环氧化酶2在食管癌中的表达及环氧化酶2抑制剂对食管癌小鼠移植瘤生长的影响

发布时间：2018-05-17 05:22

本文选题：食管癌 + 环氧化酶-2　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:食管癌是我国最常见的恶性肿瘤之一,具有很明显的地域性分布性。由于食管癌早期症状不典型,人们缺乏保健意识等原因,确诊时多已发展到晚期,所以预后差。近年来研究表明,环氧化酶-2(cyclooxygenase-2,COX-2)在恶性肿瘤的形成过程中有重要作用,它可促进肿瘤细胞增殖,抑制肿瘤细胞凋亡,促进肿瘤的侵袭、转移等。动物实验证明,对肿瘤的治疗选择性COX-2抑制剂有显著作用。因此针对肿瘤的COX-2靶点,可以选用选择性COX-2抑制剂来治疗。尼美舒利是新型选择性COX-2抑制剂,动物实验证实,尼美舒利不仅可以抑制肿瘤生长与转移、预防肿瘤形成,还可与化疗、放疗联合应用,具有增强化疗、放疗效果的作用。奥沙利铂是第三代化疗药,通过产生水化衍生物,使DNA链内和链间形成交联,从而抑制DNA的复制及合成。本实验从食管癌手术治疗的标本、人食管癌小鼠移植瘤两方面,采用多种实验方法研究COX-2在食管癌发生过程中的作用,观察COX-2抑制剂尼美舒利、化疗药物奥沙利铂的联合应用对人食管癌小鼠移植瘤生长的抑制作用,进一步探讨这两种药物联合应用可能产生的协同作用。方法:1食管癌中COX-2的表达及意义手术切除食管癌新鲜标本63例,术后立即取材,分别于10%福尔马林固定,常规包埋、切片,HE染色,由两名有经验病理科医师进行组织病理学诊断。采用免疫组织化学方法检测COX-2蛋白的表达。新鲜标本经70%乙醇处理后,采用流式细胞术(Flow cytometry,FCM)定量检测标本中的COX-2蛋白表达。2尼美舒利、奥沙利铂联合应用对人食管癌小鼠移植瘤生长的影响选取C57BL/6小鼠6-7周龄,雄性,体重25-30g,在IVC环境中以灭菌处理过的饲料和水饲养,环境温度、湿度控制在适宜范围。将人食管癌Eca-109细胞使用含10%胎牛血清、青霉素、链霉素各100U.m L-1的DMEM/F12培养基,在37℃的恒温下,用含5%的二氧化碳培养箱中常规培养。观察细胞至70%-80%融合时,用2.5g.L-1胰蛋白酶消化传代。取生长状态良好的对数生长期的人食管癌Eca-109细胞,PBS洗涤,用2.5g.L-1胰蛋白酶消化下来,制成细胞悬液。用不含胎牛血清的DMEM/F12培养基稀释,用细胞计数板进行计数,调整细胞悬液浓度为5×106.m L-1。小鼠皮肤备皮、消毒后用1ml注射器在右侧后臀部注射上述单细胞悬液1ml,建立人食管癌小鼠移植瘤模型。26只全部成瘤,定期测量肿瘤长径、短径,待肿瘤平均直径为4mm后,剔除肿瘤偏小的2只小鼠。将24只小鼠随机分为4组,每组6只,组别为对照组,尼美舒利组,奥沙利铂组,尼美舒利/奥沙利铂组。尼美舒利组小鼠给予灌胃口服尼美舒利20mg/Kg/次,每天给药一次,同时腹腔注射等量灭菌生理盐水;奥沙利铂组小鼠给予腹腔注射奥沙利铂甘露醇注射液10mg/Kg/次,每4天给药一次,同时灌胃口服等量灭菌生理盐水;尼美舒利/奥沙利铂组小鼠,同时灌胃口服尼美舒利,腹腔注射奥沙利铂甘露醇注射液,剂量与单用组相同;对照组小鼠,灌胃口服灭菌生理盐水,腹腔注射等量灭菌生理盐水。定期观察小鼠生长情况,每5天测量1次肿瘤的体积。30天后,处死小鼠切除肿瘤组织,测量4组小鼠移植瘤长径、短径及瘤重,估测瘤体体积和肿瘤生长抑制率并取材。FCM法定量检测移植瘤中COX-2蛋白的表达,FCM法检测移植瘤中细胞的凋亡率。结果:1 COX-2在食管癌变过程中的表达免疫组织化学结果显示,食管癌细胞COX-2基因表达产物主要定位于细胞浆,胞浆内出现棕黄色颗粒为阳性。在癌旁组织中表达率为20%,食管癌组织中表达率为90%。FCM定量检测COX-2含量结果显示:正常食管鳞状上皮中COX-2的FI值为1.00±0.12,随着食管鳞状上皮异型性增高,COX-2的表达升高,在高分化癌中表达最高,FI值为2.89±0.22。在食管鳞状细胞癌组织中随着癌组织的分化程度降低而COX-2的表达减少。2尼美舒利联合奥沙利铂对人食管癌小鼠移植瘤生长的影响及机制接种小鼠26只,全部成瘤,成瘤率100%,成瘤时间为细胞接种后5-8天。肿瘤细胞种植后13天,肿瘤平均直径达4mm。治疗结束后,各组体积分别为:对照组:移植瘤体积2047.42±976.55 mm3,瘤重1.41±0.49g;尼美舒利组:移植瘤体积1267.14±476.38mm3,瘤重0.97±0.51g;奥沙利铂组:移植瘤体积1054.28±102.82mm3,瘤重0.89±0.20g;尼美舒利/奥沙利铂联合用药组:移植瘤体积532.66±220.11mm3,瘤重0.45±0.31g。尼美舒利组、奥沙利铂组和尼美舒利/奥沙利铂联合用药组的抑瘤率分别为46.22%、51.93%和72.88%。方差分析组间差异显示:尼美舒利组、奥沙利铂组和尼美舒利/奥沙利铂联合用药组移植瘤生长明显较对照组缓慢(P0.05)。尼美舒利/奥沙利铂联合用药组抑瘤率明显较尼美舒利组、奥沙利铂组高(P0.05)。尼美舒利组和奥沙利铂组对移植瘤的生长的抑制作用未见明显差异(P0.05)。FCM定量检测移植瘤的细胞凋亡,对照组食管癌细胞凋亡率为(12.12±0.47)%,尼美舒利组凋亡率为(22.16±5.24)%,奥沙利铂组凋亡率为(27.33±7.25)%,尼美舒利/奥沙利铂联合用药组凋亡率为(47.26±6.73)%。方差分析组间差异显示:尼美舒利组、奥沙利铂组和尼美舒利/奥沙利铂联合用药组凋亡率较对照组升高,有显著性差异(P0.05)。尼美舒利/奥沙利铂联合用药组凋亡率明显较尼美舒利组、奥沙利铂组高,有显著性差异(P0.05)。尼美舒利组和奥沙利铂组对凋亡率的影响未见明显差异(P0.05)。FCM定量检测移植瘤中COX-2蛋白表达,尼美舒利组FI=0.598±0.090,奥沙利铂组FI=1.390±0.192,尼美舒利/奥沙利铂组FI=0.791±0.121。方差分析结果显示,尼美舒利及尼美舒利/奥沙利铂联合用药组COX-2蛋白表达水平低于对照组,并有显著性差异(P0.05)。奥沙利铂组COX-2蛋白表达水平高于对照组,并有显著性差异(P0.05)。结论:1在癌旁组织和食管癌组织中,COX-2的表达增高,表明在食管上皮癌变过程中COX-2可能起重要作用。食管鳞状细胞癌组织中,随食管癌组织分化程度降低,COX-2的表达降低。2尼美舒利及奥沙利铂均能抑制肿瘤的生长,尼美舒利/奥沙利铂联合用药组抑瘤率明显高于单用组。尼美舒利能提高了奥沙利铂的对肿瘤的抑制效果,这种效果是通过诱导肿瘤细胞凋亡来抑制肿瘤的。综上所述,本实验从食管癌手术标本,小鼠移植瘤两个方面进行研究,结果表明:COX-2在食管癌变过程中起重要作用,选择性COX-2抑制剂尼美舒利或联合奥沙利铂,均可通过诱导细胞凋亡来抑制肿瘤。尼美舒利与奥沙利铂联合应用,可提高奥沙利铂的肿瘤抑制作用。
