本文選題：眼鏡蛇毒 + 細(xì)胞毒素��； 參考：《福建醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 免疫毒素(Immuonotoxin, IT)是由能與腫瘤細(xì)胞表面受體或細(xì)胞表面靶抗原特異結(jié)合的單克隆抗體和對(duì)腫瘤細(xì)胞具有殺傷作用的毒素分子通過(guò)化學(xué)交聯(lián)構(gòu)建而成。細(xì)胞毒素(Cytotoxin, CTX)是眼鏡蛇毒中一類(lèi)重要的活性蛋白,約占蛇毒總蛋白量的40%,由60-63個(gè)氨基酸殘基組成,分子量在7000Da左右,國(guó)內(nèi)外許多報(bào)道都證實(shí)其具有很強(qiáng)的抗腫瘤作用。白介素-4受體(IL4R)已被發(fā)現(xiàn)在胰腺癌細(xì)胞表面高表達(dá),但是在正常胰腺組織和人肺癌細(xì)胞H1299則不表達(dá)或者低表達(dá),目前以IL4R作為靶點(diǎn)的免疫毒素研究已取得一系列令人鼓舞的結(jié)果。為探索細(xì)胞毒素是否能與抗IL-4R單克隆抗體(Monoclonal antibody of IL-4 receptor, MAIL4R)偶聯(lián)構(gòu)建成免疫毒素,以選擇性殺傷高表達(dá)IL-4R的胰腺癌細(xì)胞,本實(shí)驗(yàn)擬從眼鏡蛇毒中分離出CTX,將CTX與MAIL4R進(jìn)行了偶聯(lián),并觀察和比較偶聯(lián)物對(duì)胰腺導(dǎo)管上皮癌PANC-1細(xì)胞,原位胰腺癌BXPC-3細(xì)胞和肺腺癌H1299細(xì)胞的作用,為尋找胰腺癌治療新方法提供實(shí)驗(yàn)資料。 1. CTX的分離純化 眼鏡蛇毒粗毒經(jīng)過(guò)SP-Sepharose FF陽(yáng)離子交換色譜,獲得13個(gè)蛋白質(zhì)組分。其中組分XI使大鼠離體心臟標(biāo)本收縮張力增加、幅度縮小、最后停于收縮期,呈現(xiàn)明顯的心臟毒性;同時(shí)對(duì)分別刺激大鼠膈肌和膈神經(jīng)引起的收縮均有抑制作用,呈現(xiàn)明顯的細(xì)胞毒性作用,鑒定為眼鏡蛇毒細(xì)胞毒素(CTX)組分。將XI組分過(guò)Sephasil Peptide C18柱精細(xì)純化,經(jīng)SDS-PAGE(Tris-Tricine系統(tǒng))鑒定,CTX-XI呈均一蛋白區(qū)帶;分子量為7235Da左右。自動(dòng)電位滴定法測(cè)定PLA2,活力為0,得到CTX純品暫命名為CTX-XI。 2.免疫毒素MAIL4R-CTX偶聯(lián)物的制備 將CTX和MAIL4R用N-琥珀酰胺-3-(2-吡啶二硫)丙酸酯(SPDP)法進(jìn)行偶聯(lián),構(gòu)建CTX和MAIL4R的偶聯(lián)物,偶聯(lián)物MAIL4R-CTX在SDS-PAGE中為一條分子量大于100kD的單一條帶,還原后則可見(jiàn)為兩條帶;該偶聯(lián)物既能與抗眼鏡蛇毒血清產(chǎn)生較強(qiáng)的抗原-抗體反應(yīng),也能直接與羊抗小鼠的抗體產(chǎn)生反應(yīng);與該偶聯(lián)物共同孵育的人胰腺癌BXPC-3細(xì)胞被染成棕色,而不表達(dá)IL-4R的人肺腺癌H1299細(xì)胞未被染色,表明偶聯(lián)物與人胰腺癌BXPC-3細(xì)胞具有很好的結(jié)合性,而與人肺癌H1299細(xì)胞不結(jié)合。 3. CTX、MAIL4R和MAIL4R-CTX體外抗腫瘤活性 采用MTT法觀察CTX、MAIL4R和CTX-MAIL4R對(duì)體外培養(yǎng)的PANC-1細(xì)胞,BXPC-3細(xì)胞和H1299細(xì)胞增殖的抑制作用,并測(cè)定其半數(shù)抑制濃度(IC50)。CTX對(duì)PANC-1,BXPC-3和H1299細(xì)胞均有快速的殺傷作用,CTX濃度為8μg/ml時(shí),對(duì)PANC-1,BXPC-3和H1299細(xì)胞4h殺傷率分別為89.8%、88.0%和89.3%,對(duì)3株細(xì)胞的殺傷強(qiáng)度沒(méi)有明顯的區(qū)別;MAIL4R對(duì)上述細(xì)胞明顯無(wú)殺傷作用;MAIL4R-CTX濃度為9.4μg/ml時(shí),對(duì)PANC-1和BXPC-3細(xì)胞4h殺傷率分別為73.1%和84.4%,而對(duì)H1299細(xì)胞殺傷率僅為29.8%。 結(jié)論:采用SP-Sepharose FF陽(yáng)離子交換色譜和Sephasil Peptide C18反相色譜兩步得到一個(gè)不含磷脂酶A2的CTX純品。用SPDP法可以成功構(gòu)建CTX與MAIL4R免疫毒素,該免疫毒素對(duì)高表達(dá)IL4R的胰腺癌細(xì)胞具有選擇性殺傷作用。
[Abstract]:Immuonotoxin (IT) is constructed by a chemical crosslinking of a monoclonal antibody that is specific to the surface receptor or target antigen of the tumor cell surface or the target antigen of the cell surface. The cytotoxin (Cytotoxin, CTX) is one of the most important active proteins in the cobra venom, accounting for the total protein of the snake venom. The amount of 40% is composed of 60-63 amino acid residues, with a molecular weight of about 7000Da. Many reports at home and abroad have proved that it has a strong anti-tumor effect. The -4 receptor (IL4R) has been found to be highly expressed on the surface of pancreatic cancer cells, but it is not expressed or low expression in normal pancreatic tissue and human lung cancer cell H1299. At present, IL4R is used as a IL4R A series of encouraging results have been obtained in the study of the target immuno toxin. In order to explore whether the cytotoxin can be constructed with the anti IL-4R monoclonal antibody (Monoclonal antibody of IL-4 receptor, MAIL4R) to construct the immunotoxin and selectively kill the pancreatic cancer cells with high expression of IL-4R, this experiment is to separate CTX from the cobra venom and take CTX. Coupling with MAIL4R, and observing and comparing the effect of coupling on pancreatic ductal epithelial cancer PANC-1 cells, in situ pancreatic cancer BXPC-3 cells and lung adenocarcinoma H1299 cells, provide experimental data for finding new methods for the treatment of pancreatic cancer.
