抗人IgM抗体对鼻咽癌HNE-1细胞裸鼠成瘤及IgM、gp96蛋白表达的影响

发布时间：2018-06-12 23:41

  本文选题：免疫球蛋白M + 鼻咽癌　； 参考：《泸州医学院》2011年硕士论文

【摘要】：目的：尽管鼻咽癌的诊断和治疗方法都在不断进步,但对其早期诊断与预后判断还缺乏有效的手段,患者的5年生存率还有待进一步提高；因而我们需要在鼻咽癌发病机制理论研究领域取得突破性进展,寻找新的靶点,以提高其早期诊断水平和整体治疗效果。本实验通过研究抗人IgM抗体对鼻咽癌HNE-1细胞裸鼠移植瘤生长、肿瘤细胞凋亡以及免疫球蛋白M(IgM)、糖蛋白gp96 (Glycoprotein96, gp96)表达的影响,以期探讨IgM在鼻咽癌中异位表达的生物学活性以及抗人IgM抗体影响裸鼠成瘤的可能机制。方法：对鼻咽癌HNE-1细胞进行传代培养,制成细胞悬液,接种于Balb/c-nu/nu裸鼠右侧背腹部皮下,建立鼻咽癌移植瘤模型；15只荷瘤裸鼠随机分为实验组(腹腔注射抗人IgM抗体,1.5mg/裸鼠,分5次,每隔3天1次)、IgG对照组(腹腔注射IgG抗体)及PBS对照组(腹腔注射PBS),同时观察裸鼠移植瘤生长情况,定期(每四天)称量裸鼠体重并以游标卡尺测量移植瘤的长径及短径,绘制肿瘤生长曲线,计算肿瘤体积=肿瘤长径×肿瘤短径2/2。5次腹腔给药后观察1周,处死裸鼠,取瘤体,称量移植瘤重量,计算抑瘤率。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。采用TUNEL技术检测鼻咽癌HNE-1细胞裸鼠移植瘤组织中细胞凋亡情况,采用图像分析系统计算凋亡指数；同时利用免疫组化SP法检测裸鼠移植瘤组织中IgM、gp96蛋白的表达情况,同样采用图像分析系统作平均光密度值测定。结果：(1)成功构建出人鼻咽癌裸鼠移植瘤模型,三组裸鼠均有鼻咽癌原位移植瘤生长,成瘤率100%；其瘤组织经病理切片检查证实为鼻咽癌,且在实验期内,三组动物全部存活。(2)三组裸鼠饮食正常,活动自如,精神状态良好,无1只死亡；其心、肝、脾、肺、肾组织形态观察无明显差异。(3)抗人IgM抗体干预组裸鼠移植瘤体积为(26.73±16.51)mm3,较IgG对照组(204.97±151.88)mm3和PBS对照组(230.16±167.78)mm3明显减小,三组间差异有统计学意义(P0.05)；抗人IgM抗体干预组裸鼠体重为(21.10±2.35)g,而IgG对照组为(20.70±3.29)g, PBS对照组(20.01±2.22)g,三组间差异无统计学意义(P0.05)；抗人IgM抗体干预组裸鼠移植瘤重量(0.09±0.07)g,较IgG对照组(0.35±0.10)g和PBS对照组(0.38±0.08)g明显减小,经统计学分析差异有统计学意义(P0.05)；同时计算抑瘤率分别为74.45%和76.84%。(4)TUNEL法检测鼻咽癌HNE-1细胞裸鼠移植瘤组织中细胞凋亡水平,抗人IgM抗体干预组肿瘤细胞平均凋亡指数为(1.50±0.76)%,较IgG对照组(0.49±0.15)%和PBS对照组(0.42±0.20)%明显升高,三组间差异经统计学分析有统计学意义(P0.05)；(5)免疫组织化学SP法检测裸鼠移植瘤组织中IgM、gp96蛋白表达,抗人IgM抗体干预组IgM蛋白表达的平均光密度值为(0.01±0.01),与IgG对照组(0.06±0.03)和PBS对照组(0.05±0.03)相比均显著下降,三组间差异有统计学意义(P0.05)；抗人IgM抗体干预组gp96蛋白表达显著下降(0.05±0.01),经统计学分析与IgG对照组(0.10±0.03)和PBS对照组(0.11±0.02)比较差异有统计学意义(P0.05)。结论：(1)抗人IgM抗体能显著抑制人鼻咽癌HNE-1细胞裸鼠移植瘤生长,提示鼻咽癌异位表达的IgM具有生长因子样作用。(2)抗人IgM抗体能显著抑制IgM蛋白表达,通过阻断其生物学活性来抑制肿瘤生长。(3)抗人IgM抗体可抑制gp96蛋白表达,推测其机制可能与抑制IgM表达有关。(4)抗人IgM抗体能促进裸鼠移植瘤中鼻咽癌HNE-1细胞凋亡,其机制可能与抑制IgM、gp96蛋白表达有关。
[Abstract]:Objective: Although the diagnosis and treatment methods of nasopharyngeal carcinoma are constantly improving, the early diagnosis and prognosis are still lack of effective means. The 5 year survival rate of the patients still needs to be further improved. Therefore, we need to make a breakthrough in the theoretical research field of nasopharyngeal cancer pathogenesis and find new targets in order to improve their early diagnosis. The effect of anti human IgM antibody on the growth of xenografts in nude mice of nasopharyngeal carcinoma HNE-1 cells, the apoptosis of tumor cells and the expression of immunoglobulin M (IgM) and glycoprotein gp96 (Glycoprotein96, gp96) in nude mice of nasopharyngeal carcinoma, in order to explore the biological activity of ectopic expression of IgM in nasopharyngeal carcinoma and the effect of anti human IgM antibody on the effect of anti human anti human antibody on the growth of nude mice in nasopharyngeal carcinoma cells. Methods: the possible mechanism of tumor formation in nude mice. Methods: the HNE-1 cells of nasopharyngeal carcinoma were cultured and made into cell suspension and inoculated into the right dorsal abdomen of Balb/c-nu/nu nude mice to establish the model of nasopharyngeal carcinoma transplantation tumor. 15 tumor bearing nude mice were randomly divided into experimental group (intraperitoneal injection of anti human IgM antibody, 1.5mg/ nude mice, 5 times every 3 days), IgG control group (1 times every 3 days). Intraperitoneal injection of IgG antibody and PBS control group (intraperitoneal injection of PBS), and observing the growth of transplanted tumor in nude mice, weighing the body weight of nude mice regularly (every four days) and measuring the length and short diameter of the transplanted tumor with vernier caliper, plotting the tumor growth curve, and calculating the tumor volume = the tumor length and the short diameter of the tumor for 1 weeks after the intraperitoneal administration, and killing nude mice. The tumor body was taken and weighed and the tumor suppressor rate was calculated. Conventional HE