尼美舒利对喉鳞癌Hep-2细胞裸鼠成瘤的影响及机制研究

发布时间：2018-04-20 23:38

本文选题：选择性环氧化酶-2抑制剂 + 喉肿瘤　；参考：《泸州医学院》2010年硕士论文

【摘要】： 目的：尽管包括手术、放疗、化疗在内的各种方法都在不断改进,但喉鳞状细胞癌(简称喉鳞癌)患者的五年生存率仍较低,它严重威胁着人民的生命和健康；并且近年来喉鳞癌的发病率不但没有降低,反而有上升且发病年轻化的趋势,因而寻找积极有效的预防途径和治疗药物成为一项十分迫切的重要任务。本实验通过研究选择性环氧化酶-2(cyclooxyge-nase-2,COX-2)抑制剂尼美舒利(nimesulide, NIM)对喉鳞癌Hep-2细胞裸裸鼠成瘤以及COX-2、生存素(Survivin)、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)表达的影响,以探讨NIM抗肿瘤的作用和可能的机制,从而为NIM用于喉鳞癌的预防和治疗提供试验依据。方法：对喉鳞癌Hep-2细胞进行传代培养,制成细胞悬液,接种于Balb/c-nu/nu裸鼠右侧背部皮下,建立移植瘤模型,12只荷瘤裸鼠随机分为实验组(即NIM治疗组,腹腔注射NIM50mg/kg/2d,0.2ml)和对照组(腹腔注射生理盐水,0.2ml),观察裸鼠生长情况,定期(每两天)称量裸鼠体重并以游标卡尺(每两天)测量移植瘤的最长径及最短径,绘制肿瘤生长曲线,计算肿瘤体积=肿瘤最长径×肿瘤最短径2／2。用药4周后,处死裸鼠,取瘤体,常规HE染色观察移植瘤光镜下改变,并取裸鼠主要内脏行HE染色,观察NIM对裸鼠可能的毒副作用。利用免疫组织化学技术检测COX-2、Survivin和PCNA蛋白表达,显微镜下观察移植瘤细胞中COX-2、Survivin和PCNA蛋白表达情况,并采用图像分析系统作平均光密度值测定。同时采用逆转录-聚合酶链反应法(reverse transcription-polymerase chain reaction, RT-PCR)检测COX-2、Survivin和PCNA mRNA表达,Biod-rad软件分析对照组与试验组COX-2、Survivin和PCNA mRNA灰度值。结果：(1)成功构建出人喉鳞癌裸鼠移植瘤模型,两组裸鼠均有喉鳞癌原位移植瘤生长,成瘤率100%；其瘤组织经病理切片检查证实为喉鳞癌,且在实验期内,2组动物全部存活。(2)2组裸鼠饮食正常,活动自如,精神状态良好,无1只死亡；其心、肝、脾、肺、肾的重量和组织形态观察无明显差异,解剖消化道,未发现出血病灶。(3)NIM治疗组裸鼠移植瘤体积为(172.67±115.25)mm3,较对照组(478.69±341.84)mm3明显减小,两组间差异有统计学意义(P0.05)；NIM治疗组裸鼠体重为(23.22±1.78)g,对照组为(24.05±1.79)g,两组间差异无统计学意义(P0.05)；NIM治疗组裸鼠移植瘤重量(0.40±0.33)g,较对照组(0.83±0.27)g明显减小,两组间差异有统计学意义(P0.05),计算抑瘤率为51.81%。(4)免疫组织化学SP法检测裸鼠移植瘤组织中COX-2、Survivin、PCNA蛋白表达,NIM治疗组COX-2蛋白表达光密度为(38.12±15.56),与对照组(140.92±23.12)比较显著下降,经统计学分析差异有统计学意义(P0.05)；NIM治疗组Survivin蛋白表达为(46.79±10.04),经统计学分析与对照组(133.41±20.01)比较差异有统计学意义(P0.05)；NIM治疗组PCNA蛋白表达显著下降(62.97±13.83),经统计学分析与对照组(110.23±13.00)比较差异有统计学意义(P0.05)。(5) RT-PCR检测COX-2 mRNA表达NIM治疗组灰度值为、(0.70±0.18),对照组为(2.23±0.38),经统计学分析差异有统计学意义(P0.05)；NIM治疗组Survivin mRNA表达为(0.34±0.04),对照组为(1.85±0.18),经统计学分析差异有统计学意义(P0.05)；NIM治疗组PCNA mRNA表达为(0.45±0.05),对照组为(1.91±0.70),经统计学分析差异有统计学意义(P0.05)。结论：(1)选择性COX-2抑制剂NIM对喉鳞癌Hep-2细胞裸鼠移植瘤生长有明显抑制作用。(2)NIM可抑制COX-2蛋白及mRNA表达,推测NIM可通过依赖COX-2表达的方式抑制肿瘤生长。(3)NIM可在蛋白及mRNA水平抑制喉鳞癌细胞增殖、促进细胞凋亡,其机制可能与抑制COX-2、Survivin及PCNA表达有关。(4)NIM无明显毒副作用。(5)选择性COX-2抑制剂有望成为未来喉鳞癌综合治疗的一部分,它的研制开发可能为喉鳞癌预防和治疗提供全新的广阔前景。
[Abstract]:Objective: Although various methods, including surgery, radiotherapy and chemotherapy, are constantly improved, the five year survival rate of laryngeal squamous cell carcinoma (LSCC) is still low, which seriously threatens the life and health of the people; and the incidence of laryngeal squamous cell carcinoma has not decreased, but has a trend of rising and younger in recent years. It is an urgent and important task to find active and effective prophylactic and therapeutic drugs. In this experiment, Ni Mei Shug Leigh (nimesulide, NIM), a selective cyclooxygenase -2 (cyclooxyge-nase-2, COX-2) inhibitor, was used to treat nude mice of squamous cell carcinoma of laryngeal squamous cell carcinoma (nude mice), and COX-2, Survivin, and proliferating cell nuclear antigen (prolifera). The effect of Ting cell nuclear antigen, PCNA) on the anti-tumor effect and possible mechanism of NIM, thus providing experimental basis for the use of NIM for the prevention and treatment of laryngeal squamous cell carcinoma. Method: subculture of Hep-2 cells from the squamous cell carcinoma of the larynx and made into a cell suspension, inoculated on the right back of the right back of the Balb/c-nu/nu nude mice, and establishing a transplanted tumor model. 12 tumor bearing nude mice were randomly divided into experimental group (NIM treatment group, intraperitoneal injection of NIM50mg/kg/2d, 0.2ml) and control group (intraperitoneal injection of saline, 0.2ml), observing the growth of nude mice, weighing the body weight of nude mice regularly (every two days) and measuring the longest diameter and the shortest diameter of the transplanted tumor with the vernier caliper (every two days), drawing the tumor growth curve and calculating the tumor. Volume = the longest diameter of the tumor and the shortest path of 2 / 2. of the tumor for 4 weeks. The nude mice were killed and the tumor body was taken to observe the changes under the light microscopy of the transplanted tumor by routine HE staining, and