【作者】 何碧秀；

【导师】 胡成平； 陈琼； 杨红忠；

【作者基本信息】 中南大学 ， 呼吸内科， 2007， 博士

【摘要】 目前，肺癌已居人类肿瘤致死的第一名，全世界泛围内，科学家们一直积极采用各种方法来治疗肺癌，但治疗效果不能令人满意，肺癌总的治愈率仍不足14％。随着分子生物学的不断发展，人类对肺癌的发生发展转移的分子机制有了更深的认识。基因治疗已成为一种极具前景的新方法。反义脱氧寡核苷酸(antisense digodeoxynucleotides；ASODN)是一段与mRNA或DNA特异性结合，并阻断其表达的人工合成分子。ASODN可封闭或抑制肿瘤细胞关键编码基因，从而特异性抑制肿瘤细胞的增殖。反义药物是以反义核酸技术为基础开发的，以治疗为目的，安全有效的新型药物。近年来，随着对反义寡核苷酸及其结构衍生物如硫代磷酸寡聚脱氧核苷酸反义机理的阐明，大量以病毒，癌基因，细胞活性因子及其它疾病相关蛋白为靶点的反义药物相继进入临床试验阶段，使反义药物的研究再次进入蓬勃发展时期。基质金属蛋白酶家族(matrix metalloproteinases，MMPs)是一组重要的含锌离子的细胞外基质(extracellular matrix，ECM)降解酶。目前已鉴定出约20多种MMPs，基质金属蛋白酶-9是其中重要的一组酶，可在许多肿瘤组织中表达。还在肿瘤的凋亡，侵袭和转移中起着关键性作用。本研究采用人肺腺癌A549细胞株，运用脂质体介导的MMP-9ASODN转染该细胞，通过MTT法检测MMP-9ASODN转染对A549细胞生长的影响；RT-PCR观察MMP-9mRNA蛋白的表达；Western blot检测MMP-9蛋白的改变；流式细胞术检测MMP-9ASODN对A549细胞增殖和凋亡比率；MTT比色法和划痕迁移试验观察MMP-9ASODN对A549细胞体外粘附以及迁移的能力；最后观察MMP-9ASODN的体内抑瘤效果，进而为肺癌的基因治疗提供一种新方法。肺癌靶向治疗药物的研究已进入开发阶段，目前有众多的反义药物已进入临床实验阶段。本研究旨在证明反义寡核苷酸MMP-9转染A549细胞后，可抑制肿瘤细胞生长，并诱导其凋亡，从而为肺癌的反义基因治疗提供了可靠的实验依据。方法1．脂质体介导的寡核苷酸转染A549细胞人肺腺癌A549细胞在1640完全培养基中常规培养，进入对数生长期后接种于96孔板，分四组进行实验，即MMP-9ASODN，MMP-9SODN，脂质体组和空白对照组，转染后24h以荧光倒置显微镜观察，随机选取5个高倍镜视野，计数全部细胞数和发绿色荧光细胞数，计算转染率。2．MTT法检测MMP-9ASODN转染对A549细胞生长的影响ASODN转染组加设不同浓度组即200nmol／L，400nmol／L，600nmol／L和800nmol／L的ASODN转染。每组设3个复孔，分别于转染0h，4h，8h，12h，24h，48h，60h后行MTT实验。酶标仪上测定波长为570nm，计算A549细胞的存活率。3．流式细胞术检测细胞增殖和凋亡比率收集经处理后的各组细胞于离心管内，离心，洗涤，固定，染色，置流式细胞仪上，在激发波长为488nm处进行检测，计算细胞凋亡比率。4．RT-PCR法检测MMP-9mRNA的表达转染后收集各组细胞，进行RNA提取，定量后以其为模板逆转录合成cDNA链，然后用引物进行PCR扩增，产物用琼脂糖凝胶电泳进行检测。5．Western blot检测MMP-9蛋白的改变收集转染24h细胞提取细胞中总蛋白，测定出样品的蛋白质含量，然后将蛋白质在不连续聚丙烯酰胺凝胶电泳，染色，转膜观察结果。6．MMP-9ASODN转染对A549细胞体外粘附及体外迁移的影响检测包被及水化基底膜，接种及培养细胞，然后MTT比色法检测，计算细胞粘附率。同样包被及水化基底膜，制备培养单层细胞，用人工划痕法，在相差显微镜下随机选择5个100×视野内计算迁移到划痕空隙中的细胞总数。7．裸鼠模型抑瘤率观察构建移植瘤裸鼠模型后，MMP-9ASODN直接移植瘤内注射，观察其抗移植瘤生长效应，包括一般状况，局部实体瘤生长抑制率的测定以及病理组织学检测。结果1．MM-9ASODN组，MMP-9SODN组和Lip组转染效率分别为(65.85±9.61)％，(56.38±9.82)％，(53.62±6.08)％。2．MMP-9 ASODN对A549细胞存活率抑制呈浓度和时间依赖性(反义寡核苷酸浓度为600nmol／L作用48 h时抑制作用最明显)。3．600nmol／L MMP-9ASODN作用48h，经RT-PCR扩后，MMP-9mRNA的相对表达量为0.573±0.071，明显低于其他三组0.924±0.044，0.863±0.054，0.765±0.072。4．600nmol／L MMP-9ASODN作用48h，经Western检测后，MMP-9蛋白表达水平0.53±0.068，明显低于其他三组0.76±0.052，0.79±0.075，0.81±0.126。5．MMP-9ASODN转染A549细胞后，G1期细胞数明显增多，S期细胞数相对减少，G2期细胞数则无明显变化，提示MMP-9ASODN可能延缓肿瘤细胞由G1期向S期的过渡。MMP-9ASODN转染A549细胞后，A549细胞凋亡百分比明显高于其他三组。6．粘附实验表明MMP-9ASODN转染A549细胞后显著降低其粘附力，粘附率为(15.21±0.81)％，低于其他三组(38.92±1.23)％，(36.55±2.32)％及(32.61±1.41)％，差异有显著性。(p＜0.01)。7．划痕法迁延实验表明MMP-9ASODN转染A549细胞后其迁延力显著降低，迁延细胞数为74.09±4.48个，显著低于其他三组124.18±9.77个，120.14±8.50个及112.82±9.87个(p＜0.05)。8．MMP-9ASODN组肿瘤生长指数为3.20±0.14，明显低于其它三组5.93±0.20，5.87±0.02及6.40±0.33(P＜0.01)。MMP-9ASODN组的肿瘤重量为1.25±0.03，明显低于其它三组2.15±0.07，2.31±0.04及2.43±0.04(P＜0.01)，MMP-9ASODN组的抑瘤率为(33.41±1.18)％，而且心肝肾组织观察，结构均基本正常，未见有明显的组织损伤改变。结论1．MMP-9ASODN能够有效地抑制肺癌A549细胞的体外增殖，作用方式呈时间及浓度依赖性。2．MMP-9ASODN能够下调MMP-9mRNA和MMP-9蛋白的表达。3．MMP-9ASODN通过影响肺癌A549细胞增殖周期的分布及促进A549细胞凋亡，从而抑制A549细胞增殖。4．MMP-9ASODN在体外能够降低A549细胞粘附及迁移能力5．MMP-9ASODN瘤体注射可以显著抑制裸鼠移植瘤的生长，且安全，无不良反应。更多还原

【Abstract】 Recently, the mortality of lung cancer is the first in human cancers. All of the world, the scientists have been trying kinds of methods to treat lung cancer, but the effect is not satisfactory. The total cure rate is less than 14%. With the development of molecular biology, people have known more about the molecular mechanisms of tumorigenesis, progression and metastasis. Gene therapy becomes a new prospective therapy. ASODN is a paragraph of artificial synthesis molecules which specifically bind to mRNA or DNA and suppress their expression. ASODN may specifically suppress the proliferation of tumor cells by blocking or suppressing the key coding gene in tumor cell.Antisense drug is a new type of safe effective drug which based