【作者】 陈泓；

【导师】 唐步坚； 李力；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 卵巢上皮癌是妇科恶性肿瘤中发病率占第三位,死亡率却居第一的恶性疾病。由于卵巢解剖上隐匿于盆腔深处,在发病早期缺乏特异性症状和体征,很难在早期发现和诊断,约60%-70%的卵巢癌患者确诊时己属于晚期。近30年来,虽然经过全世界妇科肿瘤专家的不懈努力,不断改进手术方式,发现更新更好的化疗药物,增加新的化疗方案,但是5年生存率一直徘徊在20%-30%左右。所以,寻找新的诊断方法和新的治疗手段是目前卵巢癌研究的重点。作为一种与粘附和渗出等肿瘤浸润转移必须过程密切相关的物质,乙酰肝素酶(HPSE)在卵巢癌的浸润转移中起着举足轻重的作用。本课题通过一系列体外实验,对HPSE在卵巢癌浸润转移过程中的作用及其机制做了深入的研究,并且构建了HPSE基因慢病毒表达和RNAi载体,为今后的基因靶向治疗奠定了基础。乙酰肝素酶基因全长扩增和携带其基因的真核表达载体的构建目的:通过基因工程技术,扩增出乙酰肝素酶基因全长,构建其真核载体。为进一步研究乙酰肝素酶与卵巢癌浸润转移的关系打下基础。方法:选取浆液性卵巢癌组织作为模板提取人卵巢癌组织总mRNA,RT-PCR技术扩增cDNA,利用PCR技术进行乙酰肝素酶基因全长的扩增。将其与真核表达载体pcDNA3.1连接,进行克隆及酶切和测序鉴定。结果:成功扩增出目的基因全长,并经测序验证其同源性达100%。结论:成功从恶性卵巢癌组织中扩增出HPSE全长并构建了其真核表达载体。乙酰肝素酶介导的卵巢上皮癌细胞系浸润转移体外实验研究目的:了解乙酰肝素酶对卵巢癌细胞功能的影响,分析HPSE在卵巢癌浸润转移过程中所起的作用;探索其作为早期诊断、治疗靶向的价值。方法:成功构建HPSE基因真核表达载体后,将其转染无HPSE基因表达的卵巢癌细胞株A2780,G418筛选后经RT-PCR及western-blot鉴定确认获得稳定转染的HPSE/A2780细胞株。然后进行细胞生长特性、细胞周期变化、黏附能力、细胞侵袭转移能力的实验。结果:通过转染筛选,获得能够稳定表达HPSE蛋白的卵巢癌细胞株。流式细胞仪对细胞检测结果显示A2780-HPSE组处于增殖状态(S+G2+M)的细胞为46.5%,G1期为53.5%,对照组则相应为54.5%和45.6%,其处于增殖周期的细胞有减少的趋势。HPSE组和对照组的生长曲线几近重合。两组的克隆形成率差异无统计学意义。A2780细胞在转染前后粘附率分别为0.5183±0.0796和0.7283±0.08931,比较有统计学意义(p=0.002)。Transwell小室实验发现转染HPSE基因后的A2780细胞的侵袭能力明显提高,与对照组比较有统计学意义(p=0.003)。而其迁移能力未受影响(p=0.618)。结论:细胞功能实验结果表明,HPSE能够增强卵巢癌A2780细胞的侵袭能力,降低其黏附能力;对增殖能力和迁移能力均无影响。RNAi阻断乙酰肝素酶介导的卵巢上皮癌细胞系浸润转移体外体实验研究目的:通过构建人乙酰肝素酶(HPSE)基因的shRNA表达载体,转染卵巢癌细胞,检测其对HPSE基因的干扰效率。继而筛选出稳定干扰的细胞株,进行一系列功能实验,反面验证HPSE基因的功能。方法:针对HPSE mRNA的不同区域构建不同的shRNA表达载体,脂质体法转染HPSE高表达的人卵巢癌细胞系SKOV3,实时定量PCR检验干扰效率,选择抑制率最高的一组细胞进行抗性筛选,获得稳转细胞株,western-blot检验HPSE蛋白受抑制情况,然后进行细胞功能实验。结果:用荧光定量PCR对不同shRNA干扰载体的干扰效果进行检测结果显示,pGPU6/GFP/Neo-HPSE-1222组相对拷贝数为0.936±0.417,与其他3组相比有统计学意义,抑制率为48.63%。获得的该组稳定干扰表达株western-blot检验其HPSE蛋白表达明显减少。细胞生长曲线显示干扰组细胞倍增时间延长。干扰组与对照组集落形成率分别为0.0433±0.0451和0.0397±0.00687,p=0.059,比较无统计学意义。侵袭能力实验中,干扰组吸光值为0.5697±0.106,对照组为0.8097±0.333,干扰组与对照组相比无统计学意义(p=0.233)。细胞粘附能力检测,干扰组0.7533±0.07685,对照组0.5833±0.11994,两者相比有统计学意义(p=0.015)。侵袭能力实验干扰组为0.69±0.085,对照组为1.091±0.277,两者相比有统计学意义(p=0.015)。结论:采用构建shRNA干扰载体对HPSE基因进行干扰,有效地抑制了HPSE基因的活性,使卵巢癌细胞侵袭和黏附能力降低。HPSE重组慢病毒转基因系统和重组慢病毒干扰系统的构建目的:采用第二代和第三代慢病毒载体,分别构建HPSE基因的慢病毒表达载体和shRNA慢病毒干扰载体,摸索出较传统转染方法更为高效、可靠的转基因方法,为今后进一步的基因靶向治疗研究打下良好的基础。方法:首先扩增HPSE全长序列,将其与慢病毒载体pWPI连接后测序BLAST检索,并将重组慢病毒质粒转染293T细胞,48小时后提取mRNA,RT-PCR检验HPSE基因表达情况。其次,选取干扰效果最佳的siRNA,设计shRNA结构,退火反应形成双链后与慢病毒载体pSico连接并送测序。结果:重组慢病毒质粒HPSE-pWPI测序结果经BLAST,与HPSE基因的同源性达99%。转染293T后有HPSE基因的表达。重组慢病毒干扰载体pSico/HPSE测序结果与设计的shRNA序列完全一致。结论:成功的构建了HPSE基因的重组慢病毒系统和慢病毒干扰系统。为今后的HPSE靶向治疗奠定了基础。更多还原

【Abstract】 The morbility of epithelial ovarian is the third in malignant gynecology tumors while the mortality is the first.Ovarian conceals cavum pelvis deeply.On the pathogenetic early stage, deficiency of characteristic symptom and physical symptom make the discovery and diagnosis difficult.About 60-70%of ovarian cancers is advanced stage when they are diagnosed.In recent 30 years,chemotherapeutics modus were discovered,and operandi has been modified.but the 5 years survival rate is just 20-30%.Finding new diagnostic method and therapeutic tool is the focal point in research of ovarian cancer.Heparanase