制胆囊癌移植瘤的生长。目的：以VEGF-A的生物序列为基础,对其进行生物信息学分析。方法：首先以Phylip和Treeview软件绘制该VEGF-A系统进化树；以ExPASY网站提供的生物信息学分析模块和分析软件对VEGF-A的理化性质进行分析；预测VEGF-A的二级结构；使用ExPASy网站上SWISSMODEL模块进行VEGF-A蛋白的三级结构预测,使用STRING网站上Protein-Protein Interactions模块进行VEGF-A蛋白的四级结构(蛋白质相互作用)预测分析。结果：①从VEGF-A系统进化树可以看出,斑马鱼分支起源最早,然后依次是狗,马,猪等分支,人VEGF-A的进化发源得比较晚；②VEGF-A蛋白所带总负电荷残基数为2,所带总正电荷残基数为37,VEGF-A的等电点分别是12.38和12.9,这说明VEGF-A是一种带正电的蛋白质。VEGF-A蛋白的亲水指数为-0.605,这说明VEGF-A蛋白是一种疏水性的蛋白质。VEGF-A的不稳定指数为31.26,可以看出VEGF是比较稳定蛋白；③GOR4、HNN、SOPMA三种方法预测的二级结构都认为第55-60、65-70、98-108位等区域可能是VEGF-A的功能结构域。分析VEGF-A的亲水性、极性以及折返系数可以发现,第6-12、55-65、132-138、150-157位等区域可能是VEGF-A的功能结构域。与VEGFA有相互作用联系的蛋白质有十种,分别是Flt-1,KDR,NRP-1,NRP-2,免疫球蛋白K轻链基因片段C区,Src,N0S3, ARNT, VTN, HIF1A。结论：VEGF-A蛋白是一种带正电荷的稳定的疏水性蛋白。第55-60位区域可能是VEGF-A的功能结构域。更多还原

【Abstract】 Primary gallbladder carcinoma ranked first in malignant tumors of biliary system,and ranked fifth in malignant tumors of digestive tract. The degree of malignancy of gallbladder carcinoma is very high. Because of the early non-specific clinical manifestations, many patients at the time of diagnosis has progressed to the late, and 5-year survival rate of stage III and IV patients is only 1% and 5%. The 5-year survival rate of gallbladder cancer in United States and Europe is only 5% -13%. The main treatment for gallbladder carcinoma is surgery, but only 20%-30% of patients can be radical resection. The fact that chemotherapy and radiotherapy for gallbladder carcinoma are not sensitive has brought great difficulties to the clinical treatment for gallbladder carcinoma. It is important to explore new therapy.Solid tumor is vascular-dependent, and the tumor growth, invasion and metastasis can not be separated from angiogenesis. Tumor angiogenesis is a key factor in tumor growth. Among all tumor angiogenesis factors, only VEGF is highly specific vascular endothelial cell mitogen which directly stimulated endothelial cell division and proliferation.VEGF was highly expressed in gallbladder carcinoma. VEGF expression is different in gallbladder size, lymph node infiltration in various stages. VEGF participates in not only the occurrence of gallbladder carcinoma, but also the invasion, metastasis. The anti-angiogenic treatment targeting VEGF is very important.How to effectively inhibit VEGF gene expression is the key factort of cancer treatment. The way of inhibiting VEGF gene expression includes:antisense nucleic acid technology, ribozyme technology, and RNA interference technology. The gene suppression effect of antisense technology and ribozyme technology is very short and weak, which is not suitable for long-term study of gene function. The RNAi technology is far superior to them in suppressing specific gene expression. It has strong inhibitory effect, simple technical process, short-cycle, stability, efficiency and other unique advantages.The plan of this experiment is:we observe the impact of VEGF gene silencing on growth and invasion of gallbladder carcinoma GBC-SD cell line; we use the gallbladder carcinoma GBC-SD cells to establish nude mice xenograft model and observe the change of VEGF expression after intratumoral injection of recombinant plasmid shRNA-VEGF2 in xenografts in nude mice and effects on tumor growth and angiogenesis in order to provide experimental and theoretical basis for the clinical development and application of cancer treatment. Objective:To construct VEGF-targeted small interfering RNA and its expression vectorMethods:Complementary oligo DNA strands targeting VEGF gene were designed and synthesized according to the principles of designing siRNA. After annealing, oligo DNAs were inserted into pCY/U6/GFP/Neo vector, then enzyme digestion analysis and DNA suquencillg were performed.Results:The enzyme digestion analysis and DNA sequencing