本文選題：噬菌體展示 + 人源抗體�。� 參考：《南京醫(yī)科大學》2010年博士論文

【摘要】：鼻咽癌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構,并較早發(fā)生頸部淋巴結轉移和遠處轉移。鼻咽癌表現(xiàn)為種族和地區(qū)分布的特點,是我國十大高發(fā)惡性腫瘤之一,其發(fā)病率為20/100,000,已引發(fā)嚴重健康問題。鼻咽癌是由Epstein-Barr病毒(EBV)的潛伏感染、環(huán)境因素和宿主的基因遺傳因素共同參與的多步驟交互作用而逐步形成的復雜性疾病。所有的鼻咽癌細胞都含有EBV基因組,并表達EBNA1,EBER1,EBER2,和LMP1,LMP2A,LMP2B,這使得這些病毒蛋白成為理想的腫瘤標志物,其中LMP1(latent membrane protein1,LMP1)在鼻咽上皮細胞的轉化和癌變過程中的作用倍受關注,是目前公認的病毒癌蛋白。它以其獨特的蛋白分子結構參與鼻咽癌發(fā)生和發(fā)展的全過程。以LMP1為靶標,設計并制備各種具有中和作用或靶向功能的抗體將會為鼻咽癌的靶向治療提供新的方法。 本研究擬以噬菌體抗體展示技術篩選人源抗LMP1胞外區(qū)抗體Fab(命名為hleaFab),對篩選獲得的hleaFab進行表達、純化以及功能鑒定。進一步通過體外實驗顯示該抗體與鼻咽癌細胞HNE2-LMP1胞外區(qū)特異性結合特性和靶向功能。在此基礎上,制備免疫毒素hleaFab-絲裂霉素C(hleaFab-MMC),并通過體內和體外實驗研究其靶向抗鼻咽癌作用。 方法 1.通過全人源抗體庫篩選人源抗LMP1胞外區(qū)抗體Fab(hleaFab)。利用細胞與抗原固相篩選相結合的方法進行篩選人源噬菌體抗體庫(Fab抗體庫)。第1、2、3、6輪通過細胞的差減篩選,第4、5、7輪通過固相篩選。其中,細胞差減篩選以鼻咽癌細胞HNE2為陰性細胞,鼻咽癌細胞HNE2-LMP1為陽性細胞;固相篩選以包被GST-LMP1融合蛋白為篩選抗原。篩選7輪后經噬菌體酶聯(lián)免疫吸附反應(phage enzyme linked immunosorbent assay,phage ELISA)進行陽性克隆鑒定。 2.原核表達hleaFab抗體, hleaFab的純化和hleaFab的功能鑒定。HleaFab噬菌體感染大腸桿菌Top 10 F’,培養(yǎng)細菌至對數生長期,IPTG誘導表達;表達蛋白產物使用Protein L親和層析柱純化;純化產物經ELISA、流式細胞技術以及免疫熒光技術進行鑒定。 3.靶向鼻咽癌細胞LMP1胞外區(qū)人源抗體Fab-絲裂霉素C免疫毒素的制備及鑒定。HleaFab轉換緩沖體系,活化抗hleaFab和絲裂霉素(Mytomycin C),將兩者混合反應(hleaFab:Mytomycin C=1:1);脫鹽,轉換緩沖體系至PBS,純化得到免疫毒素hleaFab-MMC。以體外培養(yǎng)的鼻咽癌細胞HNE2-LMP1為研究對象,以相同的hleaFab-MMC、MMC、hleaFab摩爾濃度(1600、800、400、200、100、50、25、12.5、6.25、3.125、1.563nmol/L)分別作用于鼻咽癌細胞系HNE2-LMP1 , MTT法檢測hleaFab-MMC對人鼻咽癌細胞系HNE2-LMP1生長的抑制作用;流式細胞術檢測hleaFab-MMC對鼻咽癌細胞HNE2-LMP1細胞凋亡和細胞周期的影響并結合熒光顯微鏡觀察藥物干預前后細胞形態(tài)學的變化。 4.觀察靶向鼻咽癌細胞LMP1胞外區(qū)人源抗體Fab-MMC免疫毒素體內抑瘤效應。首先建立人鼻咽癌細胞HNE2-LMP1裸鼠移植瘤模型。二步法建模:細胞注射移植法,采用人鼻咽癌細胞HNE2-LMP1 2×10~6/ml注射于7只裸鼠一側肩后腿外側皮下,成瘤4只;微型瘤塊移植法,將瘤塊移植于43只裸鼠一側后腿外側皮下,成瘤40只。病理學及免疫組化檢測移植瘤。挑選腫瘤大小基本一致、生活習性正常的32只。32只鼻咽癌HNE2-LMP1裸鼠動物模型隨機分為四組,每組8只:hleaFab- MMC組、hleaFab組、MMC組、NS(生理鹽水)組。腫瘤局部注射給藥,間隔3天用藥一次,每次200μl,連續(xù)3次,共9天。分別注射抗體hleaFab (0.8mg/ml,1.6×10~(-5) mol/kg)、hleaFab- MMC(0.8mg/ml ,1.6×10~(-5)mol/kg)、MMC(0.005mg/ml ,1.6×10~(-5)mol/kg)、NS(生理鹽水)。每五天測量腫瘤直徑一次,觀察腫瘤體積大小并進行相關檢測。測量5次后處死裸鼠,將移植瘤完整切除,稱其重量,計算抑瘤率,在光學顯微鏡觀察移植瘤細胞形態(tài)變化,采用免疫組化法檢測各組瘤組織中潛伏膜蛋白1 (latent membrane protein 1,LMP1)的表達情況。 結果 1.基因測序顯示本實驗獲得Fab抗體基因序列第30號和36號2株輕鏈為κ鏈的抗LMP1胞外區(qū)抗體Fab(命名為hleaFab)。 