on the antisensenucleic acids techniques and is on the purpose of treatment. Recent years,with the elucidation of antisense mechanism of the antisense oligonucleotides and its constructive ramifications like phosphorothioic acid oligodeoxynucleotide, many antisense drugs which target for the proteins of viruses , oncogene, cytoactive factors and other disease association proteins successively enter clinical trial, which make the research on the antisense drugs reenter rising development period.MMPs is a group of important ECM catabolic enzymes containing zinc ion. There have been more than 20 kinds of MMPs identified. MMP-9 is a important group within the MMPs and expressed in many tumor tissues.It has the key effects on the apoptosis, invasion and metastasis of tumor.Using human lung adenocarcinoma A549 line which has been transfected by MMP-9ASODN with liposome, this study is to detect the effect of MMP-9ASODN transfection on the A549 cell growth by MTT, observe the expression of MMP-9mRNA proteins by RT-PCR, detect the changes of MMP-9 proteins by Westem blot, detect the effect of MMP-9ASODN on the rate of cell proliferation/apoptosis;observe the effect of MMP-9ASODN on the adhesive and migratory ability of cell in vitro, observe the inhibition effect of MMP-9ASODN on the tumor in vivo, which supply a new method for the gene therapy for the lung cancer.The research on the targeted therapy drugs for the lung cancer have entered the exploitated phase. Resently, there have been many types of antisense drugs which have enter clinic trial period. This study demonstrated that antisense oligonucleotides MMP-9 may inhibit the tumor cell growth and induce apoptosis by transinfecting the A549 cell, which supply reliable experimental evidence for the antisense gene therapy for the lung cancer.Methods1. Liposome mediate the oligonucleotide transfeetion of A549 cell.Human lung adenocarcinoma A549 cell is conventional cultured in 1640 complete medium, and logarithmic-phase growth cells were inoculated to 96 well plates. The cells were divided into 4 groups: MMP-9ASODN, MMP-9SODN, liposome and control. And they were observed by fluorescence microscope after transfection 24hrs, select 5 high power fields randomly, count all cell population and green fluorescence cell population, and calculate transfection efficiency. 2. Detect the effect of MMP-9ASODN transfection on the A549 cell growth by MTTASODN transfection group is divided into different concentration groups: 200nmol/L, 400nmol/L, 600nmol/L and 800nmol/L. There are 3 holes in each group. Detect the effect 0h,4h,8h,12h,24h,48h,60h after transinfection by MMT, respectively.the wavelength detected by ELIASA is 570nm, calculate the survival rate ofA549 cell.3. Detect the cell proliferation/apoptosis rate by flow cytometryCollect the cells treated in every group into the centrifuge tube, centrifuge, wash, fix, stain, be laid on the flow cytometer, detect in the excitation wavelength of 488nm, and calculate the cell apoptosis rate4. Detect the expression of MMP-9mRNA by RT-PCR.Collect the cells in each group after transinfection, extract the RNA, synthetize the cDNA by reverse transcription with the template after quantitation, then amplification by PCR with primer, detect the products by agarose gel electrophoresis.5. Detect the changes of MMP-9 protein by Western blotCollect the cells after 24h transinfection and extract the total proteins within the cells, detect the protein content in the sample, then detect the proteins by discontinuation PGE, stain, trarsmembrane and observe the result.6. Dectet the effect of MMP-9ASODN transinfection on the adhesive and migratory ability of A549 cell in vitroCoat and hydrate the basal membrane, inoculate and culture cell, then detect by MMT chromatometry, calculate the adhesive rate of cell. Likewise, coat and hydrate the basal membrane, prepare culture cell monolayer, select 5 field of vision of 100×randomly in contrast phase microscope by artificial scratch method and calculate the total cellular score migrating into the scratch space. 