脂质体转染survivin反义寡核苷酸对人胰腺癌细胞凋亡的影响

目的探讨survivin反义寡核苷酸(antisense oligonucleotide,ASODN)对人胰腺癌细胞PANC-1增殖的影响。方法设计并合成特异性靶向ASODN及正义寡核苷酸(sense oligodeoxynucleotides,SODN)。ASODN经FAM标记,以阳离子脂质体为载体转染至体外培养的PANC-1细胞中(ASODN组),另设空白对照组(正常培养的细胞)、脂质体空转染组(脂质体转染的为正常的培养基)及SODN组作为对照组。采用流式细胞仪(flow cytometry,FCM)测转染率,依次筛选最佳细胞接种数量、ASODN浓度和脂质体的量;荧光显微镜观察转染后的细胞;MTT法测定转染后细胞的抑制率;透射电镜观察转染后细胞的超微结构变化;吖叮橙(AO)与溴化乙啶(EB)染色观察转染后细胞凋亡的形态学改变;DNA凝胶电泳观察转染后细胞凋亡情况;FCM测转染后细胞凋亡率及细胞周期;免疫组织化学技术检测转染后survivin蛋白的表达。结果①ASODN浓度为50、100、150、200、250、400、600及800 nmol/L时,转染率分别为31.9%、37.4%、41.4%、52.6%、24.2%、11.4%、16.1%和15.5%;接种细胞数量为2×104、2×105及2×106个/孔时,转染率分别为12.0%、50.8%和11.2%;转染所用的脂质体量为2、3及4μl时,转染率分别为58.8%、34.0%和23.6%。②转染ASODN后荧光显微镜发现转染后细胞之间空隙加大,形态变圆,贴壁细胞生长较少,部分漂浮于培养液中。③ASODN组24、36及48 h后细胞抑制率明显高于各对照组(P0.05),随着时间延长,细胞抑制率增高(P0.05)。④ASODN组电泳图上凋亡细胞呈明显的"梯状"条带。⑤AO与EB染色观察ASODN组细胞出现凋亡的形态学改变,其核染色质均高度聚集或核染色呈新月形聚集于核膜一边,核仁消失。⑥ASODN组细胞凋亡率为(38.10±3.4)%,明显高于SODN组的(4.16±1.7)%(P0.05)。⑦ASODN组细胞周期阻滞于G2/M期。⑧转染后ASODN组survivin蛋白表达明显低于各对照组(P0.05)。结论 survivin ASODN转染至胰腺癌细胞最佳转染条件为接种细胞数量为2×105个/孔,ASODN的浓度为200 nmol/L,脂质体的量为2μl;survivin ASODN能明显下调survivin蛋白的表达,抑制胰腺癌PANC-1细胞的增殖,并诱导细胞凋亡。     

survivin反义寡核苷酸诱导胆囊癌细胞凋亡的研究

目的研究survivin反义寡核苷酸(ASODN)对胆囊癌细胞凋亡的诱导作用。方法采用脂质体介导survivinASODN转染人胆囊癌细胞株GBC SD,采用流式细胞仪技术检查细胞凋亡变化,采用电镜技术观察细胞的形态变化,采用逆转录聚合酶链反应(RT PCR)检测survivin基因表达的变化。结果脂质体介导转染survivinASODN后,胆囊癌细胞内survivinmRNA表达相对系数为0.512±0.011,明显低于未转染者的0.980±0.012(P<0.01),平均下调了47.83%(P<0.01),凋亡细胞比率为(26.28±3.91)%,明显高于未转染者的(0.50±0.23)%,P<0.01,电镜下可见胆囊癌细胞呈典型凋亡样改变。结论survivinASODN转染后能下调survivin的表达,可有效诱导GBC SD细胞凋亡。     

脂质体介导的聚集素反义寡核苷酸对膀胱癌EJ细胞的作用

目的探讨clusterin对膀胱癌EJ细胞生物学特征的影响. 方法应用脂质体包裹的clusterin反义寡核苷酸转染EJ细胞,阻断其表达,通过3′端末端标记、MTT试验、伊红排斥实验等方法观察clusterin反义基因对EJ细胞增殖、凋亡、坏死等生物学行为的影响. 结果转染clusterin反义寡核苷酸组(反义组)EJ细胞凋亡增加,第5天达峰,凋亡率80%,与转染clusterin正义寡核苷酸组(正义组)比较,差异有显著性意义(P<0.05).反义组细胞死亡率增加,以转染后1～4 d明显(13.8%～33.3%),显著高于相应正义组(P<0.05).反义组细胞增殖活性降低,以转染后第6、7天明显,显著高于相应正义组(P<0.05). 结论 clusterin是重要抗凋亡因子,其表达与膀胱癌细胞的生存、增殖、抗凋亡发展密切相关.     