2. HleaFab(36號)在原核表達系統(tǒng)內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抗體hleaFab并保留了抗原結合能力。ELISA、流式細胞技術以及免疫熒光技術檢測結果顯示,hleaFab能夠與GST-LMP1胞外區(qū)融合蛋白及鼻咽癌細胞HNE2-LMP1胞外區(qū)特異性結合,而不與鼻咽癌細胞HNE2結合。 3. HleaFab-MMC組在1600-200nmol/L時保持在70%以上的細胞生長抑制率,其后的細胞抑制率隨著濃度的遞減而遞減,呈劑量依賴關系,其半數細胞生長抑制率IC50約為3.7μg/ml;hleaFab組中,細胞生長抑制率隨著hleaFab抗體濃度的遞減而遞減,呈劑量依賴關系,其細胞生長半數抑制率IC50約為44μg/ml,MMC在本實驗的濃度范圍內最大的細胞生長抑制率為37.9%,未能達到細胞的半數抑制。熒光顯微鏡觀察hleaFab-MMC作用于鼻咽癌細胞HNE2-LMP1后可見細胞凋亡特征的形態(tài)學改變:細胞懸浮增多,細胞質內含物增多,細胞體積縮小、變圓、核固縮、碎裂,細胞折光性減弱,細胞內出現(xiàn)顆粒樣物質,培養(yǎng)液中有較多的碎片。流式細胞儀分析發(fā)現(xiàn), hleaFab-MMC組、MMC組、hleaFab組、PBS組細胞凋亡的比率分別13.88%、3.04%、2.78%、2.10%。HleaFab-MMC組與MMC組、hleaFab組、PBS組相比較,hleaFab-MMC在相同濃度作用下表現(xiàn)出較高的細胞凋亡率,同時hleaFab-MMC組細胞相應的S期細胞比率增加為76.35%,MMC組、hleaFab組、PBS組的S期細胞比率僅為34.94%、41.51%、28.38%。 4.細胞注射移植法7只裸鼠,成瘤4只,成瘤率57%;微型瘤塊移植法43只裸鼠,成瘤40只,成瘤率93%。病理學及免疫組化檢測移植瘤具備鼻咽癌細胞HNE2-LMP1表型。治療后第10天、第15天、第20天測量腫瘤體積顯示hleaFab-MMC組、MMC組腫瘤較hleaFab組、NS組生長緩慢,腫瘤體積差異有統(tǒng)計學意義(P0.05)。在療程的第20天hleaFab-MMC組與MMC組相比,腫瘤體積差異有統(tǒng)計學意義(P0.05)。HleaFab組、hleaFab-MMC組、MMC組的抑瘤率分別為12%、36.7%、19.7%,hleaFab-MMC抑瘤率明顯增加。hleaFab-MMC組與其它三組相比,平均瘤重明顯減輕,差異有統(tǒng)計學意義(P0.05)。MMC組與NS組相比,平均瘤重差異有統(tǒng)計學意義(P=0.0200.05);免疫組化結果顯示hleaFab-MMC組與對照組NS組相比,LMP1的表達量明顯減少,差異有統(tǒng)計學意義(P=0.0290.05)。 結論 1.通過全人源抗體庫,采用差減篩選法可以獲得抗LMP1胞外區(qū)抗體hleaFab。 2. HleaFab可以在原核生物大腸桿菌Top 10 F’中表達,并組裝具有生物活性LMP1胞外區(qū)特異性抗體hleaFab。 3.在較低的濃度100 nmol/l作用下,免疫毒素hleaFab-MMC對鼻咽癌細胞HNE2-LMP1有明顯的細胞抑制作用;免疫毒素hleaFab-MMC對鼻咽癌細胞HNE2-LMP1的生長抑制作用的機制之一是促進細胞凋亡;免疫毒素hleaFab-MMC能夠影響鼻咽癌細胞HNE2-LMP1細胞周期變化,增加S期的比率。 4.免疫毒素hleaFab-MMC明顯的抑制人鼻咽癌HNE2-LMP1 BALB/C荷瘤裸鼠體內腫瘤生長;免疫毒素hleaFab-MMC下調腫瘤細胞LMP1的表達。 總之,本研究制備免疫毒素hleaFab-MMC具有靶向抑制LMP1陽性的裸鼠鼻咽癌移植瘤效應。其抑瘤機制可能為:下調HNE2-LMP1鼻咽癌腫瘤細胞LMP1的表達;誘導細胞凋亡;增加S期HNE2-LMP1鼻咽癌細胞比例,提高鼻咽癌細胞對化療藥物敏感性。因此本研究表明人源抗鼻咽癌細胞LMP1胞外區(qū)抗體hleaFab-MMC具有潛在的靶向治療鼻咽癌臨床應用價值。
[Abstract]:Nasopharyngeal carcinoma is a highly malignant tumor derived from the nasopharyngeal epithelium. The progression of the tumor is easily infringed on the important structures such as the base of the skull, and the cervical lymph node metastases and distant metastases occur earlier. Nasopharyngeal carcinoma is characterized by ethnic and regional distribution. It is one of the ten major malignant tumors in China. Its incidence is 20/100000, which has caused serious health problems. Cancer of