三氧化二砷对人子宫肉瘤MES-SA细胞系裸鼠皮下移植瘤的作用研究

发布时间：2018-02-22 03:57

  本文关键词： 三氧化二砷 子宫肉瘤 凋亡　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:采用体内实验研究的方法,选用人子宫肉瘤MES-SA细胞系,建立人子宫肉瘤裸鼠皮下移植瘤模型,研究三氧化二砷(Arsenic Trioxide,As_2O_3)对人子宫肉瘤细胞系(MES-SA)是否具有抗肿瘤作用并初步研究其可能机制。方法:人子宫肉瘤细胞株MES-SA在含体积分数0.10标准胎牛血清的RPM I-1640培养液中进行细胞培养,并获取单细胞悬液;实验动物为40只4-6周龄的裸鼠,MES-SA单细胞悬液皮下注入裸鼠体内,产生瘤结节,建立人子宫肉瘤细胞裸鼠皮下移植瘤模型。随机分为5组:As_2O_3低、中、高剂量组(剂量分别为1.0、2.5、5.0mg/kg)、生理盐水(NS)空白对照组、异环磷酰胺(IFO)阳性对照组,每组8只,连续10天腹腔注射相应药物,观察裸鼠一般情况及瘤结节生长情况。停药后处死裸鼠剥离瘤组织,检测以下指标:称瘤质量并计算抑瘤率;流式细胞仪检测细胞凋亡率;提取瘤组织中RNA,采用荧光标记的特异性Taq探针法,进行PCR反应,测定CT值,进行半胱天冬氨酸蛋白酶(Caspase3)基因表达的相对定量分析,检测并比较caspase-3基因表达;制备肿瘤组织石蜡切片,进行p-ERK抗体免疫组化染色,观察着色情况,测定p-ERK表达(免疫组化SP法)。进行数据处理及统计学分析。结果:1.一般情况:所有裸鼠均有瘤结节长出,成功建立移植瘤模型。用药后各As_2O_3组裸鼠一般状态良好,精神饮食可、大小便正常、正常活动、生长良好、无明显消瘦,与NS组差别不大;IFO阳性对照组裸鼠用药后一般状态不佳,出现坐立不安等疑似腹膜刺激症状,且精神差,饮食少,反应迟钝,活动少,并有腹泻,消瘦等。2.移植瘤质量及抑瘤率:三组As_2O_3组及IFO组的瘤质量均小于NS组瘤质量,差异均有统计学意义,(P0.05);As_2O_3高剂量组及IFO组瘤质量最低,较其他组有显著差异(P0.05);As_2O_3高剂量组与IFO组瘤质量差别不大,As_2O_3中、低剂量组差别也不明显。结果提示As_2O_3有抑制肿瘤作用,且高剂量As_2O_3与IFO有相似的抑瘤作用,抑瘤作用最强。3.凋亡率:三组As_2O_3组及IFO组均有典型凋亡峰出现;与NS组相比,As_2O_3组及IFO组凋亡率均升高;且As_2O_3组凋亡率高于IFO组(P0.05)。As_2O_3组凋亡率随剂量增加而升高,中、低剂量组差别不明显(P0.05),高剂量组凋亡率高于中、低剂量组。可见As_2O_3能促进细胞凋亡,且高剂量As_2O_3组促凋亡作用最明显。4.caspase-3基因表达:各As_2O_3组caspase-3基因表达高于NS组及IFO组(P0.05),提示As_2O_3可上调caspase-3基因表达;高剂量As_2O_3组表达高于中、低剂量组,但差异无统计学意义(P=0.192)。5.p-ERK表达:各剂量As_2O_3组及IFO组p-ERK的表达均低于NS组,但无显著差异;高剂量As_2O_3组p-ERK表达率最低,NS组最高,高剂量As_2O_3组显著低于NS组(Z=-2.063,P=0.0390.05)。提示As_2O_3可下调p-ERK的表达,且高剂量组作用最明显。结论:1.As_2O_3对人子宫肉瘤裸鼠皮下移植瘤增殖发挥出较好的抑制生长作用,能诱导肿瘤细胞凋亡;As_2O_3可上调Caspase-3、下调p-ERK基因的表达;促凋亡可能与上调Caspase-3基因表达有关,抑瘤作用还可能与下调p-ERK表达影响ERK-MAPK信号传导通路有关。2.与IFO相比,As_2O_3不良反应相对较小,耐受可,相对安全有效,有望应用于除白血病外的多种实体瘤的治疗,为肿瘤的治疗带来新的希望。
[Abstract]:Objective: To study the in vivo, the human uterine sarcoma cell line MES-SA, establish the model of subcutaneous sarcoma of uterus in nude mice, to study the effect of arsenic trioxide (Arsenic Trioxide, As_2O_3) on human uterine sarcoma cell lines (MES-SA) have antitumor effect and study its possible mechanism. Methods: human uterine sarcoma cell line MES-SA the volume fraction of 0.10 standard fetal bovine serum RPM I-1640 culture medium for cell culture, and to obtain single cell suspension; experimental animal for 40 4-6 week old mice, MES-SA single cell suspension into nude mice subcutaneous nodules, produce, establish the model of subcutaneous human uterine sarcoma xenografts were randomly. Divided into 5 groups: As_2O_3, low and high dose group (the dose was 1.0,2.5,5.0mg/kg), normal saline (NS) control group, ifosfamide (IFO) positive control group, 8 rats in each group, intraperitoneal injection for 10 days The corresponding drugs, observe the growth of nude mice in general and tumor nodules were sacrificed. Peel the tumor tissue after discontinuation, detection of the following indicators: quality of tumor and the inhibition rate was calculated; cell apoptosis was detected by flow cytometry; extraction of tumor tissue RNA, using specific Taq probe fluorescence labeling, PCR