survivin反义寡核苷酸对SMMC-7721细胞增殖和凋亡的影响

目的 观察survivin反义寡核苷酸(ASODN)对人肝癌细胞株SMMC-7721增殖和凋亡的影响.方法 人工合成survivin基因ASODN和正义ODN(SODN),并行硫代磷酸化修饰,通过脂质体途径转染SMMC-7721;分别用RT-PCR和Western blot检测survivin mRNA和蛋白表达;用MTT法检测ASODN对SMMC-7721增殖的影响;流式细胞仪检测细胞周期变化及细胞凋亡率;倒置显微镜观察细胞形态变化.结果 SMMC-7721可强表达survivin mRNA和蛋白;ASODN呈浓度依赖性抑制survivin mRNA和蛋白表达及SMMC-7721增殖,诱导细胞凋亡,使细胞阻滞于G2/M期.SODN对survivin mRNA和蛋白及SMMC-7721的增殖、细胞周期无明显抑制作用.结论 脂质体介导转染survivin ASODN可抑制细胞增殖、使细胞阻滞于G2/M期,从而促进细胞凋亡.     

COX-2抑制剂联合survivin反义寡核苷酸抗胰腺癌BxPC-3细胞的效应

目的:探讨COX-2抑制剂和survivin反义寡核苷酸联合应用对胰腺癌BxPC-3细胞的抗肿瘤效应及其可能机制.方法:应用胰腺癌BxPC-3细胞进行研究,将BxPC-3细胞分为4组:A组(对照组),B组(Celecoxib 80μmol/L)组,C组(300 nmol/L survivin ASODN),D组(80μmol/L celecoxib 300 nmol/L survivin ASODN).采用MTT检测细胞增殖.流式细胞仪检测细胞凋亡率,caspase-3试剂盒检测caspase-3活性,并用RT-PCR检测Bcl-2、survivin和Mcl-1的mRNA国的变化.结果:将80μmol/L celecoxib和300 nmol/L survivin反义寡核苷酸单独或联合作用于胰腺癌BxPC-3细胞24h和48h,D组细胞的存活率明显低于B,C组(24h:41.0%±0.4% vs 71.0%±2.2%,63.3%±4.5%;48h:34.2%±1.1% vs 61.6%±1.7%,55.0%±3%;P<0.01).作用24h后,流式细胞仪检测细胞凋亡率显示,D组细胞的凋亡率明显高于B,C组(30.33%±3.49% vs 11.93%±1.17%,22.07%±0.93%;P<0.01).caspase-3活性在B,C,D组明显高于对照组(0.04867±0.0021,0.02967±0.0021,0.08767±0.0042 vs 0.007±0.0001;P<0.01),D组细胞的caspase-3活性明显高于B,C组(0.08767±0.0042 vs 0.04867±0.0021,0.02967±0.0021;P<0.01).用100μmol/L塞来昔布作用于胰腺癌BxPC-3细胞24h后,survivin/β-actin和Mcl-1/β-actin的mRNA的比值明显低于对照组(0.68±0.05 vs 1.05±0.06,P<0.01),而Bcl-2/β-actin的mRNA的比值无明显变化(0.99±0.02 vs 1.07±0.06,P>0.05).结论:联合应用COX-2抑制剂塞来昔布和survivin反义寡核苷酸可明显诱导胰腺癌细胞的凋亡,抑制细胞增殖,并能够明显提高caspase-3活性.COX-2抑制剂塞来昔布诱导细胞凋亡可能通过survivin和Mcl-1途径,而非Bcl-2途径.     

乙酰肝素酶反义寡核苷酸对裸鼠人食管鳞癌移植瘤的抑制作用

目的 研究乙酰肝素酶(Hpa)抑制剂反义脱氧寡核苷酸(ASODN)对食管癌裸鼠移植瘤的体内抑瘤作用.方法采用低分化食管鳞癌细胞TE-13接种裸鼠,构建食管癌皮下侵袭模型,随机分为1mg/kg ASODN组(A组)、2mg/kg ASODN组(B组)和生理盐水(NS,2ml/kg)对照组(C组),每组5只,分别在肿瘤接种区皮下注射相应剂量的ASODN和NS.观察肿瘤体积和肿瘤微血管密度(MVD),采用RT-PCR和免疫组化染色检测移植瘤中Hpa的表达.结果接种后第6天,所有裸鼠在接种部位均长出肿瘤.接种后第21天,A、B组肿瘤体积较C组明显缩小(P<0.01),A、B组抑瘤率分别为50.27%和49.67%.A、B组MVD较c组明显降低(P<0.01).A、B、C组Hpa mRNA阳性表达分别为0.43±0.01、0.45±0.12、1.33±0.05,Hpa蛋白阳性表达分别为45.60±4.57、43.40±7.80、121.20±11.90.A、B组Hpa mRNA、蛋白表达均较C组降低(P<0.01).结论应用ASODN可以抑制肿瘤生长,其作用机制可能在于抑制肿瘤Hpa mRNA、蛋白合成,从而抑制肿瘤血管新生.2mg/kg与1mg/kg Hpa ASODN比较,未能显示更强的抑瘤效果.     

