本文關(guān)鍵詞： 免疫球蛋白M 喉腫瘤 癌 鱗狀細(xì)胞 移植 異種的 糖蛋白 凋亡　出處：《瀘州醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：喉鱗癌是人類常見的惡性腫瘤,目前主要的治療方法是手術(shù)和放射治療,常用的化療藥物對喉鱗癌的治療效果均不十分滿意,人們一直在為尋找新的化療方法和化療藥物而努力。同時,上皮來源的惡性腫瘤細(xì)胞表達(dá)免疫球蛋白(immunoglobulin, Ig)已引起學(xué)者們的關(guān)注,對于包括喉鱗癌在內(nèi)的各種惡性腫瘤異位表達(dá)Ig這一現(xiàn)象進行深入、系統(tǒng)的研究已成為今后腫瘤免疫研究的熱點與難點,并可為各類惡性腫瘤發(fā)生、發(fā)展的腫瘤生物學(xué)研究提供新的思路。本實驗通過研究抗人IgM抗體對喉鱗癌Hep-2細(xì)胞裸鼠成瘤、IgM、gp96蛋白表達(dá)以及腫瘤細(xì)胞凋亡的影響,以探討抗人IgM抗體的抗腫瘤作用和可能的機制,從而為將抗人IgM抗體用于喉鱗癌的預(yù)防和治療提供實驗依據(jù)。方法：將喉鱗癌Hep-2細(xì)胞進行傳代培養(yǎng),制成細(xì)胞懸液,接種于Balb/c-nu/nu裸鼠右側(cè)背部皮下,建立移植瘤模型,15只荷瘤裸鼠隨機分為實驗組(瘤內(nèi)注射抗人IgM抗體,1.5mg/只,分五次注射,每3天一次)、IgG對照組(以羊IgG代替抗人IgM抗體)和PBS對照組(以PBS代替抗人IgM抗體)。觀察裸鼠生長情況,定期(每3天)稱量裸鼠體重并以游標(biāo)卡尺測量移植瘤的最長徑和最短徑,繪制腫瘤生長曲線,計算腫瘤體積=(腫瘤最長徑×腫瘤最短徑2)/2。最后 一次用藥3天后,處死裸鼠,取瘤體,常規(guī)HE染色,觀察抗人IgM抗體對裸鼠可能的毒副作用。利用免疫組織化學(xué)技術(shù)檢測IgM、gp96蛋白表達(dá),顯微鏡下觀察移植瘤中IgM、gp96蛋白表達(dá)情況,并采用圖像分析系統(tǒng)進行平均光密度值測定。同時采用TUNEL-AP法檢測移植瘤中凋亡細(xì)胞表達(dá),并采用圖像分析系統(tǒng)計算TUNEL陽性細(xì)胞比率。結(jié)果：(1)成功構(gòu)建出人喉鱗癌裸鼠移植瘤模型,3組裸鼠均有喉鱗癌原位移植瘤生長,成瘤率100%；其瘤組織經(jīng)病理切片檢查證實為喉鱗癌,且在實驗期內(nèi),3組裸鼠全部存活。(2)3組裸鼠飲食正常,活動自如,精神狀態(tài)良好,無1只死亡；其心、肝、脾、肺、腎的組織形態(tài)觀察無明顯差異。(3)實驗組裸鼠移植瘤平均體積為(64.58±26.74)mm3較IgG對照組(204.89±110.55)mm3,PBS對照(258.36±71.07)mm3明顯減小,差異有統(tǒng)計學(xué)意義(P0.05)；實驗組裸鼠平均體重為(22.40±1.92)g,IgG對照組(25.62±4.00)g,PBS對照(22.42±1.93)g,差異無統(tǒng)計學(xué)意義(P0.05)；實驗組裸鼠移植瘤平均重量為(0.07±0.05)g,較IgG對照組(0.26±0.20)g,PBS對照(0.25±0.09)g明顯減小,差異有統(tǒng)計學(xué)意義(P0.05)；計算抑瘤率1(即與非特異對照組比較)為73.63%,抑瘤率2(即與空白對照組比較)為73.28%。(4)免疫組織化學(xué)SP法檢測裸鼠移植瘤組織中IgM、gp96蛋白表達(dá),實驗組IgM蛋白的表達(dá)平均光密度值為(0.04±0.03),與IgG對照組(0.10±0.03),PBS對照組(0.11±0.04)比較顯著下降,經(jīng)統(tǒng)計學(xué)分析差異有統(tǒng)計學(xué)意義(P0.05)；實驗組gp96蛋白的表達(dá)平均光密度值為(0.04±0.01),與IgG對照組(0.08±0.02),PBS對照組(0.10±0.03)比較顯著下降,經(jīng)統(tǒng)計學(xué)分析差異有統(tǒng)計學(xué)意義(P0.05)。(5)TUNEL-AP法檢測裸鼠移植瘤中凋亡細(xì)胞表達(dá),實驗組陽性細(xì)胞比率為(2.32±1.03)%,與IgG對照組(0.47±0.23)%,PBS對照組(0.38±0.23)%比較顯著升高,經(jīng)統(tǒng)計學(xué)分析差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論：(1)抗人IgM抗體對喉鱗癌Hep-2細(xì)胞裸鼠移植瘤生長有明顯的抑制作用。(2)抗人IgM抗體可以抑制IgM蛋白表達(dá),推測抗人IgM抗體可能通過抗原抗體反應(yīng)與喉鱗癌組織產(chǎn)生的IgM結(jié)合來抑制腫瘤生長。(3)抗人IgM抗體可以抑制gp96蛋白表達(dá),其機制可能與抑制IgM蛋白表達(dá)有關(guān),也可能與其促進腫瘤細(xì)胞的凋亡相關(guān)聯(lián)。(4)從蛋白水平證實抗人IgM抗體可以促進喉鱗癌裸鼠移植瘤細(xì)胞的凋亡,其機制可能與抑制IgM、gp96表達(dá)有關(guān)。(5)抗人IgM抗體無明顯毒副作用。(6)抗人IgM抗體可能成為未來喉鱗癌綜合治療的一部分,對IgM異位表達(dá)進行深入研究可為喉鱗癌預(yù)防和治療提供全新的廣闊前景。
[Abstract]:Objective: laryngeal carcinoma is a common malignant tumor. The main treatment is surgery and radiation therapy, the treatment effect of chemotherapy drugs commonly used in laryngeal carcinoma were not very satisfied, people have been looking for a new method of chemotherapy and chemotherapy drugs work. At the same time, the expression of immunoglobulin in malignant tumor cells on skin source the (immunoglobulin, Ig) has