Survivin反义寡核苷酸诱导肝癌细胞凋亡及细胞结构的变化

目的采用反义寡核苷酸封闭肝癌细胞中Survivin基因的表达，研究其诱导细胞凋亡过程中对细胞超微结构与细胞骨架的作用及其机理。方法采用脂质体介导Survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞，Western—blot及原位杂交方法检测Survivin蛋白及mRNA表达，流式细胞仪检测细胞凋亡比率，透射电子显微镜观察细胞超微结构变化，激光共聚焦显微镜观察细胞骨架微丝系统变化，激酶活性检测方法测定细胞内Caspase-3活性变化。结果脂质体介导Survivin反义寡核苷酸转染肝癌细胞后Survivin蛋白及mRNA表达分别由69．59及75．60降低至10．71及22．90，Caspase-3活性由0．0153升高至0．0992，同时细胞结构呈典型凋亡改变，细胞内微丝形态结构破坏，细胞凋亡比率由0．7％增加至31．4％。结论脂质体介导转染Survivin反义寡核苷酸可以有效降低细胞内Survivin基因的表达，并激活Caspase-3。活化的Caspase-3可以切割破坏细胞内骨架微丝系统的结构，进而诱导细胞凋亡。     

存活素反义寡核苷酸诱导肾癌细胞凋亡及机制的实验研究

目的观察存活素反义寡核苷酸封闭存活素的表达对肾癌细胞凋亡的影响。方法采用脂质体介导的存活素反义寡核苷酸技术转染肾癌细胞786-0。电镜观察细胞超微结构,免疫组织化学、Western blot及RT-PCR方法检测存活素蛋白及mRNA表达,流式细胞仪检测细胞凋亡率,四甲基偶氮唑盐比色法检测癌细胞抑制率。酶标免疫测定caspase-3活性。结果存活素反义寡核苷酸能显著降低存活素蛋白和mRNA的表达,并呈浓度和时间依赖效应。转染后的肾癌细胞出现了大量凋亡小体。400、600、800 ng／ml反义寡核苷酸细胞凋亡率分别为（10．54±0．72）％,（12．80±0．38）％,（22．30±1．23）％,较空白对照组[（6．05±0．48）％]和正义对照组[（5．70±0．41）％]显著增加（P〈0．05）。反义寡核苷酸各组caspase-3的相对活性分别为0．052±0．009,0．078±0．004和0．092±0．002,与空白对照组（0．027±0．008）和正义对照组（0．028±0．001）相比,差异有统计学意义（P〈0．05）。结论存活素反义寡核苷酸能显著下调存活素基因表达,明显促进肾癌细胞786-0凋亡并抑制其增殖;可能是通过上调caspase-3表达来促进凋亡。     

Survivin反义寡核苷酸对人骨肉瘤细胞的抑制作用

我们以骨肉瘤OS 73 2细胞为对象 ,在明确其高表达Survivin基础上 ,尝试通过合成靶向Survivin的反义寡核苷酸(ASODN)转染OS 73 2细胞 ,以封闭Sur vivin表达 ,探讨ASODN对骨肉瘤细胞凋亡 /增殖的影响及其对化疗药物的促进作用。一、材料与方法1.材料 :人骨肉瘤细胞株OS 73 2购自北京积水潭医院骨科研究所。针对Survivin基因翻译起始密码区 ,设计合成ASODN (上海生工公司 ) ,序列为 5′CCCAGCCTTCCAGCTCCTTG 3′ ,并设正义 (senseoligonucleotide ,SODN )对照 ,序列为 5′GTTCCTCGACCTTCCGACCC3′ ,两组寡核苷酸均…     