存活素与血管内皮生长因子反义寡核苷酸对人甲状腺髓样癌裸鼠模型的作用

目的探讨存活素与血管内皮生长因子（VEGF）反义寡核苷酸（ASODN）对人甲状腺髓样癌（MTC）裸鼠模型的作用。方法构建的28只人甲状腺髓样癌裸鼠模型，随机分配到对照组和治疗组。对照组包括未处理组，正义寡核苷酸（SODN）组（VEGF SODN组和存活素SODN组）和SODN联合组；治疗组包括ASODN组（VEGF ASODN组和存活素ASODN组）和ASODN联合组。各组每日一次瘤内注射相应处理液，连续7d。治疗结束后l周末，杀死全部瘤鼠，切取瘤体，测算瘤重和抑瘤率以评价肿瘤生长情况，瘤组织石腊切片分别行HE、免疫组化和DNA末端原位标记法（TUNEL法）染色以评价组织病理构造、VEGF与存活素蛋白表达和细胞凋亡的变化。结果HE染色显示，治疗组血管生成明显降低。此外，相对于对照组，治疗组具有显著的VEGF和存活素低表达、高凋亡、低瘤重和高抑瘤率特点（均P〈0．01），并且ASODN联合组较ASODN组更为显著（均P〈0．05），而对照组间和SODN组间未见上述各特点的统计学差异。结论在裸鼠MTC模型，人甲状腺髓样癌存活素和VEGF的单或双靶向基因沉默治疗能够达到明显抑瘤效果。并且联合治疗更为突出。这为发展甲状腺髓样癌的基因治疗提供了实验依据。     

反义端粒酶寡核苷酸对胰腺癌细胞株生长的体外抑制作用

目的探讨反义端粒酶寡核苷酸片段对胰腺癌细胞株端粒酶活性及细胞株生长的抑制作用.方法人工合成端粒酶反义寡核苷酸片段,电转移法将反义端粒酶序列导入胰腺癌细胞株Aspc-1、Bxpc-3中,RT-PCR-ELISA法检测端粒酶活性,MTT法检测细胞活性.结果 Aspc-1、Bxpc-3细胞株的端粒酶活性分别由0.551±0.013、0.540±0.022降低至0.222±0.021、0.224±0.024,两株细胞株的A580值分别由0.420±0.006、0.322±0.013降低至0.169±0.025、0.129±0.026,P＜0.01;细胞生长显著受抑.结论反义端粒酶寡核苷酸片段具有抑制胰腺癌端粒酶活性的作用,并抑制胰腺癌细胞的生长.     

Survivin反义寡核苷酸抑制肝癌移植瘤生长的作用

目的:检测Survivin反义寡核苷酸(ASODN)在人肝癌耐药细胞株裸鼠皮下移植瘤中的表达情况．方法:将30只裸鼠建立人肝癌耐药细胞系 SMMC-7721／ADM皮下移植瘤模型,随机分成6组:空白对照组(A)、脂质体转染对照组 (B)、正义链对照组(C)、200(D)、400(E)和 600 μg/L(F)反义链组(ASODN组),用不同的转染液注射后2,4,8,12,16,20 d,用逆转录聚合酶链反应技术(RT-PCR)和Western blot蛋白免疫印迹法检测治疗后各组肿瘤组织中Survivin mRNA和蛋白表达的变化．结果:注射后20 d,ASODN组肿瘤细胞生长明显抑制,空白对照组、脂质体转染对照组和正义链对照组裸鼠的mRNA和蛋白表达无明显差异,而ASODN组mRNA和蛋白表达随着时间和浓度的增加,Survivin表达减弱,E,F组与其余个组(A,B,C,D)相比有显著差异(mRNA: 0．33±0．04,0．28±0．03 vs 0．82±0．02,0．78± 0．05,0．72±0．04,0．57±0．03,P<0．05;蛋白: 34．9±3．89,21．2±3．65 vs 72．14±6．53,69．31 ±5.34,68．29±4．98,53．8±5．23,P<0．05)．结论:Survivin反义寡核苷酸能够下调 Survivin mRNA和蛋白的表达,抑制裸鼠皮下移植瘤的生长．     