the pharynx is the latent infection of the Epstein-Barr virus (EBV), the multi step interaction of the environmental factors and the genetic factors of the host. All the nasopharyngeal carcinoma cells contain the EBV genome, and the expression of EBNA1, EBER1, EBER2, and LMP1, LMP2A, LMP2B, which make these viral proteins ideal The role of LMP1 (latent membrane protein1, LMP1) in the transformation of nasopharyngeal epithelial cells and the carcinogenesis of the nasopharyngeal epithelial cells has attracted much attention. It is recognized as a viral oncoprotein. It participates in the whole process of nasopharyngeal carcinoma with its unique protein molecular structure. With LMP1 as the target, it is designed and prepared with various neutralization effects. Targeted antibody will provide a new method for targeted therapy of nasopharyngeal carcinoma.
In this study, a phage antibody display technique was designed to screen the human anti LMP1 extracellular domain antibody Fab (named hleaFab), and to express, purify and functional identification of the selected hleaFab. The specific binding and targeting function of the antibody to the HNE2-LMP1 extracellular region of nasopharyngeal carcinoma cells were further demonstrated by in vitro experiments. On this basis, the preparation of the antibody was prepared. Immunotoxin hleaFab- mitomycin C (hleaFab-MMC) was used to study its targeting anti nasopharyngeal carcinoma function in vivo and in vitro.
Method
1. the human anti LMP1 extracellular domain antibody Fab (hleaFab) was screened by the whole human source antibody library. The human phage antibody library (Fab antibody library) was screened by the combination of cell and antigen solid-phase screening. The 1,2,3,6 wheel was screened by cell differentiation, and the 4,5,7 wheel was screened by solid phase screening. The cell differential screening was used to screen the HNE2 of nasopharyngeal cancer cells to be negative. The HNE2-LMP1 of nasopharyngeal carcinoma cells was positive cells, and the solid phase screening was screened by GST-LMP1 fusion protein as the screening antigen. After screening 7 rounds, the positive clones were identified by the phage phage enzyme linked immunosorbent assay, phage ELISA.