survivin反义寡核苷酸诱导胃癌原代细胞凋亡的实验研究

目的探讨survivin反义寡核苷酸对人胃癌原代细胞凋亡的影响，为胃癌的靶向治疗提供实验依据。方法利用人胃癌实体瘤新鲜标本培养胃癌原代细胞，胃癌细胞分为空白对照组、脂质体组、正义寡核苷酸组及反义寡核苷酸转染组。用脂质体介导survivin反义寡核苷酸转染胃癌原代细胞，48h后，观察肿瘤细胞的形态变化，Westernblot检测survivin蛋白的表达，流式细胞术检测细胞的凋亡。结果反义寡核苷酸转染组肿瘤细胞体积变小，细胞碎裂，出现凋亡小体；survivin蛋白量下降；流式细胞术检测到肿瘤细胞的凋亡率增高。与其他各组相比，其差异有统计学意义（P〈0．05），而空白对照组、脂质体组、正义寡核苷酸组之间的差异无统计学意义（P〉0．05）。结论survivin反义寡核苷酸在脂质体介导下转染人胃癌原代细胞，可以诱导肿瘤细胞发生凋亡，抑制细胞生长，反义survivin可能成为一个促进胃癌细胞凋亡的手段。     

Bcl-2反义寡核苷酸诱导增生性瘢痕细胞凋亡的研究

目的：探讨反义寡核苷酸(ASODN)对bcl-2基因的表达调控及诱导增生性瘢痕成纤维细胞凋亡的作用。方法：采用RT-PCR法和流式细胞术检测ASODN对bcl-2蛋白和mRNA表达量的调控；通过MTT法比较两条人工合成ASODN直接作用及脂质体D0’FAt，转染对增生性瘢痕成纤维细胞的抑制效果；用相差显微镜、电子显微镜观察bcl-2ASODN诱导增生性瘢痕成纤维细胞凋亡效果。结果：ASODN降低bcl-2蛋白和mRNA表达；两条ASODN抑制增生性瘢痕成纤维细胞增殖的作用呈浓度和时间依赖性，且靶位点于mRNA蛋白编码区(ASODN2)和以DOTAP为载体的ASODN对成纤维细胞的增殖抑制效果最佳。Bcl-2ASODN作用于成纤维细胞后，成纤维细胞缩小、凋亡小体和染色质浓缩等特征性改变出现。结论：bcl-2ASODN能抑制bcl-2mRNA和蛋白表达，诱导成纤维细胞凋亡。     

125I偶联Ki-67反义核酸对人肾癌细胞生长及凋亡的调控作用

目的　探讨12 5I偶联Ki 67基因反义寡核苷酸 (12 5I ASODN)对人肾癌细胞生长及凋亡的调控作用。方法　采用氯胺 T法将 10 μmol/L浓度的Ki 67反义寡核苷酸 (ASODN)与12 5I偶联 ,12 5I ASODN放射浓度为 2 .5 9MBq/L ,脂质体包装后转导肾癌 786 0细胞系。采用免疫组织化学、Western印迹技术检测Ki 67表达 ,四甲基偶氮唑蓝 (MTT)法检测细胞增殖 ,免疫组织化学脱氧核苷酸末端转移酶介导的缺口末端标记 (TUNEL)法检测细胞凋亡。结果　12 5I ASODN处理组肾癌细胞Ki 67表达阳性率 (2 1.5± 1.2 ) %降低 ,Ki 67蛋白 (4 9.9± 4.5 ) %降低 ,与ASODN处理组(2 9.9± 0 .4) %、(82 .1± 1.9) %比较差异有非常显著性 (P均 <0 .0 1)。细胞增殖抑制率12 5I A SODN处理组 (4 7.9± 3 .6) %与ASODN处理组 (2 1.8± 2 .5 ) %比较差异有非常显著性 (P <0 .0 1)。凋亡细胞阳性率12 5I ASODN处理组 (2 8.9± 1.3 ) %与ASODN处理组 (15 .7± 0 .5 ) %比较差异有非常显著性 (P <0 .0 1)。结论　12 5I ASODN较之ASODN具有更强的抑制肾癌细胞增殖、促进凋亡作用。     

