论文部分内容阅读
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(P<0.05),随着时间延长,细胞抑制率增高(P<0.05)。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)%(P<0.05)。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(P<0.05)。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。
Objective To investigate the effect of survivin antisense oligonucleotide (ASODN) on the proliferation of human pancreatic cancer cell line PANC-1. Methods Specific targeting ASODN and sense oligodeoxynucleotides (SODNs) were designed and synthesized. ASODN was labeled with FAM and transfected into PANC-1 cells cultured in vitro with cationic liposome as vector (ASODN group), another blank control group (normal cultured cells), lipofectin group Body transfected normal medium) and SODN group as a control group. The transfection rate was determined by flow cytometry (FCM), and the optimal number of cells inoculated, the concentration of ASODN and the amount of liposomes were screened sequentially. The transfected cells were observed under a fluorescence microscope. The ultrastructural changes of the cells were observed by transmission electron microscopy. The morphological changes of apoptotic cells were observed by AO staining and EB staining. The DNA gel electrophoresis was used to observe the transfection efficiency. The apoptotic rate and cell cycle of transfected cells were detected by FCM. The expression of survivin protein was detected by immunohistochemistry. Results ① The transfection rates of ASODN were 31.9%, 37.4%, 41.4%, 52.6%, 24.2%, 11.4%, 16.1% at ASODN concentrations of 50,100,150,200,250,400,600 and 800 nmol / L respectively 15.5%. The number of transfected cells was 12.0%, 50.8% and 11.2% when the number of inoculated cells was 2 × 104, 2 × 105 and 2 × 106 cells / well, respectively. At 4 μl, the transfection rates were 58.8%, 34.0% and 23.6%, respectively. ② After transfection with ASODN, fluorescence microscopy showed that the gap between the cells after transfection increased, the morphology became round, adherent cells grew less, and some floating in the culture medium. (3) The inhibition rate of ASODN group was significantly higher than that of control group at 24, 36 and 48 h (P <0.05). With the prolongation of time, the cell inhibition rate increased (P <0.05). ④ apoptotic cells inASODN group showed obvious “ladder” bands. ⑤ AO and EB staining observed ASODN cells apoptosis morphological changes, the nuclear chromatin were highly aggregated or nuclear staining were crescent-shaped gathered in the nuclear membrane side, the nucleolus disappeared. ⑥ The apoptosis rate of ASODN group was (38.10 ± 3.4)%, which was significantly higher than that of SODN group (4.16 ± 1.7)% (P <0.05). ⑦ ASODN cell cycle arrest in G2 / M phase. ⑧ Survivin protein expression in ASODN group was significantly lower than that in the control group (P <0.05). Conclusion The optimal transfection conditions of survivin ASODN to pancreatic cancer cells are 2 × 105 cells / well, the concentration of ASODN is 200 nmol / L and the amount of liposome is 2μl. Survivin ASODN can downregulate the expression of survivin protein , Inhibit the proliferation of pancreatic cancer PANC-1 cells and induce apoptosis.