抗人IgM抗体对喉鳞癌Hep-2细胞裸鼠成瘤的影响及机制研究

发布时间：2018-01-24 03:34

  本文关键词： 免疫球蛋白M 喉肿瘤 癌 鳞状细胞 移植 异种的 糖蛋白 凋亡　出处：《泸州医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：喉鳞癌是人类常见的恶性肿瘤,目前主要的治疗方法是手术和放射治疗,常用的化疗药物对喉鳞癌的治疗效果均不十分满意,人们一直在为寻找新的化疗方法和化疗药物而努力。同时,上皮来源的恶性肿瘤细胞表达免疫球蛋白(immunoglobulin, Ig)已引起学者们的关注,对于包括喉鳞癌在内的各种恶性肿瘤异位表达Ig这一现象进行深入、系统的研究已成为今后肿瘤免疫研究的热点与难点,并可为各类恶性肿瘤发生、发展的肿瘤生物学研究提供新的思路。本实验通过研究抗人IgM抗体对喉鳞癌Hep-2细胞裸鼠成瘤、IgM、gp96蛋白表达以及肿瘤细胞凋亡的影响,以探讨抗人IgM抗体的抗肿瘤作用和可能的机制,从而为将抗人IgM抗体用于喉鳞癌的预防和治疗提供实验依据。方法：将喉鳞癌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,15只荷瘤裸鼠随机分为实验组(瘤内注射抗人IgM抗体,1.5mg/只,分五次注射,每3天一次)、IgG对照组(以羊IgG代替抗人IgM抗体)和PBS对照组(以PBS代替抗人IgM抗体)。观察裸鼠生长情况,定期(每3天)称量裸鼠体重并以游标卡尺测量移植瘤的最长径和最短径,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。最后 一次用药3天后,处死裸鼠,取瘤体,常规HE染色,观察抗人IgM抗体对裸鼠可能的毒副作用。利用免疫组织化学技术检测IgM、gp96蛋白表达,显微镜下观察移植瘤中IgM、gp96蛋白表达情况,并采用图像分析系统进行平均光密度值测定。同时采用TUNEL-AP法检测移植瘤中凋亡细胞表达,并采用图像分析系统计算TUNEL阳性细胞比率。结果：(1)成功构建出人喉鳞癌裸鼠移植瘤模型,3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,3组裸鼠全部存活。(2)3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、肾的组织形态观察无明显差异。(3)实验组裸鼠移植瘤平均体积为(64.58±26.74)mm3较IgG对照组(204.89±110.55)mm3,PBS对照(258.36±71.07)mm3明显减小,差异有统计学意义(P0.05)；实验组裸鼠平均体重为(22.40±1.92)g,IgG对照组(25.62±4.00)g,PBS对照(22.42±1.93)g,差异无统计学意义(P0.05)；实验组裸鼠移植瘤平均重量为(0.07±0.05)g,较IgG对照组(0.26±0.20)g,PBS对照(0.25±0.09)g明显减小,差异有统计学意义(P0.05)；计算抑瘤率1(即与非特异对照组比较)为73.63%,抑瘤率2(即与空白对照组比较)为73.28%。(4)免疫组织化学SP法检测裸鼠移植瘤组织中IgM、gp96蛋白表达,实验组IgM蛋白的表达平均光密度值为(0.04±0.03),与IgG对照组(0.10±0.03),PBS对照组(0.11±0.04)比较显著下降,经统计学分析差异有统计学意义(P0.05)；实验组gp96蛋白的表达平均光密度值为(0.04±0.01),与IgG对照组(0.08±0.02),PBS对照组(0.10±0.03)比较显著下降,经统计学分析差异有统计学意义(P0.05)。(5)TUNEL-AP法检测裸鼠移植瘤中凋亡细胞表达,实验组阳性细胞比率为(2.32±1.03)%,与IgG对照组(0.47±0.23)%,PBS对照组(0.38±0.23)%比较显著升高,经统计学分析差异有统计学意义(P0.05)。结论：(1)抗人IgM抗体对喉鳞癌Hep-2细胞裸鼠移植瘤生长有明显的抑制作用。(2)抗人IgM抗体可以抑制IgM蛋白表达,推测抗人IgM抗体可能通过抗原抗体反应与喉鳞癌组织产生的IgM结合来抑制肿瘤生长。(3)抗人IgM抗体可以抑制gp96蛋白表达,其机制可能与抑制IgM蛋白表达有关,也可能与其促进肿瘤细胞的凋亡相关联。(4)从蛋白水平证实抗人IgM抗体可以促进喉鳞癌裸鼠移植瘤细胞的凋亡,其机制可能与抑制IgM、gp96表达有关。(5)抗人IgM抗体无明显毒副作用。(6)抗人IgM抗体可能成为未来喉鳞癌综合治疗的一部分,对IgM异位表达进行深入研究可为喉鳞癌预防和治疗提供全新的广阔前景。
[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)瀹為獙缁勮８榧犵Щ妞嶇槫骞冲潎浣撶Н涓，

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