Survivin反义寡核苷酸对乳腺癌荷瘤裸鼠的抑瘤作用研究

目的探讨survivin反义寡核苷酸（ASODN）对乳腺癌荷瘤裸鼠的抑瘤作用。方法30只裸鼠建立人乳腺癌MCF-7细胞移植瘤动物模型。成瘤后动物随机分3组，每组10只。分别于瘤周及瘤体注射阳离子脂质体（1ipofectin，Lip），ASODN／Lip及NODN／Lip，每5天1次，共3次。观察肿瘤抑制率、肿瘤体积和裸鼠体重的变化。用TUNEL法，透射电镜观察体内瘤体的细胞调亡情况；用Western—blot检测各组肿瘤的survivin蛋白表达情况。结果成功建立了裸鼠皮下移植瘤模型。ASODN／Lip治疗组的肿瘤体积在治疗期内减少，抑瘤率达70．10％；而NODN／Lip治疗组和Lip对照组肿瘤体积增加。各组裸鼠体重无明显变化。ASODN／Lip组细胞凋亡率为38．6％明显高于其他两组，差异有显著性（P〈0．05）。ASODN／Lip组肿瘤的survivin蛋白表达明显降低。结论survivin反义寡核苷酸可有效地抑制人乳腺癌裸鼠皮下肿瘤的生长速度，并促进诱导肿瘤细胞的凋亡。     

An antisense oligonucleotide to cIAP-1 sensitizes prostate cancer cells to fas and TNFalpha mediated apoptosis

BACKGROUND: The inhibitors of apoptosis (IAP) proteins are a family of structurally homologous caspase inhibitors. We synthesized an antisense oligonucleotide (AO) to target a region within the BIR domain of cIAP-1 and examined its ability to facilitate apoptosis in prostate cancer cells. METHODS: We transfected the IAP AO into PC3 and DU145 cells and determined alterations in IAP expression using Western blotting. Apoptosis and viability were assessed using propidium iodide (PI) DNA incorporation with flow cytometry. Pacitaxel, caffeic acid phenethyl ester (CAPE), Fas antibody, and TNFalpha were used as 'second hit' agents in association with the AO. RESULTS: Western blotting showed a down-regulation in cIAP-1 expression and higher levels of spontaneous apoptosis in both cell types with no alteration in overall cell viability. AO sensitized PC3 cells, to Fas antibody and TNFalpha-mediated apoptosis, but not to apoptosis mediated by paclitaxel or CAPE. CONCLUSIONS: cIAP-1 down-regulation increased spontaneous apoptosis in prostate cancer cells and sensitized PC3 cells to receptor-mediated apoptosis.     

The anti-proliferative effect of proliferating cell nuclear antigen-specific antisense oligonucleotides on human gastric cancer cell lines

The proliferating cell nuclear antigen (PCNA) is a nuclear protein that leads DNA synthesis by the DNA polymerase delta. As the PCNA gene is strongly expressed in invasive gastric cancer cells with high proliferative activity, PCNA is suspected of playing an important role in the proliferation and advancement of gastric cancer. Thus, the effects of antisense oligonucleotides specific for PCNA mRNA were examined in seven gastric cancer cell lines. It was found that treatment with antisense oligonucleotides at concentrations of 10–40 M dose-dependently inhibited the growth of all cell lines; however, random sequence oligonucleotides did not modify the proliferation of any type of cells. These results indicate that PCNA is essential for cell proliferation in gastric cancer cells, and that the growth inhibitory effect results from the inhibition of PCNA gene expression. Therefore, PCNA-specific antisense oligonucleotides may be effective in the treatment of gastric cancer.     

反义结缔组织生长因子对人瘢痕疙瘩成纤维细胞的作用

目的：研究结缔组织生长因子（CTGF）反义寡核苷酸（ASODN）对人瘢痕疙瘩成纤维细胞CTGFmRNA的表达和细胞增殖及胶原合成的影响,探讨瘢痕疙瘩的基因治疗。方法：体外分离、培养人正常皮肤成纤维细胞（NSF）和瘢痕疙瘩成纤维细胞（KF）,以脂质体介导方法将CTGFASODN转染KF中,用逆转录-聚合酶链式反应（RT-PCR）方法检测细胞中CTGFmRNA的表达;采用MTT方法测细胞增殖3;H-脯氨酸掺入法检测细胞的胶原合成量。结果：CTGFmRNA在NSF中几乎无表达,在KF中表达增高,CTGFASODN可以抑制其增殖和胶原合成（P〈0.05）。结论：CTGFASODN能够抑制成纤维细胞CTGFmRNA表达和细胞增殖及胶原合成,表明阻断CTGF可能是延缓瘢痕纤维化的有效手段。     