反义寡核苷酸治疗淋巴瘤的研究

随着对淋巴瘤细胞遗传学和分子生物学的深入研究 ,人们对其发病机制有了深入的了解。近年来 ,人们对淋巴瘤的基因治疗产生了浓厚的兴趣 ,并取得了一定进展。反义寡核苷酸 (ＡＯ)是与表达基因互补的 ,短小的 (通常是 13～ 2 0对碱基 )单链ＤＮＡ或ＲＮＡ。ＡＯ被合成后 ,通常需加以修饰 ,以防止其被酶降解。ＡＯ的作用是抑制特定癌基因的表达 ,继而抑制其蛋白产物的产生。众所周知 ,淋巴瘤最常见的染色体重排是ｔ(8;14 ) ,ｔ(14 ;18)等[1] 。ｔ(8;14 )累及癌基因ｃ ｍｙｃ[2 ] ,ｔ(14 ;18)累及癌基因ｂｃｌ 2 [3 ] 。目前研究ＡＯ治疗淋巴瘤…     

硫代反义寡核苷酸对裸鼠异植瘤HCV 5′NCR基因表达的抑制作用

目的 在HCV 5′NCR转基因细胞模型HepG2.9706细胞建立成功的基础上,采用裸鼠异植瘤模型进行HCV特异性硫代反义寡核苷酸（HCV363、HCV279及HCV349）的体内活性评价。方法 将HepG2.9706细胞接种于4－6周龄的雌性BALB／C裸鼠的侧腹皮下,动物随机分组,约1周后,采用腹腔注射途径,开始给药。结果 针对HCV 5′NCR的硫代反义寡核苷酸HCV363、HCV279及HCV349对异植瘤细胞内HCV 5′NCR调控荧光素酶表达具有明显的序列特异性抑制作用,抑制率分别为80．4％、78．6％及47．9％。三个不同浓度的HCV363作用结果表明随着药物浓度的增加,HCV363对荧光素酶基因的表达抑制活性明显增强,最大抑制率可达82．7％。结论 该研究提示反义寡核苷酸有可能成为HCV感染治疗的新途径。     

survivin反义寡核苷酸转染肝癌细胞可诱导caspase-3活化

目的:观察survivin反义寡核苷酸(antisense oligonucleotide,AS ODN)诱导肝癌细胞凋亡的可能途径。方法:培养survivin表达阳性的肝癌细胞株SMMC-7721。设计合成特异性靶向survivin的反义寡核苷酸。将SMMC-7721细胞接种于6孔培养板内,分为6组:①空白对照组;②脂质体转染对照组;③正义链转染对照组;④200nmol/L AS ODN转染组;⑤400nmol/L AS ODN转染组;⑥600nmol/L AS ODN转染组。作用20小时后收获各组细胞,并进行后续实验:①倒置显微镜下观察细胞形态变化;②western blot法检测各组细胞survivin蛋白的表达情况;③流式细胞术检测细胞增殖和凋亡指数;④比色法检测各组细胞caspase-3的活性变化。结果:①AS ODN转染组细胞survivin表达减弱,以600nmol/L转染组最为明显,各对照组survivin蛋白表达无明显改变;②AS ODN转染组细胞形态出现变圆、折光增强、漂浮、细胞碎片形成等变化,而各对照组细胞生长良好;③各AS ODN转染组细胞凋亡指数明显高于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义(P>0.05);④各AS ODN转染组细胞增殖指数明显低于各对照组(P<0.05),以600nmol/L转染组最为明显,而各对照组间差异无显著性意义;⑤在各AS ODN转染组中,可见到不同程度的caspase-3的活化,而对照组细胞内无明显的变化。结论:survivin AS ODN转染能下调survivin蛋白表达,诱导肝癌细胞凋亡,抑制细胞增殖,其作用途径可能涉及抑制survivin表达,诱导caspase-3活化,启动凋亡信号传导。     

重视肾脏病反义寡核苷酸治疗的研究

重视肾脏病反义寡核苷酸治疗的研究陈香美程庆砾反义寡核苷酸是指序列与特定ｍＲＮＡ互补的含有１５～２０个核苷酸的ＤＮＡ合成片段，其主要作用机制是通过与靶序列结合后激活ＲＮａｓｅＨ裂解ｍＲＮＡ、抑制ｍＲＮＡ成熟及向胞浆转运、阻抑翻译而阻断相关蛋白的表达。在...     