reaction determination of CT value of caspase (Caspase3) gene expression relative quantitative analysis, comparison of Caspase-3 gene expression and detection; preparation of paraffin sections of tumor tissues, p-ERK immunohistochemical staining, observe the staining, the expression of p-ERK was determined (SP immunohistochemical method). Analysis of data processing and statistics results: 1.. General: all nude mice have long nodules, xenografts were established successfully. After the treatment of As_2O_3 mice were generally in good condition, the spirit of diet, urine normal, normal activity, good growth, ignorance Significant weight loss, and NS group had no significant difference; IFO positive control group of nude mice after administration of general condition, appeared on tenterhooks suspected peritoneal irritation symptoms, and poor spirit, eating less, unresponsive, less activity, and diarrhea, weight loss and quality.2. tumor inhibition rate: three group As_2O_3 group and IFO group. Tumor mass was less than that of NS group in quality, the differences were statistically significant (P0.05); high dose of As_2O_3 group and IFO group of minimum quality than other groups have significant difference (P0.05); high dose of As_2O_3 group and IFO group, tumor mass difference, As_2O_3 in low dose group, the difference is not obvious. The results suggest that As_2O_3 has inhibitory effect on tumor, and high doses of As_2O_3 and IFO have similar anti-tumor effect, the antitumor effect of.3. was the strongest apoptosis rate: three group As_2O_3 group and IFO group showed typical apoptosis peak; compared with NS group, As_2O_3 group and IFO group. The apoptosis rate was increased and the apoptosis rate of As_2O_3 group is high; In group IFO (P0.05).As_2O_3 group apoptosis rate increased along with the increase of the dose, low dose group, the difference was not significant (P0.05), the apoptosis rate in high dose group than in low dose group. The As_2O_3 can promote cell apoptosis, and apoptosis of high dose of As_2O_3 group the most obvious expression of.4.caspase-3 gene by As_2O_3 group the expression of Caspase-3 gene was higher than that of NS group and IFO group (P0.05), suggesting that As_2O_3 can upregulate the expression of Caspase-3 gene; high dose of As_2O_3 group was higher than that in the low dose group, but the difference was not statistically significant (P=0.192).5.p-ERK expression: the expression of As_2O_3 in each dose group IFO and group p-ERK were lower than NS group, but no significant difference; high dose group As_2O_3 p-ERK expression was the lowest and the highest in NS group, high dose of As_2O_3 group was significantly lower than that of group NS (Z=-2.063, P=0.0390.05). It is suggested that As_2O_3 can downregulate the expression of p-ERK, and the most obvious effect in the high dose group. Conclusion: 1.As_2O_3 of uterine sarcoma nude 榧犵毊涓嬬Щ妞嶇槫澧炴畺鍙戞尌鍑鸿緝濂界殑鎶戝埗鐢熼暱浣滅敤,鑳借�卞�艰偪鐦ょ粏鑳炲噵浜，

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