人源抗EB病毒LMP1抗体Fab联合丝裂霉素C对鼻咽癌裸鼠移植瘤生长的抑制作用

发布时间：2018-02-03 06:46

本文关键词： 抗LMP1抗体Fab 丝裂霉素C(MMC) 潜伏膜蛋白1(LMP1) 鼻咽癌移植瘤　出处：《南京医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：鼻咽癌(Nasopharyngeal Carcinoma,NPC)是来源于鼻咽上皮的高度恶性肿瘤,肿瘤进展极易侵犯颅底等重要结构,并较早发生颈部淋巴结转移和远处转移。鼻咽癌具有种族和地区分布的特点。在我国发病率20/100,000,已引发人类的严重健康问题。目前鼻咽癌的治疗方法主要为放疗及辅助化疗。然而鼻咽癌放、化疗总的五年生存率为60%,晚期鼻咽癌治疗后五年生存率小于40%,而且放、化疗的毒性反应不可避免地导致严重的局部和全身并发症。大量研究表明,鼻咽癌是由EB病毒(Epstein Barr Virus,EBV)的潜伏感染、环境因素和宿主的遗传因素共同参与的多步骤交互作用而逐步形成的。所有鼻咽癌细胞都含有EBV基因组,并表达多种病毒蛋白如EBNA1、EBER1、EBER2和LMP1、LMP2A、LMP2B。其中LMP1(latent membrane protein1,LMP1)在鼻咽上皮细胞的转化和癌变过程中的作用倍受关注,是目前公认的病毒癌蛋白,它以其独特的分子结构参与鼻咽癌的发生和发展的全过程,这使得它成为理想的肿瘤靶向治疗标志物。因此本实验设计并制备针对LMP1的Fab抗体片段,特异性Fab抗体片段将会为鼻咽癌的诊断与靶向治疗提供新的思路。转移性复发性鼻咽癌对原有化疗药物的耐药和再次放化疗抵抗是鼻咽癌死亡的主要原因。因此,选择新的敏感化疗药物和治疗方法对转移性和复发性鼻咽癌具有重要的临床意义。丝裂霉素C(mitomycin C,MMC)是从头状链霉菌培养液中分离提取的一种广谱抗肿瘤抗生素,对多种实体肿瘤有效,已经用于临床多种肿瘤的治疗,但对鼻咽癌的抑瘤作用鲜有报道。其作用机理是通过烷化基团和双螺旋形成交联,破坏DNA和RNA的结构,引起细胞凋亡。有研究表明MMC在缺氧环境仍有较高活性,对G0期细胞比较敏感,有助于减少肿瘤药物的抗药性。另外MMC结构特殊,含有包括醌环、吲哚、氮丙啶环以及甲氧甲酰胺侧链,适合与抗体等偶联,在肿瘤的靶向治疗中有良好的应用前景。因此选择MMC作为治疗鼻咽癌的候选化疗药或联合化疗药,具有重要的临床意义。本课题组前期体外实验已证实MMC对鼻咽癌细胞生长具有明显抑制作用。本研究在前期课题组已经完成筛选全人源噬菌体Fab抗体库并制备出抗LMP1胞外区抗体Fab的基础上,重新表达、纯化及鉴定抗体Fab,观察Fab联合MMC腹腔注射治疗鼻咽癌裸鼠移植瘤的作用并初探其可能机制。方法: 1.原核表达抗体Fab、抗体Fab的纯化和功能鉴定:Fab噬菌体感染大肠杆菌Top10F’,培养细菌至对数生长期,IPTG诱导表达;表达蛋白产物使用Protein L亲和层析柱纯化;纯化产物经SDS-PAGE技术进行鉴定; 2.建立LMP1鼻咽癌裸鼠皮下移植瘤模型:于20只BALB/C裸鼠背部皮下注射鼻咽癌LMP1细胞约0.2 ml、约5×10~6/ml的细胞悬液。将20只荷瘤裸鼠随机分为4组,即Fab组、MMC组、Fab+MMC组及对照组,每组5只,腹腔注射药物治疗,每次0.3 ml,每3天1次,共5次。治疗药物及剂量为Fab组3 mg/kg、MMC组2 mg/kg、Fab+MMC组Fab 3 mg/kg+MMC 2 mg/kg、对照组为生理盐水; 3.观察肿瘤体积、瘤重,计算抑瘤率,绘制肿瘤的生长曲线; 4.免疫组化法检测肿瘤组织中血管内皮生长因子(VEGF)表达情况; 5.流式细胞术(FCM)检测肿瘤组织的细胞凋亡情况; 6.统计学分析:用SPSS10.0统计软件包对数据进行T检验,方差齐性检验及方差分析;正态分布资料用X士S表示,凋亡用率表示,率的两两比较采用方差分析,半定量资料两组间比较采用Mann-Whitney秩和检验,P 0. 05认为差异有统计学意义。结果: 1.抗体Fab在原核表达系统内能够表达并正确组装;使用Protein L亲和层析柱纯化获得了纯度较高的Fab; 2.成功建立LMP1鼻咽癌细胞裸鼠皮下移植瘤模型,成瘤率100%; 3.肿瘤体积(mm3):Fab组462.71±42.7916、MMC组407.846±51.1506、Fab+MMC组266.851±46.3747,对照组561.975±47.7266,Fab+MMC组与其它各组比较均有统计学差异(P0.01);瘤重(g):Fab组0.37±0.0332、MMC组0.324±0.0385、Fab+MMC组0.232±0.0259,对照组0.456±0.0488,Fab+MMC组与其它各组比较有统计学差异(P0.01);抑瘤率:Fab组18.90%、MMC组28.95%、Fab+MMC组49.12%; 4.肿瘤组织中血管内皮生长因子(VEGF)表达情况:Fab组、Fab+MMC组中VEGF均低表达,明显低于对照组(P0.05);MMC组VEGF呈高表达,与对照组相比无显著差异(P0.05);VEGF阳性染色主要定位于肿瘤细胞胞浆,部分巨噬细胞、血管内皮细胞、成纤维细胞等也表达VEGF; 5.流式细胞术检测肿瘤组织的细胞凋亡情况:Fab组7.337%±2.6755%、MMC组11.843%±1.5022%、Fab+MMC组17.55%±3.2058% ,对照组3.9%±0.5456%。MMC组和Fab+MMC组移植瘤细胞凋亡率明显高于对照组,差异有显著性(P0.01),而Fab组凋亡率和对照组相比,无显著性差异(P0.05)。结论: 1.成功对人源抗LMP1胞外区抗体Fab进行表达、纯化以及鉴定; 2. Fab及MMC均有抑瘤作用,联合使用效果优于单独使用; 3. Fab及MMC二者抑制肿瘤的作用途径不完全相同:Fab可能与抑制肿瘤血管生成有关,MMC可能与诱导肿瘤细胞凋亡增加有关。