2. prokaryotic expression hleaFab antibody, hleaFab purification and hleaFab function identification,.HleaFab phage infection of Escherichia coli Top 10 F ', cultured bacteria to logarithmic growth period, IPTG induced expression; expression protein products were purified by Protein L affinity chromatography column, and the purified product was identified by ELISA, flow cytometry and immunofluorescence technology.
3. target Fab- mitomycin C immuno toxin of human nasopharyngeal carcinoma cell LMP1 and identification of.HleaFab conversion buffer system, activation of anti hleaFab and Mitomycin (Mytomycin C), mixed reaction (hleaFab:Mytomycin C=1:1), desalination, conversion buffer system to PBS, purification of immunotoxin hleaFab-MMC. in vitro culture In nasopharyngeal carcinoma cell HNE2-LMP1, the same hleaFab-MMC, MMC, and hleaFab molar concentration (1600800400200100,50,25,12.5,6.25,3.125,1.563nmol/L) were used respectively in the nasopharyngeal carcinoma cell line HNE2-LMP1, and the MTT method was used to detect the inhibitory effect of hleaFab-MMC on the HNE2-LMP1 growth of human nasopharyngeal carcinoma cell lines; flow cytometry was used to detect hleaFab-M. Effects of MC on apoptosis and cell cycle of nasopharyngeal carcinoma cell line HNE2-LMP1 were observed, and the morphological changes of cells were observed by fluorescence microscope.
4. to observe the anti tumor effect of human antibody Fab-MMC immuno toxin in the extracellular region of nasopharyngeal carcinoma cell LMP1. First, the model of human nasopharyngeal carcinoma HNE2-LMP1 nude mice transplanted tumor was established. Two step method modeling: cell injection transplantation, HNE2-LMP1 2 x 10~6/ml of human nasopharyngeal carcinoma cells were injected into the lateral posterior leg of the shoulder of the human nasopharyngeal carcinoma cells, and the tumor was 4. The tumor block transplantation method was used to transplant the tumor to the lateral hind leg of 43 nude mice and 40. The tumor was detected by pathology and immunohistochemistry. 32.32 nasopharyngeal carcinoma HNE2-LMP1 nude mice were randomly divided into four groups: hleaFab- MMC group, hleaFab group, MMC group, and NS (physiological salt). Water group. Local injection of the tumor at a interval of 3 days, 200 L each time, 3 times for 9 days. The antibody hleaFab (0.8mg/ml, 1.6 x 10~ (-5) mol/kg), hleaFab- MMC (0.8mg/ml, 1.6 x 10~ (-5) mol/kg) were injected, and the diameter of the tumor was measured every five days to observe the size of the tumor. After 5 times of measurement, the nude mice were killed, the transplanted tumor was completely removed, the weight was measured, the tumor suppressor rate was calculated, the morphological changes of the transplanted tumor cells were observed in the optical microscope, and the expression of the latent membrane protein 1 (latent membrane protein 1, LMP1) in the tumor tissues was detected by immunohistochemistry.
Result
1. gene sequencing showed that the Fab antibody gene sequence thirtieth and 36 were obtained from 2 light chain LMP1 chain Fab antibodies (named hleaFab).
2. HleaFab (No. 36) could be expressed and correctly assembled in the prokaryotic expression system. The purified antibody hleaFab was obtained by using Protein L affinity chromatography column and the antigen binding capacity.ELISA was retained. The results of flow cytometry and immunofluorescence assay showed that hleaFab could be used with the fusion protein of GST-LMP1 extracellular domain and nasopharyngeal carcinoma. The extracellular domain of HNE2-LMP1 cells specifically bind to HNE2 but not with nasopharyngeal carcinoma cell line.