7. Observe the inhibition rate of tumor by athymic mouse modelConstuct the transplantation tumor athymic mouse model, then inject the MMP-9ASODN into the transplantation tumor, observe the growth effect of anti-transplantation tumor including general state, the detection of growth inhibiting rate of regional solid tumor and pathohistology.Results1. The transinfection rate of MMP-9ASDN, MMP-9SODN and Lip group is respectively (65.85±9.61) %, (56.38±9.82) %, (53.62±6.08) %。2. Among certain range, the inhibiton of A549 cell survival rate by MMP-9ASODN is concentration and time dependent (inhibitory effect is most obvious when the antisense oligonucleotides concentration is 600nmol/L and the action time is 48h.)3. When the antisense oligonucleotides concentration is 600nmol/L and the action time is 48h, by RT-PCR amplification, the relative expression level of MMP-9mRNA is 0.573±0.071 which is obviously less than other 3 groups(0.924±0.044, 0.863±0.054, 0.765±0.072)4. When the antisense oligonucleotides concentration is 600nmol/L and the action time is 48h, by Westem detection, the expression level of MMP-9 protein is 0.53±0.068 which is obviously less than other 3 groups(0.76±0.052, 0.79±0.075, 0.81±0.126).5. After the A549 cell have been transinfected by MMP-9ASODN, the cell scores in G1 phase obviously increase, the cell scores in S phase obviously decrease, there is no obvious changes of cell scores in G2 phase., which suggested that MMP-9ASODN may delay the G1-S transition of tumor cell. after the A549 cell have been transinfected by MMP-9ASODN, the apoptosis percentage of A549 cell is much more than other 3 groups. 6. Adhesive experiment suggested that MMP-9ASODN transinfection may decrease the adhesive abilitiy of A549 cell, the adhesive rate is (15.21±0.81)% which is less than other 3 groups (38.92±1.23) %, (36.55±2.32) % and (32.61±1.41 ) %,the difference have significance(p＜0.01).Protraction experiment by scratch method7. Suggested that MMP-9ASODN transinfection may decrease the migratory ability of A549 cell, the migratory cell scores is 74.09±4.48 which is less than other 3 groups124.18±9.77, 120.14±8.50 and 112.82±9.87(p＜0.05).8. Tumor growth index in MMP-9ASODN group is 3.20±0.14 which is less than other 3 groups 5.93±0.20, 5.87±0.02 and 6.40±0.33 (P＜0.01).The tumor weight in MMP-9ASODN group is 1.25±0.03 which is less than other 3 groups 2.15±0.07, 2.31±0.04 and 2.43±0.04 (P＜0.01).The inhibition rate of tumor in MMP-9ASODN group is (33.41±1.18)% ,and the heart , liver and kidney’s tissue and construct are normal on the whole, there are no obvious tissue damage changes.Conclusions1. MMP-9ASODN may effectively inhibit the lung cancer A549 cell proliferation,the mode of action is concentration and time dependent.2. MMP-9ASODN may down regulate the expression of MMP-9mRNA and MMP-9 proteins33. MMP-9ASODN may inhibit the A549 cell proliferation by influencing lung cancer A549 cell generation cycle distribution and promoting the A549 cell apoptosis.4. MMP-9ASODN may depress the adhesive and migratory ability of A549 cell.5. MMP-9ASODN may obviously inhibit athymic mouse transplantation tumor growth, which is save and no adverse effect.更多还原