attracted the attention of scholars, for a variety of malignant tumors including ectopic expression of Ig in laryngeal squamous cell carcinoma, this phenomenon in-depth, systematic research has become a hot spot in the study of tumor immunotherapy in the future, and can occur for all types of malignant tumors, and provide new ideas on the development of tumor biology study of anti human IgM antibody on laryngeal squamous cell carcinoma Hep-2 cells in nude mice, IgM, gp96 protein expression and apoptosis of tumor cells, to investigate the anti-tumor effect of anti human IgM antibody And the possible mechanism, so as to provide experimental basis for the anti human IgM antibody for the prevention and treatment of laryngeal squamous cell carcinoma. Methods: human laryngeal squamous cell carcinoma Hep-2 cells were cultured, cell suspension inoculated into Balb/c-nu/nu nude mice subcutaneously in the right back, to establish xenograft model, 15 nude mice were randomly divided into experimental group (tumor injection of anti human IgM antibody, 1.5mg/, five times of injection, once every 3 days), IgG group (with IgG instead of Sheep anti human IgM antibody (PBS) and the control group with PBS instead of anti human IgM antibody). Observe the growth of nude mice, regularly (every 3 days) mice were weighed and using vernier caliper to measure the tumor size and the longest shortest path, draw tumor growth curve, calculate the tumor volume (x = the tumor diameter of tumor shortest diameter 2 /2.) finally
A medication for 3 days, mice were sacrificed, and the tumor body, conventional HE staining, to observe the anti human IgM antibody in nude mice may have side effects. Using immunohistochemistry to detect IgM, gp96 protein expression, observe the tumor in IgM under the microscope, the expression of gp96 protein, and the average optical density determination of image at the same time, using the method of TUNEL-AP analysis system. The expression of cell apoptosis of transplanted tumor, and by calculating the rate of TUNEL positive cells in the image analysis system. Results: (1) successfully constructed a xenograft model of human laryngeal squamous carcinoma in nude mice, the growth of 3 were laryngeal squamous cell carcinoma nude mice orthotopic transplantation tumor, the tumor formation rate was 100%; the tumor tissue by pathological section examination confirmed the diagnosis of laryngeal squamous cell carcinoma, and in the experimental period, 3 groups of nude mice survived. (2) 3 groups of nude mice a normal diet, free activities, a good mental state, not only 1 deaths; heart, liver, spleen, lung, kidney and histological observation showed no significant difference .(3)瀹為獙緇勮８榧犵Щ妞嶇槫騫沖潎浣撶Н涓，

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