选择性COX-2抑制剂对人喉鳞癌Hep-2细胞裸鼠移植瘤血管生成的影响及机制研究

发布时间：2018-06-04 12:33

本文选题：环氧化酶2 + 血管生成素2　；参考：《泸州医学院》2013年硕士论文

【摘要】：目的：原发性喉癌指原发部位在喉部的肿瘤,以鳞状细胞癌(90%)最为常见。喉癌治疗包括手术、放疗和化疗等多种治疗方法。根据病情的分期可选择一种,或多种方法相结合的治疗方式。环氧化酶-2(Cyclooxygenase-2, COX-2),是催化花生四烯酸合成前列腺素(Prostaglandin, PG)过程中的关键酶,在体内存在COX-1和COX-2两种同工酶。近年来大量研究表明COX-2在多种肿瘤中都存在异常增高表达,在其发病过程中发挥着重要作用。关于COX-2在肿瘤中的作用机制,目前研究表明其可以通过多种途径作用于肿瘤发病过程。前期实验已经证实选择性COX-2抑制剂尼美舒利(nimesulide, NIM)可以通过诱导细胞凋亡,抑制细胞增殖的途径抑制人喉鳞癌Hep-2细胞裸鼠移植瘤生长。本实验进一步就NIM对喉鳞癌Hep-2细胞裸鼠移植瘤微血管密度(Microvessel density, MVD)、COX-2、血管生成因子-2(Angiopoietin-2, Ang-2)和碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)表达的影响进行观察和研究,以期进一步为NIM用于喉鳞癌的治疗提供理论依据。方法：粱灼萍等在前期试验中,将喉鳞癌Hep-2细胞接种于裸鼠背部皮下,建立移植瘤模型。将裸鼠随机分为实验组和对照组各六只,实验组予裸鼠腹腔注射NIM(50mg/kg/2d,0.2ml);对照组予裸鼠腹腔注射0.2ml生理盐水。用药4周后,处死裸鼠并获得瘤体。本实验选用粱灼萍实验所获得的喉癌Hep-2细胞裸鼠移植瘤,运用免疫组织化学技术检测CD31在喉癌裸鼠移植瘤组织中表达来定位瘤体中的MVD,运用图像分析系统作平均光密度值测定,并比较两组间的差异；分别采用免疫组织化学染色技术检测喉癌裸鼠移植瘤组织中COX-2、Ang-2和bFGF蛋白的表达情况,运用图像分析系统计算平均光密度值并比较实验组与对照组中COX-2、Ang-2、bFGF蛋白表达情况；采用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法检测喉癌移植瘤组织中COX-2, Ang-2和bFGF mRNA的表达,并对两组移植瘤组织中COX-2、Ang-2和bFGF mRNA灰度值进行比较；运用直线相关分别分析实验组中COX-2、Ang-2、bFGF蛋白表达水平与MVD值的相关性。结果：(1)免疫组织化学SP法检测裸鼠移植瘤中CD31表达得到平均MVD值,实验组为12.83±3.06,与对照组25.50±4.85(t=-5.412,P0.05)相比有统计学意义,表明选择性COX-2抑制剂NIM干预后移植瘤内血管生成减少；(2)利用免疫组织化学SP法检测裸鼠移植瘤组织中COX-2、Ang-2、 bFGF蛋白表达,实验组中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,差异有统计学意义(t=-9.825,P0.05),实验组Ang-2蛋白表达为(49.02±7.67),经统计学分析与对照组(110.04±6.76)比较差异有统计学意义(t=-14.607,P0.05)；实验组bFGF蛋白表达为(62.25±7.50),经统计学分析与对照组(119.04±17.30)比较差异有统计学意义(t=-7.391,P0.05)；(3)运用RT-PCR技术检测移植瘤中COX-2、Ang-2和bFGF mRNA表达,NIM治疗组COX-2mRNA表达为(0.70±0.18),对照组为(2.20±0.38),经统计学分析差异有统计学意义(t=-9.825,P0.05)；NIM治疗组Ang-2mRNA灰度值为(0.49±0.03),对照组为(0.98±0.10),经统计学分析差异有统计学意义(t=-11.509,P0.05)；NIM治疗组移植瘤中bFGF mRNA表达为(0.65±0.08),对照组为(1.19±0.19),经统计学分析差异有统计学意义(t=-6.209,P0.05)。(4)相关性分析：运用直线相关分析方法分别检测实验组移植瘤组织中COX-2、Ang-2、bFGF蛋白表达水平与MVD相关性,经统计学分析存在相关性,其相关系数r分别为0.832,0.944,0.844(P均0.05)。结论：(1)选择性COX-2抑制剂NIM可以抑制人喉鳞癌Hep-2细胞裸鼠移植瘤中血管生成；(2)实验组中COX-2、Ang-2、bFGF蛋白表达水平分别与MVD表达水平呈正相关,后两者为血管生成促进因子,其与MVD的相关性表明其在肿瘤血管生成中作用显著；(3)使用NIM后,移植瘤组织中Ang-2和bFGF蛋白及mRNA表达水平都降低,表明NIM抑制人喉鳞癌Hep-2细胞裸鼠移植瘤血管生成的机制可能与抑制Ang-2及bFGF表达有关；(4)选择性COX-2抑制剂NIM可通过多种机制抑制人喉鳞癌Hep-2细胞裸鼠移植瘤的生长,有望为未来喉鳞癌综合治疗提供新的思路和手段。
[Abstract]:Objective: primary laryngeal carcinoma is the primary tumor of the larynx. Squamous cell carcinoma (90%) is the most common. The treatment of larynx cancer includes surgery, radiotherapy and chemotherapy. A combination of a variety of methods is selected according to the stage of the disease. Cyclooxygenase -2 (Cyclooxygenase-2, COX-2) is a catalyst for the catalysis of Arachis four enoic acid. The key enzymes in the synthesis of Prostaglandin (PG) are the two isozymes of COX-1 and COX-2 in the body. In recent years, a large number of studies have shown that COX-2 has abnormal high expression in many kinds of tumors and plays an important role in the pathogenesis of COX-2. The early experiments have confirmed that the selective COX-2 inhibitor Ni Mei Shug Leigh (nimesulide, NIM) can inhibit the growth of human laryngeal squamous cell carcinoma Hep-2 cell xenografts by inducing apoptosis and inhibiting cell proliferation, and the microvascular density of NIM in nude mice of Hep-2 cells in laryngeal squamous cell carcinoma (LSCC) is further studied. The effects of (Microvessel density, MVD), COX-2, -2 (Angiopoietin-2, Ang-2) and basic fibroblast growth factor (basic fibroblast growth factor, bFGF) expression were observed and studied in order to provide a theoretical basis for the treatment of laryngeal squamous cell carcinoma. The Hep-2 cells of squamous cell carcinoma were inoculated subcutaneously in the back of nude mice to establish a transplanted tumor model. The nude mice were randomly divided into experimental group and six control group. The experimental group was given NIM (50mg/kg/2d, 0.2ml) intraperitoneally in nude mice, and the control group was given 0.2ml physiological saline in nude mice. The nude mice were killed and the tumor body was obtained after 4 weeks of medication. This experiment was obtained by the experiment of sorghum burning The Hep-2 cells of the larynx cancer cells were transplanted in nude mice. The immunohistochemical technique was used to detect the expression of CD31 in the xenografts in nude mice of the larynx to