【作者】 杜烨；

【导师】 范志民；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 背景：TAM作为选择性雌激素受体调节剂（selective estrogen receptormodulators, SERM），在临床上被广泛应用于早晚期乳腺癌的治疗及术后预防。然而另一方面，TAM能增加病人体内VEGF的水平，促进乳腺肿瘤新生血管的形成，因而限制了其功能，改善治疗策略成为一种必然。有研究表明塞来昔布（celecoxib, CXB）与TAM联合应用后，增强了TAM的抗肿瘤能力，并且降低了TAM剂量，显示出联合治疗的前瞻性意义。然而众所周知，CXB能引起胃肠道反应、肝损伤等并发症。利用RNAi技术，能有效沉默细胞中COX-2基因，从而诱导肿瘤细胞凋亡，抑制VEGF基因的表达，同时避免塞来昔布等药物产生的副作用。目的：在体外实验中，观察LV-COX-2和TAM单独及联合应用时对人乳腺癌MCF-7细胞株恶性生物学行为的影响（包括细胞增殖、凋亡、细胞周期分布、转移能力及侵袭性等），并检测对VEGF表达的影响；在体内试验中，构建人乳腺癌MCF-7裸鼠移植瘤模型，评估LV-COX-2与TAM联合治疗对肿瘤生长的抑制效果。通过体内外研究，观察二者协同及增敏作用，为日后临床应用提供理论依据。方法：1.根据RNAi设计原理及基因重组技术，针对COX-2基因的靶点序列设计、合成COX-2shRNA慢病毒载体，将其转染入人乳腺癌MCF-7细胞株，通过RT-PCR和Western blot法检测转染COX-2shRNA后，COX-2基因mRNA及蛋白质表达水平。2.在体外实验中观察LV-COX-2、TAM治疗及二者联合作用时对人乳腺癌MCF-7细胞株生物学行为的影响，包括：MTT法检测细胞的增殖能力；AO/EB双荧光染色法检测细胞凋亡；流式细胞术检测细胞周期分布；Boyden小室及细胞划痕实验检测细胞迁移情况；Transwell法检测细胞的侵袭能力；ELISA及Western blot法检测PEG2、VEGF及VEGFR表达水平。3.建立人乳腺癌MCF-7细胞裸鼠种植瘤模型，当肿瘤体积达到100-200mm3时，对各组荷瘤裸鼠给予不同的治疗手段，21d后取出肿瘤组织，测量瘤体重量和体积，观察LV-COX-2及TAM对裸鼠皮下种植瘤生长的影响。结果：1.成功构建针对COX-2基因的shRNA慢病毒载体，能够有效抑制人乳腺癌MCF-7细胞中COX-2基因mRNA及蛋白质表达。2.体外实验证明，LV-COX-2与TAM治疗均能明显抑制人乳腺癌细胞株MCF-7细胞活力，抑制细胞增殖，诱导细胞凋亡及降低细胞的转移和侵袭力，二者联合作用时效果更为显著。3.裸鼠体内试验证明，LV-COX-2和TAM治疗均能明显抑制MCF-7裸鼠移植瘤的生长，瘤体重量及体积明显减小，二者联合应用时效果更佳，优于任何一个单独治疗组。结论：研究发现，COX-2沉默联合TAM治疗能显著抑制体外人乳腺癌细胞株MCF-7细胞增殖，诱导细胞凋亡，同时亦明显抑制了裸鼠体内肿瘤的生长。此外，LV-COX-2和TAM联合治疗能够有效抑制血管内皮生长因子的表达及新生血管的形成。这些结果表明，LV-COX-2与TAM联合治疗在乳腺癌的抗转移和促凋亡研究中是一个很有前途的手段，二者联合应用具有协同和增敏作用，沉默COX-2基因增强了TAM的抗肿瘤活性。COX-2沉默联合TAM治疗是一个有潜能的治疗方案，尤其对于VEGF高表达的乳腺肿瘤来说。更多还原

【Abstract】 Background：Tamoxifen (TAM), a selective estrogen receptor modulator, is inwide clinical use for the treatment and prevention of breast cancer.However, extended TAM administration for breast cancer inducesincreased VEGF levels in patients, promoting new blood vessel formationand thereby limiting its efficacy and highlighting the need for improvedtherapeutic strategies. A recent study showed the combination ofcelecoxib(CXB) and TAM could achieve better antitumor activity bysuppressing VEGF expression and simultaneously could reduce doses ofTAM. However, it is known that the nonsteroidal antiinflammatorydrugs(NSAIDs), such as CXB, could result in liver injury, which wouldlimit their clinical applications. Cyclooxygenase-2(COX-2) silencing viaa replication-incompetent lentivirus (LV-COX-2) could induce cancerapoptosis, suppress VEGF gene expression and avoid the side effectsinduced by celecoxib.Objective:In this study, the effect of LV-COX-2infection, either alone or incombination with TAM, was analyzed in a breast cell lines forsuppressing VEGF expression and simultaneously reducing doses ofTAM. Cell proliferation, apoptosis, angiogenesis, metastasis, cell cycledistribution, an receptor signaling were determined after LV-COX-2combination with TAM treatment. In addition, tumor growth ability innude mice was detected to define the combination treatment effect in tumorigenesis in vivo. The objective of the present study was to evaluatethe effects of LV-COX-2in combination with TAM on the biologicalbehaviour of human breast cancer cell lines, MCF-7, in vitro and in vivo,and to reveal the underlying molecular mechanisms. These results wouldprovide theoretical base for clinical applications of LV-COX-2combinedwith TAM in future.Methods:1. To inhibit the expression of COX-2, a short hairpin RNA (shRNA)targeting the COX-2transcript was designed. The