showed that VEGF-targeted small interfering RNA and its expression vector were constructed successfully.Conclusion:VEGF-targeted small interfering RNA and its expression vectors were constructed successfully. Objective:To observe the impact of VEGF-targeted shRNA on the expressions of VEGF. Luciferase reporter gene was used to verify the validity of RNA interference sites. We choose the best plasmid to establish a stable transfected cell line. We observed the expression of VEGF receptors, and study the mechanism of action between VEGF and VEGF receptors.Methods:①In order to determine the inhibiting effect of VEGF, we use semi-quantitative PCR, real-time PCR and Western blot to detect VEGF mRNA and protein expression.②We design primers containing the VEGF target sites, make it connected with the luciferase reporter gene vector. The recombinant vector and the vectors of chapter I were transfected into human gallbladder carcinoma cells to observe the changes of luciferase activity, and to verify the validity of RNA interference sites.③We use the shRNA-VEGF2 plasmid and NC plasmid to establish stable transfected cell lines by G418 selection.④The mRNA expressions of the three VEGF receptors (Flt-1, KDR, NRP-1) are detected by real-time PCR. Then we added VEGF into the cell line and detecte the mRNA expressions of Flt-1 at 24,48,72 hours in order to study the mechanism between VEGF and of its receptors.Results:①shVEGF1, shVEGF2, shVEGF3, shVEGF4 interfering vectors all inhibit VEGF mRNA expression and the inhibition rate of shVEGF2 is 64%. Detected by real-time PCR,the inhibition rate of shVEGF2 is 83%. The inhibition rate of VEGF protein of shVEGF2 is 92% detected by western blotting. ②Luciferase reporter gene through In the verification experiment, we found luciferase activity decreased 75% which imply that the target site is accurate;③We successfully establish two stable transfected cell lines, named GBC-shV2 and GBC-NC cells.④Expression of Flt-1 and KDR receptors in GBC-shV2 cell lines decreased 28% and 18%, while expression of NRP-1 receptor increased 47%. When added VEGF into the cell line, the expression of Flt-1 is 75%, 183% and 411% at 24,48 and 72 hours respectively. We can concluded that the expression of Flt-1 increased significantly with the VEGF.Conclusion:①shVEGF2 can inhibit expression of VEGF in human gallbladder carcinoma GBC-SD cells;②The luciferase reporter gene experiments show that the RNA interference target site is accurate and valid;③We established stable transfected cell lines which express shVEGF2 plasmid;④There are VEGF receptors (Flt-1, KDR, NRP-1) in human gallbladder carcinoma GBC-SD cells;⑤There is VEGF autocrine mechanism in human gallbladder carcinoma GBC-SD cells;⑥VEGF can induce expression of Flt-1 receptor. Objective:To study the effects of VEGF gene silencing on capacity of growth, adhesion, migration, and invasion of human gallbladder carcinoma GBC-SD cells.Methods:In this part, we draw the cell growth curve using MTT method; To assess the adhesion ability through adhesion experiments; To assess the migration ability through migration experiments; To assess the invasion ability through Transwell chamber invasion assay.Results:①Compared with GBC-SD cells, the cell viability decreased significantly, reaching 36%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells.②Compared with GBC-SD cells, the cell’s adhesion ability decreased significantly, reaching 55%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells.