蒿甲醚增强人结直肠癌裸鼠移植瘤放疗效果及其机制研究

发布时间：2018-05-31 04:29

本文选题：蒿甲醚(ARE) + 人结直肠癌裸鼠移植瘤　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]:大量研究发现青蒿素及其衍生物除具有抗肿瘤活性外,还能增加肿瘤的放疗敏感性,但其具体机制尚有很多未知的方面。本课题通过研究蒿甲醚(artemether,ARE)对放疗敏感人结直肠癌HCT116细胞及放化疗抵抗人结直肠癌HCT116-CRR (chemoradioresistant,CRR)细胞裸鼠移植瘤放疗效果的影响,并探讨其可能的作用机制是否与调控上皮-间质转化(epithelial mesenchymal transition,EMT)相关,为蒿甲醚作为结直肠癌放疗增敏药物提供实验依据。[方法] :1、建立人结直肠癌裸鼠移植瘤模型:培养放疗敏感人结直肠癌HCT116细胞及放化疗抵抗人结直肠癌HCT116-CRR细胞,按二代移植瘤接种的方法,分别建立BALB/c-nu裸小鼠人结直肠癌皮下移植瘤模型,待肿瘤长至约5 × 5 ×5mm3时,将各部分裸鼠随机分为四组,分别为对照组(Control)、蒿甲醚组(ARE)、放疗组(RT)、联合组(RT+ARE),按不同分组进行治疗;2、观察各组裸鼠的成瘤率、生存状态、体重变化等;3、隔日监测肿瘤长短径,计算肿瘤体积抑制率及增敏系数(efficiency factor,EF),绘制瘤体积变化曲线;4、治疗结束处死各组裸鼠取瘤块,称重并计算抑瘤率;5、通过流式细胞仪检测各组瘤组织的凋亡率及坏死率;6、取各组瘤组织10%甲醛固定,常规石蜡包埋、切片,HE染色,光镜下观察;7、免疫组化检测各组瘤组织中EMT相关因子E-cadherin、N-cadherin、Vimentin、Snail、Slug、Twist、β -Catenin 的表达情况;8、RT-PCR 检测各组瘤组织中 E-cadherin、N-cadherin、Vimentin、Snail、Slug、Twist、β-Catenin基因的表达水平;9、Western blot 检测各组瘤组织中 E-cadherin、N-cadherin、Vimentin、Snail、Slug、Twist、β -Catenin蛋白的表达水平;10、采用SPSS22.0软件进行数据统计分析,数据以均数±标准差(x±s)表示,满足方差齐性检验要求的数据采用方差分析,事后两两比较采用LSD-t检验;不满足方差齐性检验的数据采用Brown-Forsythe分析,事后两两比较采用Dunnett's T3 检验。[结果]:1. HCT116及HCT116-CRR细胞裸鼠成瘤率为100%;各组间裸鼠体重无明显差异,治疗期间放疗组及联合组裸鼠活动明显减少,精神稍差。2. HCT116细胞各组裸鼠瘤体积治疗前无明显差异,治疗后联合组裸鼠瘤体积小于放疗组(P0.05),联合组肿瘤体积抑制率91.66%明显高于放疗组88.13%,蒿甲醚对HCT116细胞裸鼠移植瘤的EF为3.20; HCT116-CRR细胞各组裸鼠瘤体积治疗前无明显差异,治疗后联合组裸鼠瘤体积小于放疗组(P0.05),联合组肿瘤体积抑制率80.84%明显高于放疗组65.04%,蒿甲醚对HCT116-CRR细胞裸鼠移植瘤的EF为2.43。3. HCT116细胞联合组裸鼠平均瘤重小于放疗组(P0.05),联合组抑瘤率91.20%明显高于放疗组88.83%; HCT116-CRR细胞联合组裸鼠平均瘤重小于放疗组(P0.05),联合组抑瘤率83.84%明显高于放疗组67.24%。4. HCT116细胞联合组裸鼠瘤组织凋亡率(41.353±2.628) %大于放疗组(30.47±0.738)%(P0.05),各组裸鼠瘤组织坏死率无明显差异;HCT116-CRR细胞联合组裸鼠瘤组织凋亡率(24.280±1.883) %大于放疗组(12.803±0.788) %(P0.05),各组裸鼠瘤组织坏死率无明显差异。5.各组瘤组织HE染色后光镜下观察,HCT116细胞对照组裸鼠瘤组织中心可见明显的大片出血坏死液化区,坏死区域内有血细胞浸润,各治疗组裸鼠瘤组织坏死区域明显扩大,以联合组最为显著;HCT116-CRR细胞对照组裸鼠瘤组织可见部分肿瘤性坏死,肿瘤间质增生明显,各治疗组裸鼠瘤组织坏死区域有不同程度的扩大,以联合组显著。6.免疫组化检测各组裸鼠瘤组织中EMT相关因子的表达情况。HCT116细胞各组裸鼠瘤组织中,上皮标志物E-cadherin、β-Catenin呈强阳性表达,与放疗组比较,联合组表达上调;间质标志物N-cadherin在各组中呈阴性表达,Vimentin、Snail、Slug、Twist呈弱阳性表达,与放疗组比较,联合组表达下调。HCT116-CRR细胞各组裸鼠瘤组织中,上皮标志物E-cadherin在放疗组呈阴性表达,在联合组呈弱阳性表达,β-Catenin在各组中呈弱阳性表达,与放疗组比较,联合组表达上调;间质标志物N-cadherin、Slug在各组中呈弱阳性表达,Vimentin、Snail、Twist呈强阳性表达,与放疗组比较,联合组表达下调。7. RT-PCR检测各组裸鼠瘤组织中EMT相关因子的基因相对表达量。HCT116细胞联合组裸鼠瘤组织与放疗组比较,β-Catenin的基因表达量较放疗组升高(P0.05),Vimentin的基因表达量较放疗组下降(P0.05)。HCT116-CRR细胞联合组裸鼠瘤组织与放疗组比较,E-cadherin、β-Catenin的基因表达量较放疗组升高(P0.05),N-cadherin、Vimentin、Snail、Slug、Twist 的基因表达量较放疗组下降(P0.05)。8. Western blot检测各组裸鼠瘤组织中EMT相关因子的蛋白表达水平。HCT116细胞联合组裸鼠瘤组织与放疗组比较,β-Catenin的蛋白表达量较放疗组升高(P0.05),Vimentin的蛋白表达量较放疗组下降(P0.05)。HCT116-CRR细胞联合组裸鼠瘤组织与放疗组比较,E-cadherin、β-Catenin的蛋白表达量较放疗组升高(P0.05),N-cadherin、Vimentin、Snail、Slug、Twist 的蛋白表达量较放疗组下降(P0.05)。[结论]:1. ARE联合放疗能显著抑制人结直肠癌HCT116细胞裸鼠移植瘤的生长,从而增强其放疗效果;ARE联合放疗能显著抑制人结直肠癌HCT116-CRR细胞裸鼠移植瘤的生长,从而增强其放疗效果,逆转其放化疗抵抗性;2. ARE联合放疗能促进人结直肠癌HCT116细胞裸鼠移植瘤的凋亡及坏死,从而增强其放疗效果;ARE联合放疗能促进人结直肠癌HCT116-CRR细胞裸鼠移植瘤的凋亡及坏死,从而增强其放疗效果,逆转其放化疗抵抗性;3. ARE联合放疗能上调人结直肠癌HCT116细胞裸鼠移植瘤上皮标志物β-Catenin的表达,下调间质标志物Vimentin的表达,提示ARE可能通过阻滞EMT增强人结直肠癌HCT116细胞裸鼠移植瘤放疗效果;4. ARE联合放疗能上调人结直肠癌HCT116-CRR细胞裸鼠移植瘤上皮标志物 E-cadherin、β-Catenin 的表达,下调间质标志物 N-cadherin、Vimentin、Snail、Slug、Twist的表达,提示ARE可能通过逆转EMT增强人结直肠癌HCT116-CRR细胞裸鼠移植瘤放疗效果,逆转其放化疗抵抗性;5. ARE能增强人结直肠癌裸鼠移植瘤放疗效果,可能成为一种低毒高效的放射增敏剂,应用于结直肠癌放射治疗当中。