[Abstract]:Nasopharyngeal carcinoma (Nasopharyngeal Carcinoma NPC) is a highly malignant tumor derived from nasopharyngeal epithelial tumor progression, easy invasion of skull base and other important structures, and early cervical lymph node metastasis and distant metastasis of nasopharyngeal carcinoma. With ethnic and regional distribution in China. The incidence rate of 20/100000, has caused serious problems in human health. Current treatment the main method of nasopharyngeal carcinoma radiotherapy and adjuvant chemotherapy for nasopharyngeal carcinoma. However, chemotherapy overall five year survival rate was 60% after treatment, advanced nasopharyngeal carcinoma five years survival rate of less than 40%, and the toxicity of chemotherapy, inevitably lead to local and systemic complications. Many studies showed that nasopharyngeal carcinoma by EB virus (Epstein Barr Virus, EBV) latent infection, multi-step interaction of genetic factors and environmental factors of the host participation gradually formed. All nasopharyngeal carcinoma cells contain EB The V genome, and the expression of a variety of viral proteins such as EBNA1, EBER1, EBER2 and LMP1, LMP2A, LMP2B. and LMP1 (latent membrane protein1, LMP1) in the transformation and carcinogenesis of nasopharyngeal epithelial cells in the role of attention, is recognized as the viral oncoprotein, it takes its unique molecular structure in nasopharyngeal carcinoma and the whole development process, which makes it an ideal target for tumor markers. Therefore this experiment design and preparation of antibody to Fab fragment of LMP1, specific Fab antibody fragment will target for the diagnosis and treatment of nasopharyngeal carcinoma to provide new ideas.
Resistant metastatic and recurrent nasopharyngeal carcinoma on the original chemotherapy and radiotherapy resistance is the main reason of NPC death. Therefore, selecting sensitive chemotherapy drugs and a new treatment method has important clinical significance in metastatic and recurrent nasopharyngeal carcinoma. Mitomycin C (mitomycin C MMC) is a broad-spectrum culture of Streptomyces alatum extraction the separated liquid antitumor antibiotic, effective in many solid tumors, has been used for clinical treatment of tumors, but the inhibitory effect on nasopharyngeal carcinoma is rarely reported. Its mechanism is by alkylating group and double helix form cross-linking, destroy the structure DNA and RNA, causing cell apoptosis. Studies have shown that MMC still has high activity in the hypoxic environment, more sensitive to G0 cells, help to reduce drug resistance. In addition MMC special structure contains quinone ring, indole, aziridine ring and methoxy methyl The amide side chain, and suitable for antibody conjugate in tumor targeting therapy has good application prospect. So the choice of MMC as a candidate of chemotherapy or combined with chemotherapy in treatment of nasopharyngeal carcinoma, has important clinical significance. Our previous in vitro experiments have confirmed that MMC has obvious inhibitory effect on nasopharyngeal carcinoma cell growth.
