摘要
目的:我们以前的研究表明,人舌鳞癌细胞系Tca8113细胞中,维甲酸受体β(Retinoic Acid Receptor beta,RakRβ)基因转录降低与其异常的增殖、分化和凋亡有关;体外转导外源RARβ基因的Tca8113细胞用全反式维甲酸(All-Trans Refinoic Acid,ATRA)处理14天,细胞凋亡率可达80.7%。本研究目的是在已有的体外研究基础上,观察RARβ基因联合维甲酸对荷口腔鳞癌鼠的体内抗肿瘤作用和安全性,并初步探讨其抗肿瘤机制。
方法:pSG5RARβ质粒转化JM109细菌,细菌扩增、抽提质粒;BamH-I、Sac-I双酶切鉴定。Tea8113细胞复苏后,用RPMI 1640培养基(含10%小牛血清,100μ/ml青霉素,100μ/ml链霉素),在37℃,5%CO_2条件下培养;取对数生长期细胞,以0.25%胰酶消化2-3分钟后,收集细胞。将1×10~7/ml密度的细胞悬液接种于裸鼠双侧臀部皮下,每个部位接种细胞悬液0.2ml。2周后,将裸鼠分为3组,对照组6只、ATRA治疗组8只、RARβ+ATRA治疗组6只。取pSG5RARβ质粒45μg、脂质体Lipofectamine 75μl,用无血清PRMI 1640培养液稀释至500μl,充分混匀后室温放置30分钟,使之形成脂质体-DNA复合物,以无血清培养基稀释至2ml备用。于肿瘤接种后第二、三周末,取RARβ+ATRA组裸鼠,在无菌条件下,沿肿瘤周径每间隔120度选取一注射点,共三个注射点,缓慢向瘤内边退针边注射DNA-脂质体复合物,每点50μl,每只注射总量300μl;ATRA治疗组及对照组用等量生理盐水,采用同样方法瘤内注射。自第二周末开始,ATRA治疗组和转RARβ基因组裸鼠以腰麻导管,管饲给予15mg/kg剂量的ATRA(以3mg/ml的浓度溶于消毒的
区A巴p基因联合维甲酸抗口腔鳞癌的动物实验研究
超纯麻油),每天1次,每周5天,共4周。每4天观察记录肿瘤最大径
扫)和最大相交径(b)1次,肿瘤体积计算公式:V=(。/6)a.b‘;每周观察
记录动物一般情况。给药4周后处死动物,取部分肿瘤组织作常规病理
切片,HE染色,显微镜下观察。取约lmm‘肿瘤组织,立即置h戊H醛
PBS固定液固定,脱水包埋、超薄切片,醛酸钠和柠檬酸铅双染色,透
射电镜观察,照相记录。取新鲜组织 5 Inln,机械法制备单细胞悬液,PI
荧光染色,流式细胞仪检测,LysiS软件分析凋亡细胞比例。
结果:pSGSRAR 6质粒被 Sacl、BalnHI双酶切产生长约 1.4kb cDNA
片断,该片断大小与 RAR p CDNA相符。将 2 X 10’细胞接种于裸鼠双侧臀
部皮下,两周后 80%的裸鼠出现皮下肿瘤结节。对照组、ATRA、RAR D+ATRA
治疗组肿瘤平均体积分别为41.4M‘,21.6M’,29.SM’。4周治疗结柬
时,三组平均体积分别为3111.ZM,1428.smm‘,653.9毗治疗后,各
组肿瘤平均体积分别增加 75.1,66.1和 ZI.9倍,经 SAS6.02版统计软
件分析,RAR p+ATRA治疗组平均体积与对照组相比差异有统计学意义
…刃.0479人而ATRA与对照组间,两处理组间无统计学差异。三组平
均瘤重分别为2.799、0.gig和0.579,经SAS6.02版统计软件分析,ATRA
和 RAR P+ATRA治疗组瘤重与对照组相比差异有统计学意义(p<0.05),
而两处理组间无明显差异。ATRA治疗组抑瘤率为 67.4%,RAR 6+ATRA治
疗组抑瘤率为79.6%。肉眼观,肿瘤剖面呈鱼肉状,中央有坏死,组织
质脆;镜下见肿瘤细胞呈不均一圆形,核浆比例大,核染色深,对照组细
胞增生旺盛,核分裂相较多;ATRA和 RAR 6+ATRA治疗组细胞核分裂相对
少见;ATRA处理组可见部分细胞核固缩;RAR p+ATM治疗组有较多细胞
出现核固缩,呈片状分布,偶见细胞片状坏死。流式细胞仪凋亡检测结
果表明,对照组肿瘤细胞平均凋亡率为 6.3%,ATRA组和 RAR 6 +ATRA组
凋亡率分别为16.4%和16.6$。两者与对照组相比凋亡率明显增高
Q狈.05人但两处理组差别不明显。透射电镜形态观察,肿瘤细胞呈多
角形,核大,核仁多,分裂相较多,凋亡细胞极少。ATRA治疗组可见散
2
RAR p基因联合维甲酸抗口腔鳞癌的动物实验研究
在较多的凋亡细胞,表现为细胞皱缩,变小、变圆,染色质固缩、边聚,
细胞浆浓缩。转基因组组织可见较多凋亡细胞,呈散在分布。
结论:1、全反式维甲酸是治疗口腔鳞癌有希望的药物,15吧/kg乃
的ATRA治疗4周,对荷口腔鳞癌裸鼠体内抑瘤率达67.4%。
2、RAR
Objective: Our previous study showed that retinoic acid receptor-beta (RAR P ) gene transcription decreased in human tongue squamous carcinoma cell line (Tca8113) was associated with abnormal cell apoptosis and differentiation . The treatment with all-trans retinoic acid (ATRA) for 14 days induced cell apoptosis by 80.7% of Tca8113 cells transfected with RAR P gene . The purpose of this study was to determine the antitumor effect of RAR 3 gene and ATRA on human oral squamous carcinoma cell xenograft in athymic nude mice and to investigate the mechanism of growth inhibition of tumor in vivo,which was depended on the basis of in vitro study.
