受强力霉素调节的自杀基因表达载体系统的构建和在乳腺癌细胞株(MCF-7)的表达

自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一,以递转录病毒载体-pRevTRE为基础进行载体构建,首先使用PCR技术对单纯疱疹病毒胸苷激酶基因（HSVtk)进行扩增,将HSVtk基因插入到pRe-vTRE,形成重组载体pRevTRE/HSVtk,用磷酸钙共沉淀法,经过两轮转染,分别将pRevTRE/HSVtk和pRevTet-On质粒导入乳腺癌细胞株（MCF-7）经过潮霉素B（HygromycinB)和G418筛选,建立了一株稳定的受四环素衍生物-强力霉素（Doxycycline,Dox)调控,表达HSVtk基因产物的人乳腺癌细胞株MCF/TRE/tk/Tet-On,HSVtk基因表达产物可以将无毒性的药物前体Ganciclovir(GCV)转变成一种有毒的代谢产物,从而杀死乳腺癌细胞株（MCF-7）,达到基因治疗的目的。     

β-榄香烯乳剂逆转多药耐药细胞株MCF-7/ADM对阿霉素耐药性研究

目的：测定中药β-榄香烯乳剂对MCF-7/ADM细胞的无毒剂量,并检测此无毒剂量是否有逆转MCF-7/ADM细胞对化疗药物阿霉素（ADM）的多药耐药（multidrug resistance,MDR）性。方法：采用四甲基偶氮唑蓝（MTT）法测定药物的细胞毒性及耐药细胞逆转倍数;荧光分光光度法测定细胞内药物浓度。结果：无毒剂量的β-榄香烯乳剂(6μg/ml）能显著降低化疗药物ADM对乳腺癌耐药细胞株MCF-7/ADM细胞的IC50,明显增加耐药细胞内药物浓度。结论：初步研究表明β-榄香烯乳剂具有逆转MCF-7/ADM细胞MDR的作用。     

MDM2反义寡核苷酸联合紫杉醇对乳腺癌MCF-7细胞株的作用

目的:探讨靶向MDM2反义寡核苷酸(ASON)联合紫杉醇对乳腺癌MCF-7细胞株的影响。方法:合成一段与MDM2 mRNA特异性结合的反义寡核苷酸和与反义寡核苷酸有4个碱基不同的的错义寡核苷酸(MON),脂质体2000介导不同浓度的MDM2ASON转染MCF-7乳腺癌细胞系,转染的乳腺癌细胞通过1μmol/L紫杉醇药物处理后,采用RT-PCR和Western Blot方法检测MDM2 ASON联合紫杉醇的协同作用及对乳腺癌MCF-7细胞株的抑制效率,MTT观察给药后MCF-7细胞的增殖能力和药物敏感性。结果:MDM2反义寡核苷酸联合紫杉醇明显下调MDM2 mRNA及MDM2蛋白表达水平,抑制MCF-7细胞的生长,随着MDM2 ASON浓度的增加,MDM2表达越来越低,协同作用越来越强,呈剂量依赖关系,A500联合紫杉醇的协同作用最明显,MTT显示紫杉醇处理的转染MCF-7细胞增殖抑制率明显增高,A500抑制增殖作用最明显,抑制率达(13.0±0.84)%。结论:不同浓度MDM2 ASON转染后的乳腺癌MCF-7细胞,等浓度紫杉醇处理后,乳腺癌MCF-7细胞MDM2表达明显降低,细胞凋亡增加,,MDM2 ASON联合紫杉醇对MCF-7细胞有协同作用,提高了乳腺癌MCF-7细胞对紫杉醇的药物敏感性。     

KAI1基因转染人乳腺癌细胞系的建立及初步研究

目的:通过将外源性KAI1基因转染入高转移性人乳腺癌细胞株,为乳腺癌基因治疗的实验室研究提供靶细胞,并初步探讨该基因对乳腺癌细胞增殖能力及细胞周期的影响。方法:采用脂质体法将pCMV-KAI1质粒转染入低表达KAIl基因的人乳腺癌细胞株MDA-MB-231中,经G418筛选后获得抗性克隆,利用RT-PCR、Western blot分析目的基因及其蛋白的表达情况,并利用MTT法和平板克隆形成实验初步探讨该基因对乳腺癌细胞体外增殖能力的影响,流式细胞术检测细胞生长周期的变化。结果:稳定转染KAI1基因的细胞株中有外源性目的基因和相应蛋白的高表达;MTT法示细胞增殖力下降,转染KAI1基因的集落形成率(25.33 2.36)％较转染前(43．17 2．75)％明显降低(P<0．05),流式细胞术显示转染KAI1基因后G1／G0期细胞数量由未转染前的(36．78 0．61)％升高至(64 7．56)％,M／G2期细胞数量则由(17．88 0．76)％降至(7．63 0．60)％,差异有显著性。结论:通过脂质体转染法获得了高表达KAI1基因及其蛋白的人乳腺癌细胞株,并发现该细胞株的体外增殖能力明显下降,这可能是KAI1基因通过调节细胞周期来实现的。     