[Abstract]:[Objective]: a large number of studies have found that artemisinin and its derivatives can increase the radiosensitivity of tumor in addition to anti-tumor activity, but there are still many unknown mechanisms in its specific mechanism. The study was conducted by the study of artemether (ARE) for HCT116 cells in colorectal cancer sensitive human colorectal cancer and HCT116-CRR (CHEM) for colorectal cancer. The effect of oradioresistant, CRR) on the radiotherapy effect of transplanted tumor in nude mice and whether its possible mechanism is related to the regulation of epithelial mesenchymal transition (epithelial mesenchymal transition, EMT), and provide experimental evidence for artemether as a radiotherapy sensitizing drug for colorectal cancer. [method] 1, a model of human colorectal carcinoma in nude mice was established. The HCT116 cells of colorectal cancer sensitive human colorectal cancer and HCT116-CRR cells of radiochemotherapy resistant human colorectal cancer were cultured in accordance with the method of inoculation of two generations of transplanted tumor. The tumor model of human colorectal carcinoma in BALB/c-nu mice was established respectively. When the tumor grew to about 5 x 5 x 5mm3, the nude mice were randomly divided into four groups, the control group (Control), Artemisia Artemisia, respectively. Methyl ether group (ARE), radiotherapy group (RT), combined group (RT+ARE), treated with different groups, 2, observe the tumor formation rate, survival state, body weight change of nude mice, 3, monitor the long diameter of tumor on the other day, calculate the tumor volume inhibition rate and increase the sensitivity coefficient (efficiency factor, EF), draw the volume change curve of the tumor; 4, take the nude mice at the end of the treatment and take the tumor. Block, weighing and calculating the tumor suppressor rate; 5, the rate of apoptosis and necrosis of the tumor tissues were detected by flow cytometry; 6, the tumor tissues were fixed with 10% formaldehyde, conventional paraffin embedding, slicing, HE staining, observed under light microscope; 7, immunohistochemistry was used to detect E-cadherin, N-cadherin, Vimentin, Snail, Slug, Twist, and beta -Catenin in each group of tumor tissues. 8, RT-PCR detected the expression level of E-cadherin, N-cadherin, Vimentin, Snail, Slug, Twist, and beta -Catenin in the tumor tissues, and 9, Western blot to detect the expression level of the tumor tissue, and 10. The data were expressed with mean standard deviation (x + s). The data that met the requirements of variance homogeneity test were analyzed by variance, and LSD-t test was used for the post 22 comparison; Brown-Forsythe analysis was used for the data that did not meet the homogeneity of variance test, and 22 compared with Dunnett's T3 test. [fruit]: 1. HCT116 and HCT116-CRR cells in nude mice were 100%. There was no significant difference in weight of nude mice in each group. The activity of the radiotherapy group and the combined group of nude mice decreased significantly during the treatment. There was no significant difference in the tumor volume of nude mice in the.2. HCT116 cells. The volume of tumor volume in