VEGF反义寡核苷酸对淋巴瘤新生血管生成的抑制作用

目的研究硫代磷酸化修饰的血管内皮生长因子(VEGF)反义寡核苷酸(ASODN)对裸鼠淋巴瘤血管生成的作用。方法将终浓度为20μmol/L的VEGFASODN、错义序列及PBS分别与人淋巴瘤细胞系Namal-wa细胞孵育24h,裸鼠随机分为VEGFASODN组、错义序列组和对照组,将上述处理的Namalwa细胞(5×106溶于PBS液20μl)接种于裸鼠皮下,4周后处死全部裸鼠,测定肿瘤大小,标本采用链酶菌抗生素蛋白-过氧化酶免疫组化法(SP法)检测肿瘤组织微血管密度(MVD)。结果第28天VEGFASODN组、错义序列组和PBS组肿瘤体积的大小分别为(212.6±196.5)、(468.2±161.3)和(473.1±166.7)mm3。VEGFASODN组微血管密度(12.26±0.78)较对照组(24.13±1.21)显著减少。结论VEGFASODN对裸鼠淋巴瘤的生长及其新生血管的生成有明显的抑制作用。     

Avidin chase reduces side effects of radioimmunotherapy in nude mice bearing human colon carcinoma

AIM: To evaluate the influence of avidin chase on the side effects of radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma and therapeutic outcome. METHODS: Purified anti-CEA monoclonal antibody (McAb) was biotinylated with NHS-biotin, and then radiolabeled with 188Re by the direct method. 188Re-labeled biotinylated anti-CEA McAb (188Re-CEA McAb-Bt) was intravenously injected followed by intravenous injection of avidin after 24 h. SPECT imaging and biodistribution study were performed at 28-48 h after the injection of 188Re-CEA McAb-Bt. Three groups of nude mice subcutaneously grafted with human colon carcinoma were treated 7 d after the graft. Mice in the avidin chase group received intravenous injection of 188Re-CEA McAb-Bt (11.1 MBq/ 20 μg) followed by intravenous injection of cold avidin (80 μg) after 24 h. Mice in the control group (treated group without avidin chase) only received the injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg), another control group (non-treated group) only received 0.1 ml normal saline solution. Toxicity was evaluated on the basis of change of body weight and peripheral WBC counts, and therapy effects were determined by variation in tumor volume. Histological analysis of tumors was also performed. RESULTS: Avidin chase markedly accelerated the clearance of 188Re-CEA McAb-Bt from the blood and normal tissues. The tumor uptakes of 188Re-CEA McAb-Bt at 28 h were 5.90 and 6.42% ID/g, respectively, in chase group and in non-chase group, while the tumor-to-background (T/NT) ratios were 3.19 and 0.56, respectively. The tumor uptake was slightly decreased by avidin chase, but the T/NT ratios were increased. In treated groups the growth rate of body weight and the number of WBC decreased after injection of 188Re-CEA McAb-Bt, and the WBC counts recovered earlier in the group with avidin chase than in the group without avidin chase. Compared to the nontreated group, treated groups with and without avidin chase showed significant anti-tumor effects. CONCLUSION: Avidin chase can effectively reduce the side effects of RIT, and improve therapeutic efficacy.     

Effects of recombinant human growth hormone on growth of human gastric carcinoma xenograft model in nude mice

AIM: To study effects of recombinant human growth hormone (rhGH) on growth of a human gastric carcinoma cell in vivo. METHODS: Experimental mice were divided into control group, rhGH group, oxaliplatin (L-OHP) group and rhGH L-OHP group. Cultured human gastric carcinoma cells BGC823 were inoculated into right axilla of nude mice and carcinoma xenograft model was established successfully. Inhibitory rate of xenograft tumor growth was estimated by measuring tumor volume; expression of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 proteins of xenograft tumor was detected using immunohistochemical S-P method. RESULTS: Tumor growth inhibitory rate, the positive expression rate of PCNA, Bax and Bcl-2 were 49.3%, 58.2%, 65.2% and 59.2% in rhGH L-OHP group respectively; 46.6%, 62.5%, 59.7% and 64.7% in L-OHP group; 5.0%, 82.7%, 23.2% and 82.2% in rhGH group and 0, 77.8%, 23.5% and 80.3% in control group. There was significant difference between rhGH L-OHP group (or L-OHP group ) and control group or rhGH group (P < 0.05), whereas there were no significant differences (P > 0.05) between L-OHP group and rhGH L-OHP group and between rhGH group and control group. CONCLUSION: rhGH does not accelerate the proliferation of human gastric cancer cell in vivo.     