[Abstract]:Objective: esophageal cancer is one of the most common malignant tumors in China. It has a distinct regional distribution. Due to the untypical early symptoms of esophageal cancer and the lack of health awareness, it has developed to a late stage, so the prognosis is poor. In recent years, the study showed that the epoxide -2 (cyclooxygenase-2, COX-2) was formed in the formation of malignant tumor. It plays an important role in promoting tumor cell proliferation, inhibiting tumor cell apoptosis, promoting tumor invasion, metastasis and so on. Animal experiments have proved to have significant effect on the selective COX-2 inhibitors for tumor treatment. Therefore, selective COX-2 inhibitors can be used to treat tumor COX-2 targets. Ni Mei Shug Leigh is a new type of selective COX-2. The inhibitor, animal experiments confirmed that Ni Mei Shug Leigh not only can inhibit tumor growth and metastasis, prevent tumor formation, but also can be combined with chemotherapy and radiotherapy, and has the effect of enhancing chemotherapy and radiotherapy. Oxaliplatin is the third generation of chemotherapeutic agents. By producing hydrated derivatives, the interchain and interchain of DNA can be formed to inhibit the replication of DNA. In this experiment, we studied the role of COX-2 in the carcinogenesis of esophageal cancer in two aspects of the surgical treatment of esophageal cancer and the transplanted tumor of human esophageal cancer in mice. The effects of COX-2 inhibitor nimesulide and the combination of oxaliplatin, a chemotherapeutic agent, on the growth of human esophageal cancer in mice were investigated. Synergetic effects of the combined application of these two drugs. Methods: the expression of COX-2 in 1 esophageal carcinoma and the significance of surgical resection of 63 fresh esophageal carcinoma specimens were taken immediately after operation, respectively, in 10% formalin fixation, routine embedding, slicing, HE staining, and histopathological diagnosis by two experienced pathologists. Immunohistochemistry was used. The expression of COX-2 protein was detected by the method. After 70% ethanol treatment, Flow cytometry (FCM) was used to detect the COX-2 protein expression of.2 nimesulide in the specimens. The effects of oxaliplatin on the growth of transplanted tumor in human esophageal cancer mice were selected for the selection of C57BL/6 mice for 6-7 weeks, male, body weight 25-30g, and IVC environment. Eca-109 cells with 10% fetal bovine serum, penicillin and streptomycin 100U.m L-1 were used as the DMEM/F12 medium containing 10% fetal bovine serum, penicillin and streptomycin at a constant temperature of 37. The cells were cultured in a 5% carbon dioxide incubator and used 2.5G. to observe the cells to 70%-80% fusion. L-1 trypsin digested the human esophageal cancer Eca-109 cells with good growth state during the logarithmic growth period, PBS washed and digested with 2.5g.L-1 trypsin to make the cell suspension. The cell suspension was diluted with the DMEM/F12 medium without fetal bovine serum, and the cell suspension concentration was 5 * 106.m L-1. mouse skin. After disinfection, the 1ml syringe was injected with the above single cell suspension 1ml on the right rear buttocks, and the mouse model of human esophageal cancer transplanted tumor model.26 was all tumor, and the length and diameter of the tumor were measured regularly. After the average diameter of the tumor was 4mm, 2 mice were eliminated. The 