staining was used to observe the changes of the transplanted tumor and the main viscera of nude mice was stained with HE. The apoptosis in the transplanted tumor tissues of nude mice of nasopharyngeal carcinoma HNE-1 cells was detected by TUNEL technique, and the apoptosis index was calculated by the image analysis system, and the nude was detected by the immunohistochemical SP method. The expression of IgM and gp96 protein in the transplanted tumor tissues of the mice was also measured by the mean optical density value of the image analysis system. Results: (1) the transplanted tumor model of human nasopharyngeal carcinoma was successfully constructed. The three groups of nude mice had the growth of nasopharyngeal carcinoma in situ and the tumor rate was 100%. The tumor tissue was confirmed by pathological section to be nasopharyngeal carcinoma and the experiment was in the experiment. During the period, all the three groups of animals survived. (2) the three groups of nude mice had normal diet, good activity, good mental state, no 1 death, and the heart, liver, spleen, lung and kidney tissue were not significantly different. (3) the volume of the transplanted tumor in the anti human IgM antibody group was (26.73 + 16.51) mm3, compared with the IgG control group (204.97 + 151.88) mm3 and PBS control group (230.16 + 167.78) M The difference between the three groups was statistically significant (P0.05), and the body weight of nude mice in the anti human IgM antibody group was (21.10 + 2.35) g, while the IgG control group was (20.70 + 3.29) g, PBS control group (20.01 + 2.22) g, and there was no statistical difference (P0.05), and the weight of nude mice in the anti human IgM antibody group was (0.09 + 0.07) g, compared with IgG control group (0.35 + 0.). 10) g and PBS control group (0.38 + 0.08) g decreased significantly, and statistically significant (P0.05). At the same time, the tumor suppressor rate was 74.45% and 76.84%. (4) TUNEL method was used to detect the cell apoptosis level in nude mice of nasopharyngeal carcinoma HNE-1 cells. The average apoptosis index of tumor cells in anti human IgM anti body intervention group was (1.50 + 0.76)%, and was more than Ig. G control group (0.49 + 0.15)% and PBS control group (0.42 + 0.20)% increased significantly, the difference between the three groups was statistically significant (P0.05); (5) immunohistochemical SP method was used to detect the expression of IgM, gp96 protein in the transplanted tumor tissues of nude mice, and the average optical density of IgM egg white expression in the anti human IgM antibody group was (0.01 + 0.01), and that of the IgG control group was 0.06 (0.06). Compared with the PBS control group (0.05 + 0.03), the difference between the three groups was statistically significant (P0.05), and the expression of gp96 protein in the anti human IgM antibody group decreased significantly (0.05 + 0.01). The difference between the IgG control group (0.10 + 0.03) and the PBS control group (0.11 + 0.02) was statistically significant (P0.05). Conclusion: (1) anti human IgM resistance (P0.05). Physical ability significantly inhibited the growth of human nasopharyngeal carcinoma HNE-1 cells in nude mice, suggesting that the ectopic expression of IgM in nasopharyngeal carcinoma has a growth factor like effect. (2) anti human IgM antibodies can significantly inhibit the expression of IgM protein and inhibit the growth of tumor by blocking its biological activity. (3) anti human IgM antibody can inhibit the expression of gp96 protein, presumably its mechanism may be inhibited. IgM expression. (4) anti human IgM antibody can promote the apoptosis of nasopharyngeal carcinoma HNE-1 cells in nude mice. The mechanism may be related to the inhibition of IgM and gp96 protein expression.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

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