Survivin反义核酸诱导乳腺癌细胞凋亡的实验研究

目的探讨Survivin反义核酸技术诱导乳腺癌细胞凋亡的作用及效果。方法本实验共分6组，分为以脂质体介导反义寡核苷酸组（ASODN／Lip）、无义寡核苷酸组（NODN／Lip）及RPMI 1640培养液的空白对照组（Lip），转染人乳腺癌MCF-7细胞系，应用Hoechst 33258／PI双重染色观察MCF-7细胞的形态学改变，透射电镜观察细胞超微结构，流式细胞仪检测细胞周期和细胞凋亡的变化，DNA凝胶电泳观察凋亡情况，研究Survivin反义寡核苷酸对MCF-7乳腺癌细胞的生长抑制作用。结果Hoechst 33258／PI染色及透射电镜观察可见转染后的细胞结构呈凋亡样改变；流式细胞仪检测见转染后细胞凋亡显著增加，并出现G2／M期阻滞现象；DNA凝胶电泳在600ng／ml，800ng／ml ASODN／Lip组的电泳图谱上呈现出明显的“梯状”现象。结论凋亡抑制基因Survivin表达下涧对乳腺癌细胞的生长抑制作用主要是通过诱导细胞发生凋亡以及阻止有丝分裂发生在G2／M阻滞期来实现，Survivin靶向反义核酸技术可能成为乳腺癌基因治疗的一种有效方法。     

MiR-622 acts as a tumor suppressor to induce cell apoptosis and inhibit metastasis in human prostate cancer

Growing evidence indicating the critical modulator roles of microRNAs (miRNAs) involved in prostate cancer (PCa) metastasis that holds great promise as therapeutic targets. Herein, we transfected the miR-622 mimic into PC3 cells and evaluated the effects of this interference on these tumour cells' growth and the expression of specific metastatic genes. Transfecting of miR-622 mimic and inhibitor, negative control (NC) inhibitor and NC was established using Lipofectamine 2000. The mRNA levels of miR-622 and metastatic genes were evaluated using the qRT-PCR and Western blot. Cytotoxic effects of miR-622 were assessed by MTT. Apoptosis was detected using an ELISA cell death assay kit. miR-622 is down-regulated in PC3 cells. As expected, cell viability effects after transfection were described as miR-622 inhibitor >NC and NC inhibitor >miR-622 mimic (p < .01). Importantly, we showed that transfected miR-622 mimic could enhance the apoptosis of PC3 cells, while transfected miR-622 inhibitor could decrease cell apoptosis (p < .01). Furthermore, miR-622 overexpression could increase significantly down-regulated the MMP2, MMP9, CXCR-4, c-Myc and K-Ras expression levels. Findings demonstrate a novel mechanism by which miR-622 modulates PCa cells' metastasis by targeting metastatic genes. These data confirm the tumour-suppressive function of miR-622 in PCa cells by enhancing apoptosis and reducing metastasis.     

Sensitization of human renal cell carcinoma cell lines to TRAIL-induced apoptosis by anthracyclines

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the tumor necrosis factor family. The present study investigated whether anthracyclines enhance TRAIL-induced apoptosis and cytotoxicity in renal cell carcinoma (RCC) cells. METHODS: Cytotoxicity was measured using the microtiter assay. Apoptosis was monitored using DNA ladder analysis. Caspase activity was determined using a quantitative colorimetric assay. RESULTS: Treatment of ACHN and Caki-1 human RCC lines with TRAIL, in combination with subtoxic concentrations of epirubicin (EPI) or pirarubicin (THP), enhanced induction of apoptosis and cytotoxicity. Sequential treatment with EPI followed by TRAIL induced significantly more cytotoxicity than the inverse treatment. The combined cytotoxicity of TRAIL and EPI was significantly inhibited by the TRAIL-neutralizing fusion protein DR5:Fc, although EPI did not affect the mRNA expression of DR4, DR5, DcR1 or DcR2. The combination treatment with TRAIL and EPI activated caspase-6 and -3, which were downstream molecules of the death receptor. Furthermore, the combined cytotoxicity of TRAIL and EPI was almost completely inhibited by Z-VAD-FMK, and partly inhibited by Ac-DMQD-CHO. CONCLUSION: These findings indicate that anthracyclines sensitize RCC cells to TRAIL-induced apoptosis and cytotoxicity through activation of caspases, suggesting that TRAIL, in combination with anthracyclines, has a therapeutic potential in the treatment of RCC.     

