眼镜蛇毒细胞毒素—白介素4受体单抗免疫毒素的制备及其抗胰腺癌作用

发布时间：2018-05-28 04:15

本文选题：眼镜蛇毒 + 细胞毒素　；参考：《福建医科大学》2008年硕士论文

【摘要】： 免疫毒素(Immuonotoxin, IT)是由能与肿瘤细胞表面受体或细胞表面靶抗原特异结合的单克隆抗体和对肿瘤细胞具有杀伤作用的毒素分子通过化学交联构建而成。细胞毒素(Cytotoxin, CTX)是眼镜蛇毒中一类重要的活性蛋白,约占蛇毒总蛋白量的40%,由60-63个氨基酸残基组成,分子量在7000Da左右,国内外许多报道都证实其具有很强的抗肿瘤作用。白介素-4受体(IL4R)已被发现在胰腺癌细胞表面高表达,但是在正常胰腺组织和人肺癌细胞H1299则不表达或者低表达,目前以IL4R作为靶点的免疫毒素研究已取得一系列令人鼓舞的结果。为探索细胞毒素是否能与抗IL-4R单克隆抗体(Monoclonal antibody of IL-4 receptor, MAIL4R)偶联构建成免疫毒素,以选择性杀伤高表达IL-4R的胰腺癌细胞,本实验拟从眼镜蛇毒中分离出CTX,将CTX与MAIL4R进行了偶联,并观察和比较偶联物对胰腺导管上皮癌PANC-1细胞,原位胰腺癌BXPC-3细胞和肺腺癌H1299细胞的作用,为寻找胰腺癌治疗新方法提供实验资料。 1. CTX的分离纯化眼镜蛇毒粗毒经过SP-Sepharose FF阳离子交换色谱,获得13个蛋白质组分。其中组分XI使大鼠离体心脏标本收缩张力增加、幅度缩小、最后停于收缩期,呈现明显的心脏毒性;同时对分别刺激大鼠膈肌和膈神经引起的收缩均有抑制作用,呈现明显的细胞毒性作用,鉴定为眼镜蛇毒细胞毒素(CTX)组分。将XI组分过Sephasil Peptide C18柱精细纯化,经SDS-PAGE(Tris-Tricine系统)鉴定,CTX-XI呈均一蛋白区带;分子量为7235Da左右。自动电位滴定法测定PLA2,活力为0,得到CTX纯品暂命名为CTX-XI。 2.免疫毒素MAIL4R-CTX偶联物的制备将CTX和MAIL4R用N-琥珀酰胺-3-(2-吡啶二硫)丙酸酯(SPDP)法进行偶联,构建CTX和MAIL4R的偶联物,偶联物MAIL4R-CTX在SDS-PAGE中为一条分子量大于100kD的单一条带,还原后则可见为两条带;该偶联物既能与抗眼镜蛇毒血清产生较强的抗原-抗体反应,也能直接与羊抗小鼠的抗体产生反应;与该偶联物共同孵育的人胰腺癌BXPC-3细胞被染成棕色,而不表达IL-4R的人肺腺癌H1299细胞未被染色,表明偶联物与人胰腺癌BXPC-3细胞具有很好的结合性,而与人肺癌H1299细胞不结合。 3. CTX、MAIL4R和MAIL4R-CTX体外抗肿瘤活性采用MTT法观察CTX、MAIL4R和CTX-MAIL4R对体外培养的PANC-1细胞,BXPC-3细胞和H1299细胞增殖的抑制作用,并测定其半数抑制浓度(IC50)。CTX对PANC-1,BXPC-3和H1299细胞均有快速的杀伤作用,CTX浓度为8μg/ml时,对PANC-1,BXPC-3和H1299细胞4h杀伤率分别为89.8%、88.0%和89.3%,对3株细胞的杀伤强度没有明显的区别;MAIL4R对上述细胞明显无杀伤作用;MAIL4R-CTX浓度为9.4μg/ml时,对PANC-1和BXPC-3细胞4h杀伤率分别为73.1%和84.4%,而对H1299细胞杀伤率仅为29.8%。结论:采用SP-Sepharose FF阳离子交换色谱和Sephasil Peptide C18反相色谱两步得到一个不含磷脂酶A2的CTX纯品。用SPDP法可以成功构建CTX与MAIL4R免疫毒素,该免疫毒素对高表达IL4R的胰腺癌细胞具有选择性杀伤作用。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R735.9

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