Materials and methods: JM-109 bacteria were transfected with pSGSRAR P plasmids. The transfected bacteria were cultured in LB media at 37.The pSGSRARP plasmids were extracted from bacteria using plasmid mini extraction kit after the proliferation of the transfected bacteria. The purified plasmid DNA was digested with restriction endonuclease BamH- I and Sac- I ,then were confirmed using 0.8% agarose electrophoresis. The Tca8113 cells were routinely cultured in RPMI-1640 media supplemented with 10% bovine calf serum,2mM 1-glutamine ,100units/ml penicillin and 100 units/ml streptomycin in 95% air and 5% CO: Cells were harvested by trypsinization and resuspended in RPMI-1640 serum-free midia at a concentration of 1 X 107cells/ml .
Athymic nude mice (6-7 weeks old, about 20 g in weight) were implanted with 2 X106 cells bilaterally into the buttocks with a 24 gauge needle/Ice tuberculin syringe. After two weeks the animals were divided randomly into three groups: control group, ATRA group and ATRA +RAR P group. There were 6,8 and 6 mice in each group, respectively. The lipofectamine-DNA compounds were prepared by mixing 75 u 1 lipofectamine and 45 u g pSGSRAR P plasmids after both of them were diluted in 500 u 1 RPMI -1640 serum-free media. The compounds were placed at room temperature for thirty minutes, so that the lipofectamine-DNA compounds could be formed. At the 14th and 21th day after the implanting of Tca8113 cells, selected three points around the tumor, there was an interval of 120?between two points .The compounds of lipofectamine-DNA (50 n 1) were injected into the tumor at each point, So that the compound can diffuse better in the tumor. The same volume of normal saline was injected into the tumors of mice in control group and ATRA group. At the same time, ATRA were administered p.o to mice in ATRA and ATRA +RAR 0 group with a 20 gauge intragastric feeding tube daily ,5 days per week, in 0.1 ml of super-refined and sterilized sesame oil at a dose of 15 mg/kg/d for 4 weeks. The sizes of the tumors were recorded every 4 days. The formula for the volume of the tumor was v- x /6(ab2) (a representing the longest diameter of the tumor ; b representing the longest diameter vertical to a). At the end of the treatment, all the animals were killed. Tumors were surgically excised. Some samples were fixed in 10% formalin for 24 to 48 hours before being embedded in paraffin. Hematoxylin and erosin staining were performed for regular pathological examination. Some samples were also prepared for electronic microscopic obseration and flow cytometry test. Lysis precedure was used to analyze the apoptotic ratio.
Results: One small fragment of DNA was released from pSGSRAR 3 plasmid after digestion. The size of this cDNA fragment was about 1.4 kb consistent with RAR P cDNA. Tumors were formed in mice after subcutaneous injection of 2X106Tca8113 cells with 80% success rate . The average volumes of the tumors in control group, ATRA group and RAR 0 +ATRA group were 41.4mm3,21.6mm3 and 29.8mm3 respectively. At the end of the treatment, the average volumes of each group were 3111.2mm3, 1428.8mm3 and 653.9mm3, respectively. The Volumes of each group increased 75.1, 66.1 and 21.9 times, respectively. The average weights of the tumors for each group were 2.79g, 0.9Ig and 0.57g, respectively. SAS6.02 version statistical procedure was used for the statistic analyse. The difference of the
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