本文選題：環(huán)氧化酶2 + 血管生成素2　； 參考：《瀘州醫(yī)學院》2013年碩士論文

【摘要】：目的：原發(fā)性喉癌指原發(fā)部位在喉部的腫瘤,以鱗狀細胞癌(90%)最為常見。喉癌治療包括手術(shù)、放療和化療等多種治療方法。根據(jù)病情的分期可選擇一種,或多種方法相結(jié)合的治療方式。環(huán)氧化酶-2(Cyclooxygenase-2, COX-2),是催化花生四烯酸合成前列腺素(Prostaglandin, PG)過程中的關(guān)鍵酶,在體內(nèi)存在COX-1和COX-2兩種同工酶。近年來大量研究表明COX-2在多種腫瘤中都存在異常增高表達,在其發(fā)病過程中發(fā)揮著重要作用。關(guān)于COX-2在腫瘤中的作用機制,目前研究表明其可以通過多種途徑作用于腫瘤發(fā)病過程。前期實驗已經(jīng)證實選擇性COX-2抑制劑尼美舒利(nimesulide, NIM)可以通過誘導(dǎo)細胞凋亡,抑制細胞增殖的途徑抑制人喉鱗癌Hep-2細胞裸鼠移植瘤生長。本實驗進一步就NIM對喉鱗癌Hep-2細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)和堿性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)表達的影響進行觀察和研究,以期進一步為NIM用于喉鱗癌的治療提供理論依據(jù)。方法：粱灼萍等在前期試驗中,將喉鱗癌Hep-2細胞接種于裸鼠背部皮下,建立移植瘤模型。將裸鼠隨機分為實驗組和對照組各六只,實驗組予裸鼠腹腔注射NIM(50mg/kg/2d,0.2ml);對照組予裸鼠腹腔注射0.2ml生理鹽水。用藥4周后,處死裸鼠并獲得瘤體。本實驗選用粱灼萍實驗所獲得的喉癌Hep-2細胞裸鼠移植瘤,運用免疫組織化學技術(shù)檢測CD31在喉癌裸鼠移植瘤組織中表達來定位瘤體中的MVD,運用圖像分析系統(tǒng)作平均光密度值測定,并比較兩組間的差異；分別采用免疫組織化學染色技術(shù)檢測喉癌裸鼠移植瘤組織中COX-2、Ang-2和bFGF蛋白的表達情況,運用圖像分析系統(tǒng)計算平均光密度值并比較實驗組與對照組中COX-2、Ang-2、bFGF蛋白表達情況；采用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction, RT-PCR)方法檢測喉癌移植瘤組織中COX-2, Ang-2和bFGF mRNA的表達,并對兩組移植瘤組織中COX-2、Ang-2和bFGF mRNA灰度值進行比較；運用直線相關(guān)分別分析實驗組中COX-2、Ang-2、bFGF蛋白表達水平與MVD值的相關(guān)性。結(jié)果：(1)免疫組織化學SP法檢測裸鼠移植瘤中CD31表達得到平均MVD值,實驗組為12.83±3.06,與對照組25.50±4.85(t=-5.412,P0.05)相比有統(tǒng)計學意義,表明選擇性COX-2抑制劑NIM干預(yù)后移植瘤內(nèi)血管生成減少；(2)利用免疫組織化學SP法檢測裸鼠移植瘤組織中COX-2、Ang-2、 bFGF蛋白表達,實驗組中COX-2蛋白表達光密度為(38.12+15.56),與對照組(140.92±23.12)比較顯著下降,差異有統(tǒng)計學意義(t=-9.825,P0.05),實驗組Ang-2蛋白表達為(49.02±7.67),經(jīng)統(tǒng)計學分析與對照組(110.04±6.76)比較差異有統(tǒng)計學意義(t=-14.607,P0.05)；實驗組bFGF蛋白表達為(62.25±7.50),經(jīng)統(tǒng)計學分析與對照組(119.04±17.30)比較差異有統(tǒng)計學意義(t=-7.391,P0.05)；(3)運用RT-PCR技術(shù)檢測移植瘤中COX-2、Ang-2和bFGF mRNA表達,NIM治療組COX-2mRNA表達為(0.70±0.18),對照組為(2.20±0.38),經(jīng)統(tǒng)計學分析差異有統(tǒng)計學意義(t=-9.825,P0.05)；NIM治療組Ang-2mRNA灰度值為(0.49±0.03),對照組為(0.98±0.10),經(jīng)統(tǒng)計學分析差異有統(tǒng)計學意義(t=-11.509,P0.05)；NIM治療組移植瘤中bFGF mRNA表達為(0.65±0.08),對照組為(1.19±0.19),經(jīng)統(tǒng)計學分析差異有統(tǒng)計學意義(t=-6.209,P0.05)。(4)相關(guān)性分析：運用直線相關(guān)分析方法分別檢測實驗組移植瘤組織中COX-2、Ang-2、bFGF蛋白表達水平與MVD相關(guān)性,經(jīng)統(tǒng)計學分析存在相關(guān)性,其相關(guān)系數(shù)r分別為0.832,0.944,0.844(P均0.05)。結(jié)論：(1)選擇性COX-2抑制劑NIM可以抑制人喉鱗癌Hep-2細胞裸鼠移植瘤中血管生成；(2)實驗組中COX-2、Ang-2、bFGF蛋白表達水平分別與MVD表達水平呈正相關(guān),后兩者為血管生成促進因子,其與MVD的相關(guān)性表明其在腫瘤血管生成中作用顯著；(3)使用NIM后,移植瘤組織中Ang-2和bFGF蛋白及mRNA表達水平都降低,表明NIM抑制人喉鱗癌Hep-2細胞裸鼠移植瘤血管生成的機制可能與抑制Ang-2及bFGF表達有關(guān)；(4)選擇性COX-2抑制劑NIM可通過多種機制抑制人喉鱗癌Hep-2細胞裸鼠移植瘤的生長,有望為未來喉鱗癌綜合治療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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R739.65

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