Adjuvant effects of antineoplastic prostaglandins to cisplatin in nude mice bearing human ovarian cancer cells

Summary Effects of antineoplastic prostaglandins (PG) on human ovarian cancer cell growth were examined by using HR cells derived from ascites of a patient with serous cystadenocarcinoma of the ovary. With regard to inhibition of cancer cell proliferation in vitro, the effects of ⁷-PGA was most marked, followed by that of ¹²-PGJ₂, PGJ₂ and PGD₂. When antineoplastic prostaglandins were administered to nude mice bearing HR cells, tumor growth in groups treated with PGJ₂ and ¹²-PGJ₂ alone was significantly inhibited 63 days after tumor inoculation, compared to that in an untreated group. Consequently, a significant prolongation of median survival was obtained with ¹²-PGJ₂, compared to that in untreated groups and in groups with cisplatin alone. In addition, when prostaglandins were administered together with cisplatin, adjuvant inhibitory effects on the tumor growth were obtained 35, 56 and 63 days after tumor inoculation. Subsequently a significant prolongation of median survival was observed when cisplatin was combined with PGD₂ or ⁷-PGJ₁, compared to the results in groups treated with PGD₂ alone, ⁷-PGJ₁ alone or cisplatin alone. Combination of PGJ₂ or ¹²-PGJ₂ and cisplatin resulted in a significant decrease of hematocrit and body weight 63 days after tumor inoculation, suggesting a deterioration of the median survival. These results suggest that combination of PGD₂ or ⁷-PGJ₁ with cisplatin may be of clinical use for ovarian cancer resistant to cisplatin.Abbreviation Cisplatin cis-diamminedichloro-platinum(II) This work was supported in part by a grant-in-aid for comprehensive ten year strategy for cancer control from the Ministry of Health and Welfare of Japan (Y.K. and M.F.)     

Effect of antidepressants on body weight, ethology and tumor growth of human pancreatic carcinoma xenografts in nude mice

AIM： To investigate the effects of mirtazapine and fluoxetine, representatives of the noradrenergic and specific serotonergic antidepressant （NaSSA） and se- lective serotonin reuptake inhibitor （SSRI） antidepres- sant respectively, on body weight, ingestive behavior, locomotor activity and tumor growth of human pancre- atic carcinoma xenografts in nude mice. METHODS： A subcutaneous xenograft model of hu- man pancreatic cancer cell line SW1990 was estab- lished in nude mice. The tumor-bearing mice were ran- domly divided into mirtazapine group [10 mg/（kg·d）], fluoxetine group [10 mg/（kg·d）] and control group （an equivalent normal saline solution） （7 mice in each group）. Doses of all drugs were administered orally, once a day for 42 d. Tumor volume and body weight were measured biweekly. Food intake was recorded once a week. Locomotor activity was detected weekly using an open field test （OFT）. RESULTS： Compared to the fluoxetine, mirtazapine significantly increased food intake from d 14 to 42 and attenuated the rate of weight loss from d 28 to 42 （t = 4.38, P 〈 0.05）. Compared to the control group, food intake was significantly suppressed from d 21 to 42 and weight loss was promoted from d 35 to 42 in the fluoxetine group （t = 2.52, P 〈 0.05）. There was a significant difference in body weight of the mice after removal of tumors among the three groups. The body weight of mice was the heaviest （13.66 ± 1.55 g） in the mirtazapine group and the lightest （11.39 ± 1.45 g） in the fluoxetine group （F（2,12） = 11.43, P 〈 0.01）. The behavioral test on d 7 showed that the horizontal and vertical activities were significantly increased in the mirtazapine group compared with the fluoxetine and control groups （F（2,18） = 10.89, P 〈 0.01）. These effects disappeared in the mirtazapine and fluoxetine groups during 2-6 wk. The grooming activity was higher in the mirtazapine group than in the fluoxetine group （10.1 ± 2.1 vs 7.1 ± 1.9 ） （t = 2.40, P ?     

西咪替丁抑制人肠癌裸鼠移植瘤

目的：研究西咪替丁对人结肠癌裸鼠移植瘤生长的抑制作用及机制。方法：建立人结肠癌裸鼠皮下移植瘤模型。随机分2组,每组5只实验鼠。肿瘤种植前3 d开始分别皮下注射生理盐水(对照组)或西咪替丁(治疗组),每天1次,观察成瘤时间及瘤体成长情况。肿瘤种植后第7周处死实验鼠,测定瘤体大小,并用免疫组化方法测定肿瘤组织内微血管密度(MVD)和血管内皮生长因子(VEGF)的表达。结果：治疗组肿瘤体积明显小于对照组;治疗组肿瘤组织中的VEGF表达程度和MVD计数亦明显低于对照组。结论：西咪替丁通过抑制VEGF表达,减少血管生成,从而抑制肿瘤生长。