the main viscera of the nude mice was stained with HE. The possible toxic and side effects of NIM on nude mice were observed. The expression of COX-2, Survivin and PCNA protein was detected by immunohistochemical technique, and the transplantation was observed under the microscope. The expression of COX-2, Survivin and PCNA protein in the tumor cells was measured by the image analysis system, and the reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) was used to detect COX-2, Survivin and PCNA mRNA table. Results: in and PCNA mRNA gray value. Results: (1) the transplanted tumor model of human laryngeal squamous cell carcinoma in nude mice was successfully constructed. The two groups of nude mice had the growth of laryngeal squamous cell carcinoma in situ, the tumor rate was 100%, and the tumor tissue was confirmed by pathological section for laryngeal squamous cell carcinoma. In the experimental period, all the 2 groups of animals survived. (2) the 2 groups of nude mice had normal diet, good activity and good mental state. No significant difference between the weight and tissue morphology of the heart, liver, spleen, lung and kidney was observed without 1 deaths. (3) the volume of nude mice transplanted in the NIM group was (172.67 + 115.25) mm3, compared with the control group (478.69 + 341.84) mm3, and the difference between the two groups was statistically significant (P0.05); the body weight of nude mice in the NIM treatment group was the weight of the nude mice. (23.22 + 1.78) g, the control group was (24.05 + 1.79) g, the difference between the two groups was not statistically significant (P0.05); the weight of the transplanted tumor of nude mice in the NIM treatment group was (0.40 + 0.33) g, compared with the control group (0.83 + 0.27) G, the difference was statistically significant (P0.05), and the tumor suppressor rate was 51.81%. (4) immunohistochemical SP method in the nude mice transplanted tumor tissue COX-2, S Urvivin, PCNA protein expression, NIM treatment group COX-2 protein expression light density (38.12 + 15.56), compared with the control group (140.92 + 23.12), a significant decrease, statistically significant difference (P0.05), NIM treatment group Survivin protein expression is (46.79 + 10.04), statistical analysis and the control group (133.41 + 20.01) the difference is statistically significant Significance (P0.05); the expression of PCNA protein in the NIM treatment group decreased significantly (62.97 + 13.83), and the difference between the control group and the control group (110.23 + 13) was statistically significant (P0.05). (5) the gray value of the COX-2 mRNA expression in the RT-PCR group was (0.70 + 0.18), and the control group was (2.23 + 0.38), and the statistical difference was statistically significant (P0.05). The expression of Survivin mRNA in the NIM treatment group was (0.34 + 0.04), the control group was (1.85 + 0.18), and the statistical difference was statistically significant (P0.05); the PCNA mRNA expression in the NIM group was (0.45 + 0.05), the control group was (1.91 + 0.70), and the difference was statistically significant (P0.05). Conclusion: (1) NIM of the selective COX-2 inhibitor NIM is Hep- on laryngeal squamous cell carcinoma The growth of 2 cells in nude mice has obvious inhibitory effect. (2) NIM can inhibit the expression of COX-2 protein and mRNA. It is suggested that NIM can inhibit the growth of tumor by relying on COX-2 expression. (3) NIM can inhibit the proliferation of laryngeal squamous cell carcinoma cells and promote cell apoptosis at the level of protein and mRNA. The mechanism may be related to the inhibition of COX-2, Survivin and PCNA expression. (4) NIM no (5) selective COX-2 inhibitors are expected to be part of the comprehensive treatment of laryngeal squamous cell carcinoma in the future. Its development and development may provide a new broad prospect for the prevention and treatment of laryngeal squamous cell carcinoma.

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65

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