靶向HER-2 mRNA 反义硫代寡核苷酸体外抗乳腺癌活性研究

 目的 研究以HER2 mRNA为靶点的反义硫代脱氧寡核苷酸（S-ODNs）HA6722对HER-2过表达乳腺癌细胞株MDA-MB-453体外增殖的抑制作用，及HA6722对肿瘤细胞HER-2表达的影响。方法 选择HER2过表达的MDA-MB-453细胞与HER2低表达的MDA-MB-231细胞，MTT法观察S-ODNs对肿瘤细胞增殖的影响，免疫细胞化学（ICC）与RT-PCR方法研究S-ODNs对细胞HER2蛋白及mRNA表达的影响。结果 HA6722可以剂量依赖方式抑制MDA-MB-453细胞的体外增殖，IC50值（41．8±8．1nmol·L^-1，n=5，mean±s）显著低于对照序列Scramble6722（IC50=489．4±12．1nmol·L^-1，n=5，P〈0．01）。HA6722在蛋白水平与mRNA水平显著抑制MDA-MB-453细胞中HER-2的表达；HA6722对MDA-MB-231细胞的体外增殖无显著影响（IC50=476．7±17．6nmol·L^-1，n=5，P〉0．05）。结论 HA6722可序列特异性地抑制HER-2过表达乳腺癌细胞的体外增殖，其抑制增殖作用与靶细胞HER-2表达下调有关。     

反义硫代寡核苷酸与榄香烯乳剂合用抗乳腺癌活性的体外研究

目的:研究以HER2mRNA为靶点的反义硫代脱氧寡核苷酸(S-ODNs)HA6722单用及与榄香烯乳剂合用时对HER-2过表达乳腺癌细胞株MDA-MB-453体外增殖的抑制作用。方法:选择HER2过表达的MDA-MB-453细胞与HER2低表达的MDA-MB-231细胞,MTT法观察HA6722单用及与榄香烯乳剂合用时对两种肿瘤细胞增殖的影响。结果:HA6722及榄香烯单用均可以剂量依赖方式抑制MDA-MB-453细胞的体外增殖,IC50值分别为41.8±8.1nmol·L-1和79.4±12.1nmol·L-1(n=3,mean±s)。加用低剂量的HA6722可增强榄香烯乳剂对MDA-MB-453细胞的抑制作用,IC50值由合用前的79.4±12.1nmol·L-1降至51.6±8.2nmol·L-1(n=3,P<0.05)。相反,在HER2低表达的MDA-MB-231细胞则没有这种增强作用;在IC50浓度下二者之间的联合指数(CI)为0.72±0.38(n=3)。结论:反义寡核苷酸HA6722与榄香烯乳剂合用对HER-2过表达乳腺癌细胞的体外增殖抑制具有协同作用。     

C-erbB-2反义寡核苷酸抑制乳腺癌细胞增殖

目的观察反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)能否降低人乳腺癌细胞株MCF-7、c-erbB-2基因的表达,以及能否抑制其细胞增殖.方法将c-erbB-2 ASODN及其对照序列(空白对照和错配序列)通过脂质体转染MCF-7细胞,应用Western blot观察c-erbB-2蛋白表达的变化,然后应用MTT观察细胞增殖.结果c-erbB-2 ASODN能特异性下调MCF-7细胞c-erbB-2蛋白,而且能抑制其细胞增殖.结论实验结果认为应用c-erbB-2 ASODN抑制乳腺癌细胞增殖具有可行性.     

bcl-2硫代反义寡核苷酸提高肺癌细胞对Fas介导细胞毒作用的敏感性

Ｆａｓ系统介导的细胞凋亡是细胞免疫反应所致细胞毒作用的重要途径。有研究证明 ,即使小细胞肺癌细胞存在Ｆａｓ蛋白的表达 ,而且肿瘤侵袭淋巴细胞中有相应的ＦａｓＬ ,也不会诱导癌细胞发生大量凋亡[1 ] 。这可能与肺癌细胞中ｂｃｌ 2基因高表达有关 ,但目前研究尚少。我们用ｂｃｌ 2硫代反义寡核苷酸抑制小细胞肺癌细胞系的ｂｃｌ 2基因表达 ,观察激活型Ｆａｓ抗体对其凋亡的诱导作用 ,以探讨ｂｃｌ 2反义寡核苷酸用于肺癌治疗的价值。一、材料与方法1 小细胞肺癌细胞系∶细胞株ＮＣＩ Ｈ6 9,同时表达ｂｃｌ 2蛋白和Ｆａｓ蛋白。2 硫代…     