Separation and purification of 1. CTX
13 protein components were obtained by SP-Sepharose FF cation exchange chromatography. The component XI made the contractile tension of the isolated heart specimens increased, narrowed and stopped at the systolic time, showing obvious cardiac toxicity, and inhibited the contraction of the diaphragmatic and phrenic nerves in rats respectively. The significant cytotoxic effect was identified as the component of cobra venom toxin (CTX). The XI group was finely purified by Sephasil Peptide C18 column and CTX-XI showed a homogeneous protein zone by SDS-PAGE (Tris-Tricine system); the molecular weight was about 7235Da. The automatic potentiometric titration method was used to determine PLA2, the activity was 0, and CTX pure product was temporarily named CTX-XI..
Preparation of 2. immunotoxin MAIL4R-CTX conjugate
The coupling of CTX and MAIL4R with N- succinamide -3- (2- pyridine two sulfur) propionate (SPDP) was used to construct a coupling between CTX and MAIL4R. The coupling MAIL4R-CTX in SDS-PAGE is a single band with a molecular weight larger than 100kD, and two bands after reduction. The conjugate can produce a stronger antigen antibody reaction with the anti cobra venom sera. The human pancreatic cancer BXPC-3 cells, which were incubated with the conjugate, were stained brown, and the human lung adenocarcinoma H1299 cells, which did not express IL-4R, were not stained, indicating that the coupling was very good with human pancreatic cancer BXPC-3 cells and was not associated with human lung cancer H1299 cells.
Antitumor activity of 3. CTX, MAIL4R and MAIL4R-CTX in vitro
The inhibitory effects of CTX, MAIL4R and CTX-MAIL4R on the proliferation of PANC-1 cells, BXPC-3 cells and H1299 cells in vitro were observed by MTT, and the median inhibitory concentration (IC50).CTX (IC50).CTX had a rapid killing effect on PANC-1, BXPC-3 and H1299 cells. When the concentration was 8 mu, the killing rate was 89.8%, 88, respectively. .0% and 89.3%, the killing intensity of 3 cells was not distinctly different. MAIL4R had no killing effect on the above cells, and the killing rate of 4H in PANC-1 and BXPC-3 cells was 73.1% and 84.4% respectively when the concentration of MAIL4R-CTX was 9.4 mu, and the killing rate of H1299 cells was only 29.8%..
Conclusion: a pure CTX without phospholipase A2 was obtained by SP-Sepharose FF cation exchange chromatography and Sephasil Peptide C18 reversed phase chromatography. The CTX and MAIL4R immuno toxin can be successfully constructed by SPDP method. The immunotoxin has a selective killing effect on the high expression of IL4R pancreatic cancer cells.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;R735.9

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