Sensitization of human colon cancer cells to trail-mediated apoptosis

TNF-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor (TMF) family, is thought to induce apoptosis preferentially in cancer cells; however, increasing evidence suggests that a number of cancers are resistant to TRAIL treatment. FLICE-like inhibitory protein (FLIP), which structurally resembles caspase-8, can act as an inhibitor of apoptosis when expressed at high levels in certain cancer cells. The purpose of our present study was to determine whether human colon cancer cells are sensitive to TRAIL treatment and, if not, to identify potential mechanisms of resistance. Colon cancer cells of different metastatic potential (KMlZC, KML4A, and KM20) were found to be resistant to the effects of TRAIL when used as a single agent. FLIP expression levels were increased in all three KM cell lines. Treatment with either actinomycin D (Act D;10 μ/ml) or cycloheximide (CHX; 10 μg/ml) decreased FLIP expression levels in all three cell lines. The decrease in cellular levels of FLIP was associated with sensitization to TRAIL-mediated apoptosis, as demonstrated by enhanced cell death and caspase-3 activity compared with either Act D or CHX alone. Our findings suggest that reduction of FLIP levels by Act D or CHX renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL along with agents such as Act D or CHX, which target proteins that prevent cell death, may provide a more effective and less toxic regimen for treatment of resistant colon cancers. Presented at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 2l–24, 2000, and published as an abstract in Gastroentmolog 118:A1025, 2000. Supported by grants R01 AG10885, R01 DK48498, PO1 DK35608, and T32 DK07639 from the National Institutes of Health, Bethesda, Md.     

反义寡核苷酸技术在胰腺癌研究中现状及展望

反义寡核苷酸技术对认识胰腺癌基因或蛋白作用机制及功能有重要作用，作为一种治疗策略具有潜在价值，为胰腺癌研究提供了新的思路。     

Livin靶向ASODN对前列腺癌PC3细胞生物学特性的影响

目的：合成Livin靶向的反义核苷酸（ASODN）,观察其对前列腺癌PC3细胞凋亡和增殖等生物学特性的影响。方法：合成全硫代磷酸化修饰的LivinASODN并通过脂质体转染前列腺癌PC3细胞。采用MTT法检测其对细胞增殖的影响,采用RT—PCR检测转染后Livin基因mRNA的表达情况,采用流式细胞仪检测转染后细胞的凋亡效应,采用体内成瘤实验比较细胞在裸鼠体内成瘤性的变化,采用激酶法测定Caspase-3活性的变化。结果：LivinASODN转染PC3细胞后,与对照组相比,实验组细胞内LivinmRNA的表达水平下调（P〈O．01）;细胞的增殖受到显著抑制（P〈O．01）;细胞凋亡率明显增高（P〈O．01）;肿瘤细胞在荷瘤鼠体内的成瘤体积小于对照组（P〈O．05）;Caspase-3活性明显增加（P〈0．05）。结论：靶向Livin基因的ASODN干扰了前列腺癌PC3细胞中Livin基因的表达,抑制了细胞的增殖并诱导其凋亡,延缓了肿瘤细胞的生长。     

反义人端粒酶RNA亚基对胆囊癌端粒酶活性及细胞生长的抑制作用

目的探讨反义RNA技术对胆囊癌细胞端粒酶活性的影响及对胆囊癌细胞生长增殖的作用。方法根据测定的胆囊癌人端粒酶RNA亚基(hTR)基因的序列结果，并根据真核表达载体多克隆酶切位点的物理图谱，体外合成反义RNA及正义RNA的基因序列，并构建入pTriEx-4真核表达载体，酶切鉴定正确后，采用脂质转染法导入胆囊癌细胞。TRAP法检测端粒酶活性。流式细胞仪检测细胞周期及凋亡情况，电镜观察细胞微观形态变化。结果实验组胆囊癌细胞的端粒酶活性较对照组明显减低，正义组、转染空载体及单纯脂质体转染的细胞端粒酶活性与未转染细胞比较则变化不明显。反义RNA基因作用后，G0／G1期细胞比例明显增高，S期细胞比例明显降低。细胞的分裂、增殖受到明显抑制。反义RNA基因转化后，10d凋亡率为11．10％。13d后凋亡率为29．02％。电镜下可见明显凋亡细胞。结论反义RNA技术对胆囊癌细胞端粒酶活性有明显的抑制作用；并抑制胆囊癌细胞的生长，促进细胞的凋亡；且克服了反义寡核苷酸作用时间短的缺点。