the combined group was less than that of the radiotherapy group (P0.05). The tumor volume inhibition rate of the combined group was 91.66% significantly higher than that of the radiotherapy group 88.13%, artemether. The EF of HCT116 cell xenografts in nude mice was 3.20, and there was no significant difference in the tumor volume of nude mice in HCT116-CRR cells. The volume of tumor volume in the combined group was less than that of the radiotherapy group (P0.05). The tumor volume inhibition rate of the combined group was 80.84% higher than that of the radiotherapy group (65.04%). The EF of artemether to HCT116-CRR cell nude mice was 2.43.3. HCT116 cells. The average tumor weight of nude mice in the combined group was less than that of the radiotherapy group (P0.05), the tumor inhibition rate in the combined group was 91.20% significantly higher than that in the radiotherapy group (88.83%), and the average tumor weight of nude mice in the HCT116-CRR cell combined group was less than that of the radiotherapy group (P0.05), and the tumor inhibition rate of 83.84% in the combined group was significantly higher than that in the radiotherapy group (41.353 + 2.628)% of the 67.24%.4. HCT116 cell group (41.353 + 2.628). Group (30.47 + 0.738)% (P0.05), the necrosis rate of tumor tissue in nude mice was no significant difference, the apoptosis rate of tumor tissue in nude mice of HCT116-CRR cell group was (24.280 + 1.883)% greater than that of radiotherapy group (12.803 + 0.788)% (P0.05). There was no significant difference in the necrosis rate of tumor tissue in each group of nude mice. The tumor tissue of each group was observed under the light microscope after HE staining, and the nude mouse tumor of HCT116 cell control group was nude mice. The tissue Center showed obvious area of hemorrhagic necrotic liquefaction, blood cell infiltration in the necrotic region, the necrotic area of tumor tissue in nude mice was obviously enlarged in the treatment group, and the most significant in the combined group. The tumor tissue of the nude mice of the HCT116-CRR cell control group was partly tumor necrosis, the tumor interstitial hyperplasia was obvious, and the necrotic region of the nude mice was necrotic area in the treatment group. The expression of EMT related factors in tumor tissue of nude mice was detected by.6. immunohistochemistry in different groups. The epithelial markers, E-cadherin, and beta -Catenin were strongly positive in the tumor tissues of each group of nude mice of each group of.HCT116 cells. Compared with the radiotherapy group, the expression of the combined group was up regulated, and the interstitial marker N-cadherin was negative in each group. The expression of Vimentin, Snail, Slug, Twist showed weak positive expression. Compared with the radiotherapy group, the joint group expressed the negative expression of the epithelial marker E-cadherin in the tumor tissue of.HCT116-CRR cells in the radiotherapy group, and was weakly positive in the combined group. The expression of beta -Catenin in each group was weakly positive, and the expression of the combined group was up regulated compared with the radiotherapy group. The interstitial marker N-cadherin and