GALNT14对人乳腺癌MCF-7细胞迁移的影响

构建真核表达载体pcDNA 3.1-Flag-T14,重组质粒经酶切分析及测序鉴定后,利用脂质体将重组质粒转染人乳腺癌细胞系MCF-7细胞,经G418筛选并建立稳定转染GALNT14细胞株.应用半定量RT-PCR、Western blot检测稳定细胞株GALNT14 mRNA及蛋白表达水平,细胞划痕修复及穿膜试验检测GALNT14基因对MCF-7迁移能力的影响,同时RT-PCR检测GALNT14对MMP-2,MMP-9,TGF-β1及VEGF等肿瘤浸润转移相关因子表达的影响.结果显示成功构建了真核重组表达载体pcDNA 3.1-Flag-T14,经RT-PCR和Western blot检测显示成功获得了稳定表达GALNT14的MCF-7细胞株;GALNT14能够提高MCF-7细胞株的迁移能力,且能增加侵袭转移相关因子MMP-2,MMP-9,TGF-β1及VEGF的表达.结论:GALNT14可明显促进MCF-7细胞的迁移,可能在肿瘤侵袭转移中起重要作用.     

人重组核苷二磷酸激酶α亚基对MCF-7/S的影响

软琼脂集落形成实验检测rhNDPK -α对乳腺癌细胞株MCF - 7 S集落形成率的影响 ,结果表明 ,MCF - 7 S集落形成率随rhNDPK -α浓度增高下降 ,提示rhNDPK -α可能具有抑制乳腺癌细胞株MCF - 7 S转移作用 ;MTT法检测rhNDPK -α分别联用丝裂霉素C、5 -氟尿嘧啶和千金藤碱对MCF - 7 S的增殖影响 ,结果显示 ,rhNDPK -α分别联用三种化疗物与这三种化疗药物单独作用于MCF - 7 S无明显差异 (P >0 0 5 ) ,提示rhNDPK -α对丝裂霉素C、5 -氟尿嘧啶和千金藤碱没有增敏作用。     

睾丸特异蛋白GGN1稳定表达细胞株的建立

GGN1是1种功能未知的睾丸特异表达蛋白,它是睾丸特异性基因Ggn的表达产物。Ggn基因的原初转录产物有多种剪接方式,并最终翻译产生3种蛋白,GGN1就是其中的1种。研究发现Ggn的表达与精子的生成过程紧密相关。GGN1还可以与多种蛋白相互作用,其中包括FANCL,GGNBP1,GGNBP2和OAZ3。为了能够在体外更方便的研究Ggn及其相关基因的功能,通过脂质体转染和G418加压法筛选获得了能稳定表达pEGFP-N2／GGN1的CHO—K1细胞株。     

槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

沉默Bmi-1基因表达稳定细胞株的建立及其生物学特性的研究

B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virus insertionsite 1,Bmi-1)基因是多梳基因家族成员,参与细胞增殖调控.研究发现Bmi-1基因可能参与肿瘤的形成,可能成为肿瘤潜在的治疗靶点.用RNA干扰(RNA interference,RNAi)沉默Bmi-1基因表达观察其对乳腺癌细胞株MCF-7侵袭和转移等生物学特性的影响,以探讨Bmi-1在乳腺癌发生发展中的作用.PT67细胞包装质粒后产生的逆转录病毒感染MCF-7细胞,嘌呤霉素筛选建立稳定细胞株,稳定抑制Bmi-1的细胞株命名为MCF-7/Bmi-1si.通过RT-PCR和Western blot分别从mRNA和蛋白水平检测Bmi-1的表达量;平板克隆形成实验检测细胞克隆形成能力;Transwell侵袭小室模型检测细胞体外侵袭和转移能力.MCF-7/Bmi-1si组与MCF-7和MCF-7/GFPsi组相比,Bmi-1 mRNA和蛋白表达量明显减少,克隆形成数及形成率也明显减少(P<0.05).侵袭和转移实验表明:与MCF-7和MCF-7/GFPsi组相比,MCF-7/Bmi-1si组细胞在Transwell侵袭小室中24 h穿膜细胞数明显减少(P<0.05).结果表明沉默Bmi-1基因表达稳定细胞株构建成功,Bmi-1基因表达的沉默能显著降低MCF-7细胞的体外增殖及侵袭转移能力.     