3. HleaFab-MMC group kept the cell growth inhibition rate above 70% at the time of 1600-200nmol/L, and the subsequent cell inhibition rate decreased with the decrease of concentration, which was dose-dependent, and the growth inhibition rate of half cells was about 3.7 mu g/ml, and the growth inhibition rate of cell growth decreased with decreasing of hleaFab antibody concentration in hleaFab group, in a dose dependent manner. The median inhibitory rate of cell growth was about 44 IC50 g/ml, and the maximum inhibitory rate of cell growth was 37.9% within the concentration range of this experiment, which failed to reach half of the inhibition of cell growth. The morphological changes of cell withering characteristics after hleaFab-MMC were observed after HNE2-LMP1 in nasopharyngeal carcinoma cells: cell suspension increased, and thin cell suspension was observed by fluorescence microscopy. The cell volume decreased, the cell volume reduced, the cell became round, the nuclear condensation, the fragmentation, the cell refraction weakened, the cells appeared granular material, and there were more fragments in the culture fluid. Flow cytometry found that the rate of cell apoptosis in group hleaFab-MMC, MMC, hleaFab, and PBS was 13.88%, 3.04%, 2.78%, 2.10%.HleaFab-MMC and MMC, hleaFa In group B, compared with group PBS, hleaFab-MMC showed higher cell apoptosis rate under the same concentration, and the ratio of S cells in group hleaFab-MMC increased to 76.35%, MMC, hleaFab, and PBS groups were only 34.94%, 41.51%, 28.38%..
4. cells were implanted in 7 nude mice, 4 tumor formation and 57% of tumor formation, 43 nude mice and 40 tumor cells with microtumor mass transplantation. The tumor rate 93%. pathology and immunohistochemistry detected the HNE2-LMP1 phenotype of nasopharyngeal carcinoma cells. Tenth days after treatment, fifteenth days and twentieth days, the tumor volume showed hleaFab-MMC group, group MMC tumor was compared to hleaFab group and NS group. The tumor volume difference was statistically significant (P0.05). The tumor volume difference between group hleaFab-MMC and MMC group was statistically significant (P0.05) in group.HleaFab, group hleaFab-MMC and group MMC on twentieth days of treatment. The tumor inhibition rate in group MMC was 12%, 36.7%, 19.7%, and hleaFab-MMC inhibition rate increased significantly compared with the other three groups, the average tumor was compared with the other three groups. The difference was statistically significant (P0.05) and the average tumor weight difference between group.MMC and NS group was statistically significant (P=0.0200.05), and the immunohistochemical results showed that the expression of LMP1 in hleaFab-MMC group was significantly decreased compared with the control group NS group, and the difference was statistically significant (P=0.0290.05).
conclusion
1. through the whole human antibody library, the anti LMP1 extracellular domain antibody hleaFab. can be obtained by the subtractive screening method.
2. HleaFab can be expressed in prokaryotic E.coli Top 10 F "and assembled with bioactive LMP1 extracellular domain specific antibody hleaFab..
3. under the action of low concentration of 100 nmol/l, immuno toxin hleaFab-MMC has obvious inhibitory effect on nasopharyngeal carcinoma cell HNE2-LMP1, and one of the mechanisms of the inhibitory effect of immuno toxin hleaFab-MMC on nasopharyngeal carcinoma cell HNE2-LMP1 is to promote cell apoptosis, and immuno toxin hleaFab-MMC can affect the HNE2-LMP1 cell cycle of nasopharyngeal carcinoma cells. Phase change, increase the ratio of S period.
4. immuno toxin hleaFab-MMC significantly inhibited the growth of human nasopharyngeal carcinoma HNE2-LMP1 BALB/C tumor bearing nude mice, and immuno toxin hleaFab-MMC downregulated the expression of LMP1 in the tumor cells.
In conclusion, the immuno toxin hleaFab-MMC prepared in this study has the effect of targeting LMP1 positive nude mice with nasopharyngeal carcinoma, which may be: down regulation of the expression of LMP1 in HNE2-LMP1 nasopharyngeal carcinoma cells, inducing apoptosis, increasing the proportion of HNE2-LMP1 nasopharyngeal carcinoma cells in S stage, and raising the sensitivity of high nasopharyngeal cancer cells to chemotherapeutic drugs. Studies have shown that human anti NPC LMP1 extracellular domain antibody hleaFab-MMC has potential clinical value in targeting nasopharyngeal carcinoma.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.63

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相關博士學位論文 前1條

1 徐潔潔;EB病毒潛伏膜蛋白2A轉染樹突狀細胞誘導特異性CTL殺傷鼻咽癌的體外、體內研究[D];南京醫(yī)科大學;2006年

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