synthesizedoligonucleotides containing specific target sequence were cloned intopGCSIL-GFP lentivirus vector. The recombinant vector plasmids weretransfected into human breast cancer cell lines, MCF-7. The expression ofCOX-2mRNA and protein were detected by RT-PCR and Western blotrespectively.2. To study the effects of COX-2gene silencing by RNAi and TAMon biological behaviour of human breast cancer MCF-7cells in vitro.MTT assay was used to detect ability of cell proliferation. Cells stainedwith acridine orange(AO) and ethidium bromide(EB) were observedunder fluorescence microscope in order to detect cell apoptosis. Flowcytometry was performed to detect the distribution of cells in thecell-cycle phases. Boyden Chamber and wound-healing assay as well astranswell filter were used to assess the migration and invasion ability ofthe cells. PGE2synthesis was determined by competitive enzyme-linkedimmunosorbent assay (ELISA). In addition, VEGF and VEGFR proteinexpression level were also measured by western blotting.3. Exponentially growing MCF7cells were harvested and atumorigenic dose of2×106cells was injected intraperitoneally into4to5week-old female BALB mice. When tumors reached100-200mm3, mice were divided randomly into five groups.The control group received1%polysorbate resuspended in deionized water. The other four groups weretreated respectively with LV-NC, ATM, LV-COX-2, or ATM plusLV-COX-2intraperitoneally on alternative days for3weeks.The tumorsize was measured using caliper before the treatment injections weregiven and on the7th,14th and21th day of treatment. On21th day, theanimals were euthanized using chloroform, tumor tissue in nude miceswere collected, their volume and weight were measured, and the impactof COX-2silence on the growth of the implanted subcutaneously tumorin nude mice was analysised.Results:1. The recombinant lentiviral carrying a based shRNA againstCOX-2, LV-COX-2, were successfully constructed. LV-COX-2suppressed effectively the mRNA and protein expression of COX-2genein human breast cancer MCF-7cells.2. The experiment results in virto demonstrated that LV-COX-2andTAM inhibited significantly the cell vibility and proliferation of MCF-7cells, induced cell apoptosis, and decreased the ability of the cellmigration and invasion. The effects of LV-COX-2in combination withTAM were higher than LV-COX-2and TAM treatment alone.3. Silencing COX-2gene expression by LV-COX-2and TAMinhibited remarkably tumor growth in nude mices, and the tumor volumeand weight were significantly decreased. The combination withLV-COX-2and TAM got the better the results, compared toLV-COX-2and TAM alone.Conclusion:It is found that LV-COX-2combination with TAM treatment inbreast cancer cell significantly inhibited the proliferation and metastasis, and induced tumor apoptosis in vitro, and tumor growth also wassuppressed in vivo. In addition, we also found that LV-COX-2combination with TAM treatment could inhibit angiogenesis and VEGFexpression. Taken together, our experimental results indicate thatLV-COX-2combination with TAM has promising outcome inanti-metastatic and apoptotic studies, COX-2gene silencing can increasethe anti-tumor activity of TAM. Furthermore, these results showed thatLV-COX-2combination with TAM is a potential drug candidate fortreatment of breast tumors expressing high levels of VEGF.更多还原