稳定表达外源性p16基因肺癌A549细胞株的建立及鉴定

为构建稳定表达外源性抑癌基因p16的肺癌A549细胞株,用脂质体介导的基因转染方法,借助真核质粒表达载体（pcDNA3)。将抑癌基因p16转移入此基因缺失的人肺癌细胞株A549细胞中,经G418筛选,获得稳定表达的细胞克隆,用逆转录聚合酶链反应（RT-PCR）及免疫组织化学鉴定p16基因的表达,同时对克隆细胞分泌蛋白进行活性检测。结果显示转染p16基因的A549细胞中可以检测到p16mRNA及蛋白的表达,说明建立的p16真核表达载体能在肺肿瘤细胞中分泌表达蛋白,表达P16抑癌蛋白的A549细胞株的建立有助于研究抑癌基因p16在肺癌发生中的作用。     

Methionine cytotoxicity in the human breast cancer cell line MCF-7

Summary Among the first nutrients to be linked to cancer were methyl group containing nutrients including methionine. Methionine and its metabolic derivatives are essential components in several indispensable biological reactions including protein synthesis, polyamine synthesis, and many transmethylation reactions. The purpose of this study was to determine the extent to which methionine excess affects the proliferation and gene expression of the human breast cancer cell line MCF-7. Cells were first grown in control medium; the medium was then replaced with either control or methionine-supplemented treatment media. We found that 5 and 10 g/L methionine significantly suppressed cell growth on day 1, and no further growth was detected after 3 d of treatment. Cell, proliferation in the methionine treated group was significantly lower than that of the control group. Northern analysis revealed that expression of p53 in methionine-treated MCF-7 cells was approximately 70% lower than that of control cells. p53 is a key cell cycle regulatory, protein that has been implicated in tumorigenesis and cancer progression. Alteration of the p53 tumor suppressor gene is the most common genetic change found in a wide variety of malignancies, including cancer. This study shows that excess methionine (5 g/L) inhibited proliferation of MCF-7 breast cancer cells, and down regulation of p53 is correlated with this inhibition. These findings may aid in the development of nutritional strategies for breast cancer therapy.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

川芎嗪逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性

目的 探讨川芎嗪（tetramethylpyrazine,TMP）逆转人乳腺癌MCF-7/ADM细胞对阿霉素（ADM）的耐药性.方法 MTT法测定细胞的药敏性,荧光分光光度法检测细胞内阿霉素浓度的变化,流式细胞术检测耐药细胞凋亡百分率的变化.结果 非细胞毒性剂量（320 mg/L）及低毒剂量（1250 mg/L）川芎嗪均能显著降低MCF-7/ADM的IC50（P＜0.01）,逆转倍数分别为2.13倍和2.82倍;均能显著增加耐药细胞内ADM的浓度（P＜0.01）.320 mg/L川芎嗪能显著增加耐药细胞的凋亡百分率（P＜0.01）.结论 川芎嗪具有部分逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性,其逆转机制与增加细胞内ADM浓度有关.     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

eIF-4E腺病毒载体构建及对乳腺癌MCF-7细胞转移能力的影响

摘要 目的：构建乳腺癌真核细胞起始因子一4E(eukaryotic initiation factor 4E,eIF-4E)基因重组腺病毒载体,并观察其对乳腺癌细胞MCF-7转移能力的影响。方法：应用基因重组技术将eIF-4E基因构建于腺病毒载体pAD-X,转染293包装细胞得到高滴度重组腺病毒,并用real-time PCR进行eIF-4E基因表达的验证。将重组腺病毒pAD-eIF-4E感染MCF-7细胞后,应用transwell小室法观察其对细胞侵袭和运动能力的影响。结果：酶切结果与预期相符,real-time PCR可检测到转染后MCF-7细胞有eIF-4E基因表达。且病毒转染后transwell小室可见,MCF-7细胞的侵袭和运动能力均受到显著的抑制（均为p<0.01）。结论：重组eIF-4E腺病毒载体正确构建,其对乳腺癌细胞MCF-7的侵袭和运动都有抑制作用。     