is associative with infiltration of tumor intimately.Heparanase plays an important role in ovarian cancer infiltration too.We discussed the mechanism and effect of heparanase on invasion and metastasis of human epithelial ovarian in vitro.And then we constructed lentivirus expressing vector and RNAi vector of HPSE gene.That can promote the gene targeted therapy. Amplification and eukaryotic vector construction of heparanase gene fromhuman ovarian cancer tissueObjective The goal of this study was to amplify the full length of human heparanase gene and construct a eukaryotic vector so as to facilitate the further study.Methods We extracted total RNA from mucus ovarian cancer tissue and then performed reverse transcription polymerase chain reaction to amplify the full length of heparanase gene.The amplicon was then cloned into the vector of pcDNA3.1.The result was confirned by enzyme digestion and sequencing.Results We successfully amplified the full length of human heparanase gene and cloned it into the vector of pcDNA3.1.The homology was 99.9%confirmed by sequencing.Conclusion We successfully amplified the full length of heparanase and cloned it into eukaryotic vector.Mammalian:heparanase ovarian cancer eukaryotic vectorAn in vitro study on heparanase mediated invasive ability of epithelial cancerObjective:To study heparanase on ovarian function in cancer cells,and analysis its affection on ovarian cancer invasive and metastasis,and pilot its role in early diagnosis, treatment targeting treatment.Method:We successfully constructed HPSE gene expression vector,and transfered it into ovarian cancer cell line A2780,After G418 selection then screening by RT-PCR and western-blot to confirm a stable transfer of the cell lines HPSE/A2780.Then proceed to test the growth of cells,cell cycle,the capacity of adhesion, cell invasion and metastasis experiment.Results:after transfer and screening,we succefully stabilize the protein expression HPSE in ovarian cancer cell line.FCM test showed that A2780-HPSE is in a proliferation of state(S + G2 + M)for 46.5%,G1 for 53.5%, corresponding to the control group,54.5%and 45.6%,respectively.HPSE group and the control group the growth curve almost coincidence.The difference of clone forming unit of the two groups was not statistically significant.A2780 cell adhesion before and after the transfer rate was 0.5183±0.0796 and 0.7283±0.08931,There was significant(p = 0.002). Transwell small room found that gene transfer HPSE after the invasion of A2780 cells markedly improved,compared with the control group were significant(p = 0.003).And its migration were not affected(p = 0.618).Conclusion:cell functions test showed that HPSE can enhance the invasive ability of A2780 ovarian cancer cell,reducing its adhesion ability of the proliferation and migration were not affected.Mammalian:heparanase transfer ovarian cancer cell functions Objective:We constructed the expression vector of shRNA and transfer it into ovarian cancer cell lines,then test its interfere rate on HPSE gene function.We