bcl-2反义寡核苷酸增强5-Fu诱导乳腺癌细胞凋亡的研究

目的　观察转染bcl -2反义寡核苷酸 (ASODN )能否增强 5 -氟脲嘧腚 ( 5 -Fu)诱导乳腺癌细胞株MCF -7凋亡。方法　通过电穿孔转染bcl -2ASODN和对照序列 ,荧光显微镜定量检测转染以后MCF -7凋亡率的变化。结果　和对照序列相比 ,转染bcl -2ASODN显著增高 5 -Fu诱导MCF -7的细胞凋亡率 (P <0 .0 1)。结论　联用bcl -2ASODN和 5 -Fu可能改善乳腺癌的治疗效果。     

bcl-2/bcl-xL反义寡核苷酸对乳腺癌细胞增殖和凋亡的影响

目的　观察靶向bcl 2 /bcl xL基因的反义寡核苷酸 (ASODN )对乳腺癌细胞株MCF 7增殖和凋亡的影响。方法　将bcl 2 /bcl xLASODN及其对照序列通过脂质体转染MCF 7细胞。采用MTT法测定MCF 7细胞增殖 ,荧光显微镜定量检测MCF 7细胞凋亡率。结果　与对照序列相比 ,bcl 2 /bcl xLASODN能抑制MCF 7增殖和诱导其凋亡 (P <0 .0 1)。结论　bcl 2 /bcl xLASODN在乳腺癌治疗方面作为 1种新的化合物值得进一步研究     

bcl-2硫代反义寡核苷酸提高胃癌细胞凋亡的敏感性

OBJECTIVE: To study the effect of bcl-2 anti-sense oligonucleotide on the sensitivity of gastric cancer cells to Fas-mediated apoptosis. METHODS: Gastric cancer cell line MKN45 was transfected with bcl-2 anti-sense oligonucleotide, and expression of bcl-2 was examined by Western blotting. Agonistic Fas antibody was used to induce apoptosis detected by TUNEL staining and flow cytometry. RESULTS: Bcl-2 expression in MKN45 cells transfected with bcl-2 anti-sense oligonucleotide was markedly inhibited. When cultured with antibody apoptosis index of the anti-sense oligonucleotide-treated MKN45 cells was 55.6% +/- 4.7% (n = 5), which was significantly higher than that of the control (8.4% +/- 2.1%, n = 5). CONCLUSION: Expression of bcl-2 in gastric cancer cells may antagonize Fas-mediated apoptosis.     

Aurora A反义寡核苷酸联合紫杉醇对体外肺癌细胞的作用及其机制

目的:探讨在体外培养的肺癌细胞A549中,AuroraA反义寡核苷酸对紫杉醇(PTX)化疗敏感性的影响,并分析其内在机制。方法:用脂质体瞬时转染法介导AuroraA反义硫代磷酸寡核苷酸(antisense oligodeoxyneucle-otides,ASODN)处理肺腺癌A549细胞6h后,给予一定浓度的PTX继续作用24h,用四唑氮蓝法(MTT)观察各组的量效反应,并计算半数抑制浓度IC50的值。Aurora A ASODN转染细胞后24及48h时应用流式细胞仪检测细胞周期分布的变化。结果:Aurora A ASODN作用于A549细胞后,细胞的生长抑制率呈剂量和时间依赖性,其中48h的IC50值约为300nmol/L;在此浓度和时间下,Aurora A反义寡核苷酸可使细胞周期阻滞于G2/M期。Aurora A ASODN转染增加了肺癌细胞A549对PTX的敏感性,ASODN+PTX组的细胞生长抑制率在30h达(70·51±1·77)%,明显高于单用ASODN的(29·98±2·05)%(P=0·000)和PTX的(33·61±1·57)%,P=0·000。结论:Aurora A ASODN增强肺腺癌细胞系A549对紫杉醇的化疗敏感性,这可能与Aurora A ASODN使细胞发生G2/M期阻滞有关。     

STAT3反义寡核苷酸对乳腺癌MCF-7细胞增殖和凋亡的影响

目的探讨STAT3反义寡核苷酸的抗肿瘤机制,为乳腺癌的基因治疗提供新的实验依据。方法应用阳离子脂质体介导STAT3反义寡核苷酸转染乳腺癌MCF-7细胞,在倒置显微镜下观察细胞生长情况及形态变化;采用MTT比色法检测细胞增殖状态;流式细胞仪检测细胞凋亡率及细胞周期;Western blot检测STAT3总蛋白和p-STAT3蛋白的表达。结果STAT3反义核苷酸转染乳腺癌细胞后,能抑制MCF-7细胞的增殖,促进MCF-7细胞的凋亡,STAT3蛋白的表达和磷酸化水平下降,出现G1期阻滞。结论STAT3反义核苷酸能抑制MCF-7细胞的增殖,促进MCF-7细胞的凋亡,这将为乳腺癌的治疗提供新的方向。     