PC-１基因表达增强C4-2B前列腺癌细胞生存

建立稳定表达外源PC-１基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC- １基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-１基因的真核表达载体pcDNA3.1PC-１稳定转染C4-2B细胞,Western 印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-１基因表达. MTT和软琼脂集落形成能力等一系列方法,研究PC-１基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化. 结果表明,PC-１基因的高表达能够诱导雄激素受体（AR）调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX31、Jagged1、EphA3、SGEF和 NOTCH3等表达发生变化. 实验结果初步证明,PC-１基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-１基因表达可调控一些重要信号通路.对PC-１基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.     

Role of the insulin-like growth factor system on an estrogen-dependent cancer phenotype in the MCF-7 human breast cancer cell line

We previously established that exposure of the estrogen receptor (ER) positive MCF-7 human breast cancer cell line to 17-β-estradiol (E₂) results in the post-confluent development of multilayered cellular aggregates (foci) which is consistent with the in vivo cancer phenotype of uncontrolled cellular proliferation. In this investigation, the interaction between the insulin-like growth factor receptor (IGF-IR) and ER-signaling systems in regard to post-confluent focus development was studied. We demonstrated that focus development requires the presence of E₂ and insulin-like growth factor I (IGF-I) or insulin-like growth factor II (IGF-II), as well as intact ER and IGF-IR.

Focus development in MCF-7 cultures, which occurs only after formation of a confluent monolayer, coincides with E₂ regulation of key members of the IGF-signaling system such as IGF-IR, IGF-II, insulin receptor substrate 1 (IRS-1), and insulin-like growth factor binding protein 3 (IGFBP-3), as demonstrated by real-time polymerase chain reaction (PCR). To establish the relevancy of an intact IGF-signaling system for foci formation, we generated stable clones from MCF-7 with IGF-IR suppressed by siRNA. Results from these studies implicate signaling through the IGF-IR to be an integral requirement for E₂-dependent post-confluent proliferation and focus formation. In summary, these studies establish the interactive roles of IGFs and E₂ in the post-confluent development of foci, and will allow subsequent identification of targets for therapeutic intervention in the control and treatment of estrogen-dependent breast cancer.     

外源高表达PC-1蛋白N端43个氨基酸可加速人前列腺癌细胞系C4-2的生长

研究PC-1蛋白N端43个氨基酸表达对人前列腺癌细胞C4—2生长的影响。用DNA重组技术将含PC—1蛋白N端43个氨基酸的DNA序列正向克隆到真核表达载体pIRES2-EGFP中,采用脂质体法将重组质粒稳定转染进C4—2细胞中,RT—PCR分析外源序列的转录情况,固相ELISA法测定PC—1蛋白N端43个氨基酸的表达,MTT实验分析细胞的生长速度。结果获得了稳定转染PC—J基因N端43个氨基酸的前列腺癌细胞株,在该细胞株中外源PC—1蛋白N端43个氨基酸得到高表达,细胞生长速度较对照细胞加快了38％。结果表明外源PC—I基因N端43个氨基酸高表达可提高人前列腺癌细胞C4—2的生长速度,推论PC—J基因高表达可能在人前列腺癌的发展中起一定的促进作用。     

Fatty acid modulation of MCF-7 human breast cancer cell proliferation,apoptosis and differentiation

Epidemiological studies suggest that dietary polyunsaturated fatty acids (PUFA) may influence breast cancer progression and prognosis. In order to study potential mechanisms of action of fatty acid modulation of tumor growth, we studied, in vitro, the influence of n-3 and n-6 fatty acids on proliferation, cell cycle, differentiation and apoptosis of MCF-7 human breast cancer cells. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited the MCF-7 cell growth by 30% and 54%, respectively, while linoleic acid (LA) had no effect and arachidonic acid (AA) inhibited the cell growth by 30% (p < 0.05). The addition of vitamin E (10uM) to cancer cells slightly restored cell growth. The incubation of MCF-7 cells with PUFAs did not alter the cell cycle parameters or induce cell apoptosis. However, the growth inhibitory effects of EPA, DHA and AA were associated with cell differentiation as indicated by positive Oil-Red-O staining of the cells. Lipid droplet accumulation was increased by 65%, 30% and 15% in the presence of DHA, EPA and AA, respectively; (p < 0.05). These observations suggest that fatty acids may influence cellular processes at a molecular level, capable of modulating breast cancer cell growth.