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当前位置: 代写毕业论文 >> 临床医学 >> 辐射诱导性自杀基因对胰腺癌体内治疗效果的实验观察

辐射诱导性自杀基因对胰腺癌体内治疗效果的实验观察

来源:华世论文网
 

【摘要】  目的 将PC-3胰腺癌细胞种植于balb/c裸小鼠皮下,建立移植瘤模型,观察pAdEgr-1-TK注入后在射线和前药GCV下对胰腺癌的治疗作用。方法 PC-3胰腺癌细胞以1×107混以0.1 ml磷酸盐缓冲液(PBS)皮下接种于balb/c裸鼠皮下,每组6只,共4组24只,待肿瘤长至约0.6 cm时,分为4组,以PC-3组作为对照,PC-3/pAdE组注射同量混以PBS的pAdEgr-1,PC-3/pAdET(不放射)和PC-3/pAdET(放射)组将纯化腺病毒pAdEgr-1-TK以1×109 pfu/ml、0.1 ml/只直接注射到治疗组肿瘤体内,1次/d,连续3 d,同时,腹腔内每日注射GCV(25 mg/kg),连续14 d,即分两次间隔48 h 行剂量为10 Gy的60Co-γ射线照射,观察各组动物肿瘤生长情况,每3天测量肿瘤体积[(长径×短径2)/2],治疗结束后,处死裸鼠,取瘤体称重,并在光镜下观察肿瘤的病理结果变化。结果 4组细胞接种后,balb/c裸小鼠均成瘤,成瘤潜伏期无差异,成瘤率为100%,治疗前(60Co-γ射线照射+GCV),四组动物瘤体大小无差异(P=0.824),治疗后,观察对照组肿瘤生长迅速,治疗组肿瘤生长明显减慢,体积和质量PC-3/pAdET(放射)组均明显小于其他3组(P=0.000)。结论 成功建立胰腺癌裸鼠移植瘤模型,体内实验证实Egr-1基因启动子在射线作用下可驱动下游TK自杀基因表达而对胰腺癌起治疗作用。

【关键词】  胰腺肿瘤 基因治疗 Egr-l启动子 大鼠

    The therapeutical effect of radio-inducible suicide gene on pancreatic cancer cell in vivo  

    Abstract   Objective  The nude mouse model of pancreatic cancer was established by implanting PC-3 cell subcutaneously into the animal, to observe the therapeutical effect of pAdEgr-1-TK with irradiation and prodrug GCV addition to the cancer cell. Methods  1×107 PC-3 cells together with 0.1 ml phosphate buffer solution (PBS) were implanted subcutaneously into 24 nude mice, which were divided equally into 4 groups. By the time when the tumor grew to 0.6 cm, the group PC-3/pAdE was injected with the mixture of PBS and pAdEgr-1, pAdEgr-1-TK was directly injected into the tumor of group PC-3/pAdET (non-irradiated) and group PC-3/pAdET (irradiated) qd for 3 days in the dosage of 1×109 pfu/ml×0.1 ml per mouse, meanwhile, GCV was injected into the abdominal cavity (25 mg/kg/day) for 14 days, when the mice would be irradiated by  60Co-γ in dosage 10Gy and once more 48 hours later. The volumes of the tumor were measured every 3 days (longer diameter × shorter diameter 2/2). At last, the tumors were taken out of the killed mice to observe the pathologic changes under light microscope. Results  Tumor successfully grew in all of the mice without variance during the latent period. No variance existed (P=0.824) before treatment (irradiation and GCV). After the treatment, the growth of tumor was very fast in the control group but markedly slowed down in the experimental group, while the volume and weight of tumor of group PC-3/pAdET were apparently less than that of the other three. All above-mentioned indicated that TK gene promoted by Egr-1 gene was activated to transcript by γ-rays, and TK suicide gene was highly expressed to inhibit the tumor cells significantly. Conclusion  The nude mouse model of pancreatic cancer is successfully established, and the downstream TK suicide gene can be highly expressed activated by the radio-inducible Egr-1 gene promoter, which provide therapy effect in pancreatic cancer in vivo.

