本文關(guān)鍵詞： 抗LMP1抗體Fab 絲裂霉素C(MMC) 潛伏膜蛋白1(LMP1) 鼻咽癌移植瘤　出處：《南京醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：鼻咽癌(Nasopharyngeal Carcinoma,NPC)是來源于鼻咽上皮的高度惡性腫瘤,腫瘤進展極易侵犯顱底等重要結(jié)構(gòu),并較早發(fā)生頸部淋巴結(jié)轉(zhuǎn)移和遠處轉(zhuǎn)移。鼻咽癌具有種族和地區(qū)分布的特點。在我國發(fā)病率20/100,000,已引發(fā)人類的嚴重健康問題。目前鼻咽癌的治療方法主要為放療及輔助化療。然而鼻咽癌放、化療總的五年生存率為60%,晚期鼻咽癌治療后五年生存率小于40%,而且放、化療的毒性反應不可避免地導致嚴重的局部和全身并發(fā)癥。大量研究表明,鼻咽癌是由EB病毒(Epstein Barr Virus,EBV)的潛伏感染、環(huán)境因素和宿主的遺傳因素共同參與的多步驟交互作用而逐步形成的。所有鼻咽癌細胞都含有EBV基因組,并表達多種病毒蛋白如EBNA1、EBER1、EBER2和LMP1、LMP2A、LMP2B。其中LMP1(latent membrane protein1,LMP1)在鼻咽上皮細胞的轉(zhuǎn)化和癌變過程中的作用倍受關(guān)注,是目前公認的病毒癌蛋白,它以其獨特的分子結(jié)構(gòu)參與鼻咽癌的發(fā)生和發(fā)展的全過程,這使得它成為理想的腫瘤靶向治療標志物。因此本實驗設計并制備針對LMP1的Fab抗體片段,特異性Fab抗體片段將會為鼻咽癌的診斷與靶向治療提供新的思路。 轉(zhuǎn)移性復發(fā)性鼻咽癌對原有化療藥物的耐藥和再次放化療抵抗是鼻咽癌死亡的主要原因。因此,選擇新的敏感化療藥物和治療方法對轉(zhuǎn)移性和復發(fā)性鼻咽癌具有重要的臨床意義。絲裂霉素C(mitomycin C,MMC)是從頭狀鏈霉菌培養(yǎng)液中分離提取的一種廣譜抗腫瘤抗生素,對多種實體腫瘤有效,已經(jīng)用于臨床多種腫瘤的治療,但對鼻咽癌的抑瘤作用鮮有報道。其作用機理是通過烷化基團和雙螺旋形成交聯(lián),破壞DNA和RNA的結(jié)構(gòu),引起細胞凋亡。有研究表明MMC在缺氧環(huán)境仍有較高活性,對G0期細胞比較敏感,有助于減少腫瘤藥物的抗藥性。另外MMC結(jié)構(gòu)特殊,含有包括醌環(huán)、吲哚、氮丙啶環(huán)以及甲氧甲酰胺側(cè)鏈,適合與抗體等偶聯(lián),在腫瘤的靶向治療中有良好的應用前景。因此選擇MMC作為治療鼻咽癌的候選化療藥或聯(lián)合化療藥,具有重要的臨床意義。本課題組前期體外實驗已證實MMC對鼻咽癌細胞生長具有明顯抑制作用。 本研究在前期課題組已經(jīng)完成篩選全人源噬菌體Fab抗體庫并制備出抗LMP1胞外區(qū)抗體Fab的基礎上,重新表達、純化及鑒定抗體Fab,觀察Fab聯(lián)合MMC腹腔注射治療鼻咽癌裸鼠移植瘤的作用并初探其可能機制。 方法: 1.原核表達抗體Fab、抗體Fab的純化和功能鑒定:Fab噬菌體感染大腸桿菌Top10F’,培養(yǎng)細菌至對數(shù)生長期,IPTG誘導表達;表達蛋白產(chǎn)物使用Protein L親和層析柱純化;純化產(chǎn)物經(jīng)SDS-PAGE技術(shù)進行鑒定; 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細胞約0.2 ml、約5×10~6/ml的細胞懸液。將20只荷瘤裸鼠隨機分為4組,即Fab組、MMC組、Fab+MMC組及對照組,每組5只,腹腔注射藥物治療,每次0.3 ml,每3天1次,共5次。治療藥物及劑量為Fab組3 mg/kg、MMC組2 mg/kg、Fab+MMC組Fab 3 mg/kg+MMC 2 mg/kg、對照組為生理鹽水; 3.觀察腫瘤體積、瘤重,計算抑瘤率,繪制腫瘤的生長曲線; 4.免疫組化法檢測腫瘤組織中血管內(nèi)皮生長因子(VEGF)表達情況; 5.流式細胞術(shù)(FCM)檢測腫瘤組織的細胞凋亡情況; 6.統(tǒng)計學分析:用SPSS10.0統(tǒng)計軟件包對數(shù)據(jù)進行T檢驗,方差齊性檢驗及方差分析;正態(tài)分布資料用X士S表示,凋亡用率表示,率的兩兩比較采用方差分析,半定量資料兩組間比較采用Mann-Whitney秩和檢驗,P 0. 05認為差異有統(tǒng)計學意義。 結(jié)果: 1.抗體Fab在原核表達系統(tǒng)內(nèi)能夠表達并正確組裝;使用Protein L親和層析柱純化獲得了純度較高的Fab; 2.成功建立LMP1鼻咽癌細胞裸鼠皮下移植瘤模型,成瘤率100%; 3.腫瘤體積(mm3):Fab組462.71±42.7916、MMC組407.846±51.1506、Fab+MMC組266.851±46.3747,對照組561.975±47.7266,Fab+MMC組與其它各組比較均有統(tǒng)計學差異(P0.01);瘤重(g):Fab組0.37±0.0332、MMC組0.324±0.0385、Fab+MMC組0.232±0.0259,對照組0.456±0.0488,Fab+MMC組與其它各組比較有統(tǒng)計學差異(P0.01);抑瘤率:Fab組18.90%、MMC組28.95%、Fab+MMC組49.12%; 4.腫瘤組織中血管內(nèi)皮生長因子(VEGF)表達情況:Fab組、Fab+MMC組中VEGF均低表達,明顯低于對照組(P0.05);MMC組VEGF呈高表達,與對照組相比無顯著差異(P0.05);VEGF陽性染色主要定位于腫瘤細胞胞漿,部分巨噬細胞、血管內(nèi)皮細胞、成纖維細胞等也表達VEGF; 5.流式細胞術(shù)檢測腫瘤組織的細胞凋亡情況:Fab組7.337%±2.6755%、MMC組11.843%±1.5022%、Fab+MMC組17.55%±3.2058% ,對照組3.9%±0.5456%。MMC組和Fab+MMC組移植瘤細胞凋亡率明顯高于對照組,差異有顯著性(P0.01),而Fab組凋亡率和對照組相比,無顯著性差異(P0.05)。 結(jié)論: 1.成功對人源抗LMP1胞外區(qū)抗體Fab進行表達、純化以及鑒定; 2. Fab及MMC均有抑瘤作用,聯(lián)合使用效果優(yōu)于單獨使用; 3. Fab及MMC二者抑制腫瘤的作用途徑不完全相同:Fab可能與抑制腫瘤血管生成有關(guān),MMC可能與誘導腫瘤細胞凋亡增加有關(guān)。
[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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.63

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