Bel-2反义寡核苷酸增强小细胞肺癌细胞化疗敏感性研究

目的：研究Bcl-2反义寡核苷酸是否能增强小细胞肺癌细胞NCI—H69对化疗药物的敏感性。方法：将NCI．H69细胞培养传代，细胞共分4组，反义寡核苷酸组、正义寡核苷酸组、无义寡核苷酸组和空白对照组，通过脂质体介导的方法将不同寡核苷酸导人细胞中，Western—Blot法检测Bcl-2蛋白的表达。不同浓度的顺铂与阿霉素作用于4组转染后的细胞，MTT法测定细胞存活分数。结果：4组细胞中，与空白对照组相比较，反义寡核苷酸组的Bcl-2蛋白表达明显受到抑制，而正义寡核苷酸组和无义寡核苷酸组细胞的Bcl-2蛋白表达则与空白对照组差别不明显。细胞分别同顺铂与阿霉素作用后，反义寡核苷酸组细胞在各个浓度的存活分数均低于空白组，其差异在统计学上有显著性意义（P〈0．01），而转染正义链和无义链组与空白组比较其差异在统计学上无显著性意义。结论：Bcl-2反义寡核苷酸能有效封闭Bcl-2基因的表达，增强小细胞肺癌细胞对化疗药物的敏感性。     

Experimental Research of Tankyrase1 Antisense Oligodeoxynucleotides on the Proliferation of Lung Cancer Cell Nodules

OBJECTIVE To observe the effects of sense and antisense oligodeoxynucleotides of tankyrase 1 (TANK1-SODN and TANK1-ASODN) on murine tumor growth following intratumoral injection, investigate the actual suppressing result and mechanism of TANK1-ASODN on cancer cell proliferation, and discuss the possibility of using it in gene therapy on human lung cancer cells. METHODS After BALB/c nude mice had been subcutaneously inoculated with human lung cancer cell line CALU and it had grown into tumor nodules, we distributed these mice randomly into 3 groups: 4 in saline treatment group, and 5 each in TANK1-SODN group, and TANK1-ASODN groups. Then multiple direct intratumoral injections of synthesized TANK1-ASODN given continuously into tumor nodules for 16 days, this was compared with TANK1-SODN and saline control groups. During the experiment we measured the tumor volume every 5 days with vernier calipers; observed the histopathological characteristics of tumor tissues under microscope; went further to detect the minute changing of ultrastructure of cancer cells by electron microscope; tested the expression levels of ki67 and hTERT protein by means of SABC immunohistochemical method; and detected the lung cancer cells' hTERT mRNA expression level by hybridization in situ (ISH) in each group. RESULTS After 16 days of continuous injection, the tumor volume in TANK1-ASODN group was significantly smaller than the other 2 groups (both P < 0.01); quite a lot of tumor cell degeneration and necrosis were observed in mice given TANK1-ASODN. The results of electron microscope also showed that TANK1-ASODN has the power to kill cancer cells in various ways. Moreover, statistically signi.cant decreases in the positive expression ratio of Ki67 Labeling Index (P < 0.01), hTERT protein (P < 0.01), and hTERT mRNA (P < 0.01) were consistently observed in the TANK1-ASODN group. CONCLUSION Human lung cancer cell line CALU expressed high telomerase activity. TANK1-ASODN had the ability to decline the high expression level of hTERT; inhibit the activity of telomerase, accelerate tumor cell degeneration and necrosis; and then suppress the proliferation of cancer cells.     

Bcl-2反义核酸对人胆管癌QBC939细胞增殖的影响

目的研究Bcl-2反义核酸（ASODN）对人胆管癌QBC939细胞增殖的影响。方法将Bcl-2ASODN与QBC939细胞共同孵育，采用台盼蓝拒染实验检测细胞存活率，采用克隆形成实验俭测克隆形成率。结果台盼蓝拒染实验和克隆形成实验检均显示Bcl-2ASODN可以部分抑制QBC939细胞增殖，经ASODN作用细胞的存活率和克隆形成率均显著低于对照组（P〈0．05）。结论Bcl-2反义核酸对人胆管癌QBC939细胞增殖有抑制作用。     

Preparation of Immunotoxin Herceptin-Botulinum and Killing Effects on Two Breast Cancer Cell Lines