This study has completed the screening of human phage antibody library of Fab and preparation of anti LMP1 extracellular domain antibody Fab, re expression in the early stage of the research group, purification and identification of antibody Fab, observation of Fab combined with MMC intraperitoneal injection in the treatment of nasopharyngeal carcinoma xenografts in nude mice and explore the possible mechanism.
Method:
1. prokaryotic expression antibody Fab, antibody Fab purification and function identification: Fab bacteriophage infection Escherichia coli Top10F ', cultured bacteria to logarithmic growth phase, IPTG induced expression; expressed protein products were purified by Protein L affinity chromatography; purified products were identified by SDS-PAGE technology.
2. models of subcutaneous LMP1 nasopharyngeal carcinoma xenografts in nude mice: 20 BALB/C subcutaneous injection of human nasopharyngeal carcinoma cell line LMP1 was about 0.2 ml, about 5 * 10~6/ml cell suspension. 20 nude mice were randomly divided into 4 groups, Fab group, MMC group, Fab+MMC group and control group, 5 rats in each group, injection drug treatment of abdominal cavity, 0.3 ml each time, 1 times every 3 days, a total of 5 times. The treatment drug and dose group Fab 3 mg/kg, MMC 2 mg/kg group, Fab+MMC group Fab 3 mg/kg+MMC 2 mg/kg, the control group normal saline;
3. the tumor volume, the tumor weight, the tumor suppressor rate were calculated, and the growth curve of the tumor was drawn.
4. immunohistochemical method was used to detect the expression of vascular endothelial growth factor (VEGF) in tumor tissues.
5. flow cytometry (FCM) was used to detect the apoptosis of tumor tissue.
6. statistical analysis: SPSS10.0 statistical software package for T test data, homogeneity test of variance and variance analysis; normal distribution data of X + S, apoptosis rate, rate of 22 compared with analysis of variance, semi quantitative data between the two groups were compared using the Mann-Whitney rank test, P 0.05 was considered statistically the difference in meaning.
Result:
The 1. antibody Fab was expressed in the prokaryotic expression system and assembled correctly, and the purity of Fab was obtained by the purification of Protein L affinity chromatography column.
2. the subcutaneous transplantation tumor model of nude mice with LMP1 nasopharyngeal carcinoma cells was successfully established, and the rate of tumor formation was 100%.
3. the tumor volume (mm3): Fab = 462.71 + 42.7916, 407.846 + 51.1506 MMC group, Fab+MMC group of 266.851 + 46.3747, 561.975 + 47.7266 in control group, Fab+MMC group and other groups were statistically significant (P0.01); tumor weight (g): Fab = 0.37 + 0.0332, 0.324 + 0.0385 MMC group, Fab+MMC group 0.232 + 0.0259, 0.456 + 0.0488 in control group, Fab+MMC group and other groups were statistically significant (P0.01); the inhibition rate was 18.90% in group Fab, group MMC 28.95%, group Fab+MMC 49.12%;
Vascular endothelial growth factor 4. (VEGF) expression in tumor tissues: group Fab, group Fab+MMC VEGF low expression was significantly lower than the control group (P0.05); high expression of MMC VEGF was compared with the control group, no significant difference (P0.05); VEGF positive staining was mainly localized in the cytoplasm of tumor cells, part macrophages, vascular endothelial cells, fibroblasts also express VEGF;
Cell apoptosis in tumor tissues were detected by flow cytometry in 5. group Fab: 7.337% + 2.6755%, 11.843% + 1.5022% MMC group, Fab+MMC group, control group 17.55% + 3.2058%, 3.9% + 0.5456%.MMC group and Fab+MMC group transplanted tumor cell apoptosis rate was significantly higher than the control group, there was significant difference (P0.01), and the apoptosis rate of Fab group and compared with the control group, no significant difference (P0.05).
Conclusion:
1. the expression, purification and identification of human anti LMP1 extracellular domain antibody Fab were successfully expressed.
2. Fab and MMC have tumor suppressor effect, and the combined use effect is better than that of single use.
3., Fab and MMC two have different ways of inhibiting tumor. Fab may be related to inhibiting angiogenesis. MMC may be associated with increased apoptosis of tumor cells.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

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