24 mice were divided into 4 groups, each group was 6 and the group was the control group, Ni Mei Shug Leigh Group, oxaliplatin group, Ni Mei Shug Leigh / oxaliplatin group. The mice in the Ni Mei Shug Leigh group were given the oral administration of Ni Mei Shug Leigh 20mg/Kg/ times, once a day, and the same dose of normal saline was injected into the abdominal cavity. The oxaliplatin group was given an intraperitoneal injection of Oxaliplatin and Mannitol Injection 10mg/Kg/ times, once every 4 days, at the same time. The nimesulide / oxaliplatin group, nimesulide / oxaliplatin group, was given the same dose of nimesulide and intraperitoneal injection of Oxaliplatin and Mannitol Injection, and the dose was the same as that in the single group. The control mice were given the normal saline by intraperitoneal injection, and the abdominal injection was used to sterilize the physiological saline. The growth of the mice was observed and the body was measured every 5 days, and the body was measured every 5 days. After.30 days, the tumor tissues were excised and the long diameter, short diameter and tumor weight of the 4 groups of mice were measured. The volume of the tumor and the inhibition rate of tumor growth were measured and the expression of COX-2 protein in the transplanted tumor was measured by.FCM. The apoptosis rate of the cells in the transplanted tumor was detected by FCM. Results: the expression of immune tissues in the process of esophageal carcinogenesis was expressed by the FCM method. The chemical results showed that the COX-2 gene expression products of esophageal cancer cells were mainly located in the cytoplasm, and the brown yellow granules were positive in the cytoplasm. The expression rate in the para cancerous tissues was 20%. The expression rate of the esophageal carcinoma tissue was 90%.FCM quantitative detection COX-2 content: the FI value of COX-2 in the normal esophageal squamous epithelium was 1 + 0.12, with the esophageal scale. The high expression of COX-2, the highest expression of COX-2, the highest expression in highly differentiated carcinoma, FI value is 2.89 + 0.22. in the squamous cell carcinoma of the esophagus with the decrease of the differentiation degree of cancer tissue, and the expression of COX-2 reduces the effect of.2 combined with oxaliplatin on the growth of the transplanted tumor of human esophageal cancer and the mechanism of the inoculation of 26 mice. The tumor formation rate was 100% and the time of tumor formation was 5-8 days after the cell inoculation. After 13 days after the tumor cells were planted, the average diameter of the tumor reached the end of 4mm. treatment. The volume of each group was as follows: the volume of the transplanted tumor was 2047.42 + 976.55 mm3 and the tumor weight was 1.41 0.49g; the nimesulide group was 1267.14 + 476.38mm3, and the tumor weight was 0.97 + 0.51g; the oxaliplatin group was moved. The tumor volume was 1054.28 + 102.82mm3 and the tumor weight was 0.89 + 0.20g; Ni Mei Shug Leigh / oxaliplatin combined group: the tumor volume was 532.66 + 220.11mm3, the tumor weight was 0.45 + 0.31g., the tumor inhibition rate of the oxaliplatin group and the Ni Mei Shug Leigh / oxaliplatin group was 46.22%, 