Background: Worldwide, breast cancer is the most common cancer diagnosed among women and a leadingcause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin,a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2-neu receptors, but it is an expensive drug with only 20% beneficial rate of survival. This study introduces anovel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinumtoxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. Materials andMethods: Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidylacetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)according to the supplier’s guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxinand Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the newbioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. Results: Herceptin-Toxinbioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared tothe control group. Conclusions: Toxin-Herceptin bioconjugation can be a potential cand     

成纤维细胞系3T3细胞来源exosome对小鼠乳腺癌细胞增殖能力的影响

目的 观察小鼠成纤维细胞系3T3来源的外泌小体（exosome）对小鼠乳腺癌细胞4T1增殖能力的影响,并探索其中可能的机制。方法 PureExo Exosome提取试剂盒提取3T3细胞上清液中的exosome,按照不同浓度及时间作用于4T1细胞,CCK8法检测4T1细胞的增殖能力,BrdU/PI双掺入法测定细胞DNA合成及细胞周期;免疫印迹法（Western blot）及荧光定量实时PCR（qPCR）检测人表皮生长因子受体2（epidermal growth factor receptor-2, EGFR2,也称HER2）及下游PI3K/AKT信号转导通路相关蛋白的变化。利用HER2单克隆抗体靶向药物赫赛汀（Herceptin）,观察exosome是否影响4T1细胞对于Herceptin敏感度。结果 exosome处理组OD450吸光度值显著高于对照组（P<0.05）,细胞增殖及细胞周期进程加快。Western blot及qPCR实验提示随着exosome浓度的增加,HER2表达逐渐升高, AKT磷酸化水平增加。而同时给予exosome可明显增加4T1细胞对Herceptin的敏感度。结论 小鼠成纤维细胞系3T3来源exosome可促进小鼠乳腺癌细胞4T1增殖及周期进程,并且可能通过HER2激活其下游PI3K/AKT信号通路发挥上述作用。     

乳腺癌干细胞临床与基础研究的进展

乳腺癌是一类高度异质性的肿瘤,包括18种组织病理学类型和5 种分子亚型。乳腺癌干细胞（CSCs）是乳腺癌组织中极少数具有自我更新能力和多向分化潜能的细胞,具有肿瘤启动作用。CD44+/CD24-/low表型为乳腺CSCs的标志,大约30% 乳腺癌组织中存在CD44+/CD 24-/low表型细胞,CD44+/CD 24-/low表型细胞与病理组织学类型有关,在呈现CD44+/CD 24-/low表型的肿瘤中,浸润性导管癌所占比例最多（78%）,其次为髓样癌（11%）和浸润性小叶癌（7%）。 CD44+/CD 24-/low表型细胞与乳腺癌的分子亚型有关,在依据基因表达谱得到的5 种分子亚型中,CD44+/CD 24-/low表型在基底样癌中最常见。乳腺癌干细胞通常为ER- 和PR- 的表型,但ER- 和PR- 的多潜能干细胞能产生ER+ ,PR+ 的肿瘤细胞。HER2 过表达能够增加CSCs的数量并促进乳腺癌的发生、进展和浸润,但乳腺癌组织中CD44+/CD 24-/low表型通常与HER2 低表达或阴性表达呈正相关。迄今有关干细胞与乳腺癌的临床基础研究提出了更多新的问题,引发了更多的思考。      

Combined EGFR and c-Src Antisense Oligodeoxynucleotides Encapsulated with PAMAM Denderimers Inhibit HT-29 Colon Cancer Cell Proliferation

Colon cancer continues to be one of the most common cancers, and the importance and necessity of newtherapies needs to be stressed. The most important proto-oncogen factors for colon cancer appear to beepidermal growth factor receptor, EGFR, and c-Src with high expression and activity leading to tumor growthand ultimately to colon cancer progression. Application of c-Src and EGFR antisense agents simultaneouslyshould theoretically therefore have major benefit. In the present study, anti-EGFR and c-Src specific antisenseoligodeoxynucleotides were combined in a formulation using PAMAM dendrimers as a carrier. Nano drug entryinto cells was confirmed by flow cytometry and fluorescence microscopy imaging and real time PCR showed geneexpression of c-Src and EGFR, as well as downstream STAT5 and MAPK-1 with the tumor suppressor geneP53 to all be downregulated. EGFR and c-Src protein expression was also reduced when assessed by westernblotting techniques. The effect of the antisense oligonucleotide on HT29 cell proliferation was determined byMTT assay, reduction beijng observed after 48 hours. In summary, nano-drug, anti-EGFR and c-Src specificantisense oligodeoxynucleotides were effectively transferred into HT-29 cells and inhibited gene expression intarget cells. Based on the results of this study it appears that the use of antisense EGFR and c-Src simultaneouslymight have a significant effect on colon cancer growth by down regulation of EGFR and its downstream genes.