    Key words   pancreatic neoplasms; gene therapy; Egr-1 promoter; rats

    将Egr-l启动子与肿瘤的自杀基因相耦联,再转染胰腺癌细胞,经辐射照射诱导后,实验证实Egr-l下游TK基因可超强表达。为进一步观察构建含 TK基因的腺病毒载体pAdEgr-1-TK对人胰腺癌细胞株PC-3的治疗作用,我们将肿瘤细胞PC-3制成体内胰腺癌模型,待成瘤后,pAdEgr-1-TK腺病毒直接注射至肿瘤,并腹腔给予GCV,在射线照射后,通过对肿瘤的体积、质量及平均肿瘤抑制率的观察比较,观察放射敏感启动子Egr-1调控的TK自杀基因在60Co-γ射线作用下对体内肿瘤的治疗效果。

    1  材料和方法

    1.1  病毒、细胞及动物  病毒为pAdEgr-1及pAdEgr-1-TK[1],人胰腺癌细胞株PC-3购自中科院上海细胞所;balb/c nu/nu裸鼠24只,雌雄不限,6~8周龄,质重(20±5)g,SPF级,购自中国科学院上海药物研究所。裸鼠均置于层流罩内饲养,所用饮水、垫料均经高温消毒,层流罩每日紫外线照射消毒。

    1.2  动物试验  将对数生长期PC-3细胞,用0.25%胰蛋白酶消化,PBS洗涤2次,调整细胞浓度至108/ml,在balb/c裸鼠前腹侧部接种细胞107细胞/只(100 μl/只),制备成裸鼠移植瘤模型,待肿瘤长至约0.6 cm时,随机分成4组,分别为PC-3组、PC-3/pAdE组(为pAdEgr-1转染组)、PC-3/pAdET(不放射)和PC-3/pAdET(放射)组(为pAdEgr-1-TK转染组)。

    待肿瘤长至约0.6 cm时,PC-3组作为对照,肿瘤体内注射PBS,PC-3/pAdE组注射同量混以PBS的pAdEgr-1,PC-3/pAdET(不放射)和PC-3/pAdET(放射)组将纯化腺病毒pAdEgr-1-TK以1×109 pfu/ml、0.1 ml/只直接注射到治疗组肿瘤体内,1次/d,连续3 d,同时,腹腔内每日注射GCV(25 mg/kg),连续14 d,即分两次间隔48 h行剂量为10 Gy的60Co-γ射线局部照射,第2次照射后12 d,采用颈椎脱臼法全部处死小鼠,取各小鼠肿瘤称重,并记录质量。取肿瘤组织,10%甲醛固定,石蜡包埋,HE染色。

    1.3  肿瘤体积与平均肿瘤抑制率的比较  每周两次观察肿瘤生长情况,用游标卡尺测量肿瘤长短径,以其最大径(A)与最小径(B)来计算肿瘤体积[1]。

    肿瘤体积(mm3)=A(mm)×B(mm)2


    肿瘤的平均肿瘤抑制率按下式计算:

    平均肿瘤抑制率=转染基因细胞平均瘤体质量-转染基因细胞平均瘤体质量

    ?????????????????????????×100%

    未转染基因细胞平均瘤体质量

    1.4  统计学方法  计量数据以x±s表示,用SPSS 11.5统计软件进行各组间方差分析。

    2  结果

    2.1  裸鼠体内试验  裸鼠成瘤后约0.6 cm,随机分成4组,分别为PC-3组、PC-3/pAdE组、PC-3/pAdET(不放射)和PC-3/pAdET(放射)组,治疗不同天数后肿瘤体积明显不同,PC-3/pAdET(放射)组的肿瘤体积明显低于对照PC-3组和PC-3/pAdE组,治疗前各组肿瘤体积大小差异无统计学意义(F=0.31,P=0.824),见表1。

    2.2  裸鼠的瘤体质量及平均肿瘤抑制率  各组动物经治疗后,PC-3/pAdET(放射)组的肿瘤质量明显低于对照PC-3组和PC-3/pAdE组(P=0.000)。平均肿瘤抑制率各组与PC-3对照组相比分别为PC-3/pAdE组(0.02%)、PC-3/pAdET(不放射)组(25.90%)和PC-3/pAdET(放射)组(90.43%),说明基因-放射治疗后肿瘤生长受到了明显的抑制,见表2。

    2.3  组织病理学检查  PC-3细胞接种动物后,分别用PC-3/pAdE、PC-3/pAdET(不放射)和PC-3/pAdET(放射)进行处理,肿瘤的HE染色病理表现PC-3/pAdE组与对照组PC-3没有明显区别,细胞的形态结构完整,细胞肥大,无坏死及出血等。用PC-3/pAdET(不放射)处理后,肿瘤内有少量淋巴细胞浸润,在用PC-3/pAdET(放射)进行处理后,肿瘤组织坏死明显,且有较多的纤维化。

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