locate the MVD in the tumor. The mean optical density was measured by the image analysis system, and the difference between the two groups was compared, and the immuno histochemical staining technique was used to detect the xenograft group in the nude mice of larynx cancer. The expression of COX-2, Ang-2 and bFGF protein in the fabric was calculated. The average optical density was calculated by the image analysis system and the expression of COX-2, Ang-2, bFGF protein in the experimental group was compared with the control group. Reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) was used to detect the COX-2 in the tumor tissue of the larynx cancer. The expression of ng-2 and bFGF mRNA and the comparison of the gray values of COX-2, Ang-2 and bFGF mRNA in the two groups of transplanted tumor tissues were compared. The correlation between the level of COX-2, Ang-2, bFGF protein expression in the experimental group and MVD value in the experimental group was analyzed by linear correlation. Results: (1) the average expression of the immunohistochemical SP method in the nude mice was detected, and the experimental group was found in the experimental group. It was 12.83 + 3.06, compared with the control group (25.50 + 4.85) (t=-5.412, P0.05), compared with the control group (t=-5.412, P0.05), which showed that the selective COX-2 inhibitor NIM was used to reduce the angiogenesis in the transplanted tumor; (2) the immunohistochemical SP method was used to detect the expression of COX-2, Ang-2, and bFGF in the transplanted tumor tissues of nude mice, and the density of the COX-2 protein expression in the experimental group was (38.12+15.56). Compared with the control group (140.92 + 23.12), the difference was statistically significant (t=-9.825, P0.05). The expression of Ang-2 protein in the experimental group was (49.02 + 7.67). The difference between the experimental group and the control group (110.04 + 6.76) was statistically significant (t=-14.607, P0.05), and the expression of bFGF protein in the experimental group was (62.25 + 7.50), and was statistically analyzed with the control group (1. 19.04 + 17.30) were statistically significant (t=-7.391, P0.05); (3) the expression of COX-2, Ang-2 and bFGF mRNA in the transplanted tumor was detected by RT-PCR, and the COX-2mRNA expression in the NIM treatment group was (0.70 + 0.18), and the control group was (2.20 + 0.38). The statistical difference was statistically significant (t=-9.825, P0.05), and the gray value of the NIM treatment group was (0.49). The control group was (0.98 + 0.10), and the difference was statistically significant (t=-11.509, P0.05). The expression of bFGF mRNA in the transplanted tumor of the NIM group was (0.65 + 0.08), the control group was (1.19 + 0.19), and the difference was statistically significant (t= -6.209, P0.05). (4) correlation analysis: linear correlation analysis was used to detect the difference. The correlation of COX-2, Ang-2, bFGF protein expression level with MVD in the transplanted tumor tissue of the experimental group was statistically analyzed, and the correlation coefficient r was 0.832,0.944,0.844 (P 0.05). Conclusion: (1) the selective COX-2 inhibitor NIM could inhibit angiogenesis in the transplanted tumor of human laryngeal squamous cell carcinoma in nude mice; (2) COX-2, Ang-2, and Ang-2 in the experimental group. The expression level of GF protein was positively correlated with the level of MVD expression, and the latter two was angiogenic promoting factor. The correlation with MVD showed that it was significant in the angiogenesis of tumor. (3) after the use of NIM, the expression level of Ang-2 and bFGF and mRNA in the transplanted tumor tissues decreased, indicating that NIM inhibited the transplanted tumor of the laryngeal squamous cell carcinoma in nude mice. The mechanism of angiogenesis may be related to the inhibition of Ang-2 and bFGF expression; (4) selective COX-2 inhibitor NIM can inhibit the growth of human laryngeal squamous carcinoma Hep-2 cell xenografts in nude mice through a variety of mechanisms. It is expected to provide new ideas and means for comprehensive treatment of laryngeal squamous cell carcinoma in the future.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.65

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