also screened some stable transfer cells and did series gene function test.Method:We designed several shRNA vector to inhibit various HPSE mRNA domains and lipofect them into SKOV3,in which HPSE is highly expression.We use real-time PCR to test the interfere rate and we test the antibiotics resistance of the highest interfere group.And we got the stable cell line and did western-blot to test the inhibition of HPSE protein.Results:fluorescence quantitative PCR to test different shRNA interference vector interference effects show that,pGPU6/GFP / Neo-HPSEo1222 goup reative copy number is 0.936±0.417,having statistically significance compared with the other three groups,inhibition rate is 48.63%.The group was stable interfere and western-blot test the expression of HPSE decreased significantly.The cell growth curve showed that cell doubling time extended.Interference group and control group colony-forming rate was 0.0433±0.0451 and 0.0397±0.00687,p = 0.059 and was not statistical significant.In the Invasion experiment,the interference groups adherence value was 0.5697±0.106,the control group was 0.8097±0.333,interference and control group was not significant(p = 0.233).Cell adhesion ability test showed that interference group was 0.7533±0.07685,the control group was 0.5833±0.11994,having statistically significant(p = 0.015). Invasion of interference group was 0.69±0.085,the control group was 1.091±0.277,two groups showed statistically significant(p = 0.015).Conclusion:we successfully constructed shRNA interference HPSE gene vector,effectively curbed HPSE gene activity,reduced the invasion and metastasis ability of ovarian cancer cell.Mammalian:heparanase RNAi shRNA interference vector cell functions Objective:Using the second and thkd generation lentivkal vector to construct HPSE expression lentivirus vector and shRNA interference lentivirus vector,exploring a more efficient and reliable method than traditional method of gene transfer,laying a good foundation for further research of targeted therapy.Method:First of all we amplified HPSE full-length sequence,and cloned it into lentiviral vector pWPI and them sequencing,and used BLAST to search the similarity.We transferred reconstruction lentivirus plasmid into 293 T cells,after 48 hours extracted mRNA,RT-PCR test HPSE gene expression.Second,we selected the best interference siRNA,and design shRNA,annealing to a double-stranded and cloned into lentiviral vector pSico then sent to sequencing.Results:BLAST searched the reconstruction of lentivirus vector HPSE-pWPI sequencing results,we found that homology of the vector sequence and HPSE gene is 99%.293 T cell line transfected by HPSE gene can expression HPSE protein.The recombinant of the slow-interference vector pSico / HPSE sequence and the design of shRNA sequence is exactly the same.Conclusion:we successfully constructed the HPSE lentivirus system and the lentivirus interference system laying the foundation for further study of HPSE targeted therapy.Mammalian:heparanase lentiviralvector transfer siRNA更多还原