③Compared with GBC-SD cells, the cell’s migration ability decreased significantly, reaching 61%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells (p<0.05)④Compared with GBC-SD cells, the cell’s invasion ability decreased significantly, reaching 51%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells (p<0.05)Conclusion:shRNA-VEGF interfering plasmid significantly inhibited the, cell, and ability of gallbladder cancer cells. In vitro, it validate that the VEGF gene can be a good therapeutic target. Objective:We use the GBC-SD, GBC-NC and GBC-shV2 cells to established mice xenograft models to observe the change of tumorigenic capacity; We intratumoral inject recombinant plasmid shRNA-VEGF2 into the tumors to observe the VEGF expression in tumor tissue and the change of tumor growth and angiogenesis.Methods:In this study, the experiment were divided into two parts: tumor formation experiment and treatment of tumor-bearing mice experiment.①Tumor formation experiment:18 health nude mice were randomly divided into three groups of six. The GBC-SD, GBC-NC and GBC-shV2 cells are injected into the nude mice to establish mice xenograft models. We observe tumor formation time, rate of tumor formation, change of tumor volume, and draw the growth curve.②Treatment of tumor-bearing mice experiment:24 healthy nude mice were randomly divided into four groups of six. When the xenograft models are established, we inject different 200ul reagents into the tumors: group A:liposome; group B:RPMI1640; group C:empty plasmid and liposome; group D:shRNA-VEGF2 plasmid and liposome.After the animal experiments, we remove the tumor from the mice and take it for VEGF and CD34 immunohistochemistry. We use the computer-assisted image analysis system to analyze IOD and MVD to assess the expression of VEGF and angiogenesis in tumor tissue.Results:①Tumor formation experiment:The tumor formation rate of GBC-shV2 group is 67%, and the tumor volume inhibition rate is 70%. Compared with GBC-SD cells, the IOD value of VEGF decrease 59% and the MVD value decrease 64%. There is no difference between the GBC-NC group and GBC-SD group.②Treatment of tumor-bearing mice experiment:The tumor volume inhibition rate of group D is 45%. Compared with group A, the IOD value of VEGF of group D decrease 50% and the MVD value decrease 59%. There is no difference between the group A、B and C.Conclusion:RNA interference can inhibit the expression of VEGF in tumor tissue of gallbladder carcinoma. VEGF silencing can inhibit gallbladder carcinoma cell growth and angiogenesis. Objective:Making bioinformatics analysis with VEGF-A biological sequences to obtain the structure and function-related information.Methods:First of all, we draw phylogenetic trees of VEGF-A by Phylip and Treeview software; Then, analyze the amino acid composition, isoelectric point, the signal peptide cleavage point and the titration curve of VEGF-A by Anthprot bioinformatics analysis software and ExPASY website; We predict the secondary structure of VEGF-A by GOR4, HNN, SOPMA methods and analyze hydrophilicity, polarity, and return coefficient by ExPASY website; Finally, these results are compared and analyzed. We predict the tertiary and fourth structure of VEGF-A by STRING website.Results:①we can see from the phylogenetic tree that:zebra fish is the origin of the first branch, followed by dogs, horses, pigs and other branches;②VEGF is a positively charged, hydrophobic and stable proteins;③GOR4, HNN, SOPMA predict the secondary structures that the 55-60,65-70,98-108 regions may be VEGF-A function domain. Analysis of VEGF A hydrophilicity, polarity, and return coefficient can be found, the 6-12,55-65,132-138,150-157 regions may be VEGFA function of the regional domain. There are ten interacting proteins associated with VEGF:Flt-1, KDR, NRP-1, NRP-2, HIF1A, Immunoglobulin Kappa light chain C gene segment, SRC, NOS3, ARNT, VTN.Conclusion:VEGF is a positively charged, hydrophobic and stable proteins. The 55-60 regions may be VEGF-A functional domain。更多还原