51.93% and 72.88%. variance analysis group showed: neimego The growth of transplanted tumor in group, oxaliplatin group and Ni Mei Shug Leigh / oxaliplatin group was significantly slower than that of the control group (P0.05). The tumor inhibition rate of Ni Mei Shug Leigh / oxaliplatin group was significantly higher than that of the Ni Mei Shug Leigh group and the oxaliplatin group (P0.05). The inhibitory effect of the Ni Mei Shug Leigh group and oxaliplatin group on the growth of the transplanted tumor was not significantly different. (P0.05).FCM quantitative detection of the apoptosis of the transplanted tumor cells, the apoptosis rate of the control group was (12.12 + 0.47)%, the apoptosis rate of the Ni Mei Shug Leigh group was (22.16 + 5.24)%, the apoptosis rate of the oxaliplatin group was (27.33 + 7.25)%, and the rate of apoptosis in the combination group of Ni Mei Shug Leigh / oxaliplatin was (47.26 + 6.73)%. The difference between the groups of variance analysis showed that in the Ni Mei Shug Leigh group, The apoptosis rate of oxaliplatin group and Ni Mei Shug Leigh / oxaliplatin group was significantly higher than that of the control group (P0.05). The apoptosis rate of the combination group of Ni Mei Shug Leigh / oxaliplatin was significantly higher than that of the Ni Mei Shug Leigh group and the oxaliplatin group (P0.05). The effect of the nimisulli group and oxaliplatin group on the apoptosis rate was not obvious. Differential (P0.05).FCM quantitative detection of COX-2 protein expression in transplanted tumor, Ni Mei Shug Leigh group FI=0.598 + 0.090, oxaliplatin group FI=1.390 + 0.192, Ni Mei Shug Leigh / oxaliplatin group FI=0.791 + 0.121. variance analysis results showed that the expression level of COX-2 protein in the combination group of Ni Mei Shug Leigh and Ni Mei Shug Leigh / oxaliplatin was lower than that of the control group, and it was significant. The difference (P0.05). The expression of COX-2 protein in the oxaliplatin group was higher than that in the control group, and there was a significant difference (P0.05). Conclusion: 1 the expression of COX-2 is increased in the para cancerous and esophageal cancer tissues, indicating that COX-2 may play an important role in the carcinogenesis of the esophagus. The degree of differentiation of esophageal squamous cell carcinoma is reduced, COX-2 The expression of.2 Ni Mei Shug Leigh and oxaliplatin inhibited the growth of the tumor. The tumor inhibition rate of the combination of Ni Mei Shug Leigh / oxaliplatin was significantly higher than that in the single use group. Ni Mei Shug Leigh could improve the inhibitory effect of oxaliplatin on the tumor. This effect is to inhibit tumor by inducing tumor cells to wither. Two aspects of tumor operation specimens and mice transplanted tumor were studied. The results showed that COX-2 played an important role in the carcinogenesis of the esophagus. The selective COX-2 inhibitor Ni Mei Shug Leigh or oxaliplatin could inhibit the tumor by inducing apoptosis. The combination of Ni Mei Shug Leigh and oxaliplatin could improve the tumor suppression of oxaliplatin. Use.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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