Slug were weakly positive in each group, Vimentin, Snail, and Twist were strongly positive. Compared with the radiotherapy group, the expression of.7. RT-PCR in the joint group was down regulated to detect the relative expression of EMT related factors in the tumor tissues of the nude mice. The tumor tissue of the group of.HCT116 cells in the combination group of the group of.HCT116 cells was compared with the radiotherapy group, and the gene table of the beta -Catenin was expressed. Compared with radiotherapy group, the expression of Vimentin was lower than radiotherapy group (P0.05). Compared with radiotherapy group, the gene expression of E-cadherin and beta -Catenin was higher than that of radiotherapy group (P0.05), N-cadherin, Vimentin, Snail, Slug, and the gene expression of Twist was lower than that of radiotherapy group. The protein expression level of EMT related factors in the tumor tissues of nude mice by blot was compared with the radiotherapy group. The protein expression of beta -Catenin was higher than that of the radiotherapy group (P0.05), and the protein expression of Vimentin was lower than that of the radiotherapy group (P0.05) the tumor tissue of the nude mice was compared with the radiotherapy group, E-cadhe, E-cadhe. Rin, the protein expression of beta -Catenin was higher than that of radiotherapy group (P0.05), the protein expression of N-cadherin, Vimentin, Snail, Slug, Twist decreased (P0.05). [Conclusion]: 1. ARE combined radiotherapy could significantly inhibit the growth of human colorectal carcinoma HCT116 cell xenografts, and enhance the effect of radiotherapy; ARE combined radiotherapy can significantly inhibit human knot. The growth of HCT116-CRR cell xenografts in rectal carcinoma of rectal cancer can enhance the effect of radiotherapy and reverse its chemoradiotherapy. 2. ARE combined with radiotherapy can promote the apoptosis and necrosis of human colorectal HCT116 cell xenografts in nude mice and enhance the effect of radiotherapy. ARE combined with radiotherapy can promote the withering of human colorectal HCT116-CRR cells in nude mice. Death and necrosis can enhance the effect of radiotherapy and reverse its chemoradiotherapy. 3. ARE combined with radiotherapy can regulate the expression of the epithelial marker of the tumor HCT116 cells in colorectal cancer and down regulate the expression of the interstitial marker Vimentin, suggesting that ARE may increase the efficacy of ARE by blocking EMT in nude mice transplanted with HCT116 cells in colorectal cancer. 4. ARE combined radiotherapy can regulate the expression of E-cadherin, the expression of beta -Catenin, the expression of N-cadherin, Vimentin, Snail, Slug, Twist of the tumor HCT116-CRR cells in nude mice of colorectal cancer, suggesting that ARE may reverse the effect of radiotherapy on the xenografts in nude mice of human colorectal cancer by reversing EMT. Chemoradiotherapy, 5. ARE can enhance the effect of radiotherapy for human colorectal cancer in nude mice, and may be a low toxic and efficient radiosensitizer used in the radiotherapy of colorectal cancer.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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