【作者】 王鸿程；

【导师】 黄鹤光； 应敏刚； 许云禄；

【作者基本信息】 福建医科大学 ， 内科学， 2008， 博士

【摘要】 一研究目的:为探索一种新的免疫毒素治疗胰腺癌的方法,通过SPDP化学偶联法,我们将眼镜蛇毒细胞毒素(CTX)与抗IL-4R单克隆抗体(MAIL4R)构建成免疫毒素MAIL4R-CTX,观察其是否具有导向性杀伤胰腺癌细胞的作用。二材料与方法1.眼镜蛇毒细胞毒素的分离纯化:先采用SP-Sephadex C-25离子交换柱层析、再用Superdex 75凝胶过滤及Phenyl- Sepharose High Performance疏水层析两步从舟山眼镜蛇粗毒中精细纯化分离CTX。2.免疫毒素的制备:应用SPDP偶联法,在室温下,先将CTX与SPDP反应生成CTX-PDP;再将MAIL4R与SPDP反应生成MAIL4R-PDP,后者在DTT的作用下还原为MAIL4R-SH;最后CTX-PDP与MAIL4R-SH在室温下充分反应至少24小时后,生成抗白介素4-受体单抗-细胞毒素免疫毒素(MAIL4R-CTX)。3.采用SDS-聚丙烯酰胺凝胶电泳、双向免疫扩散和免疫斑点试验检测免疫毒素的组成。4.采用免疫组织化学染色检测胰腺癌组织中IL-4R的表达。采用免疫细胞化学染色检测体外培养人胰腺癌细胞株PANC-1、BxPC-3中IL-4R的表达。5.采用DAB法检测免疫毒素MAIL4R-CTX对高表达IL-4R细胞BxPC-3、PANC-1和不表达IL-4R人肺腺癌细胞H1299的结合能力。6.采用MTT法分别测定CTX、MAIL4R及MAIL4R-CTX对体外培养胰腺癌细胞和肺癌细胞的作用。三结果1.眼镜蛇毒粗毒的SP-Sephadex C-25柱层析分离共得14个蛋白峰;组分XIII鉴定为细胞毒素,再经Superdex 75凝胶过滤和Phenyl-Sepharose HP疏水层析获得单一对称蛋白峰,测其分子量为:77.381kDa。将其命名为CTX。2.CTX与SPDP反应物经Superdex30凝胶柱后获得的第一峰为CTX-PDP;MAIL4R与SPDP反应物经Superdex30凝胶柱获得的第一峰为MAIL4R-PDP;MAIL4R-PDP经DTT还原后再经凝胶柱获得的第一峰为MAIL4R-SH;最后将过量的CTX-PDP与MAIL4R-SH反应物经Superdex30凝胶过滤,获得2个蛋白峰,第一峰即为免疫毒素MAIL4R-CTX。3.SDS-聚丙烯酰胺凝胶电泳、双向免疫扩散和免疫斑点试验显示该免疫毒素分子中既含有CTX也含有MAIL4R。4.免疫组织化学染色显示在胰腺癌细胞中,细胞质呈现均匀着色的棕黄色颗粒,而正常胰腺组织细胞均为阴性着色。免疫细胞化学显示在胰腺癌PANC-1、BxPC-3细胞中,细胞质呈现棕黄色颗粒着色,而H1299细胞为阴性着色。5.高表达IL-4R的BXPC-3和PANC-1细胞DAB染色为棕色,而不表达IL-4R细胞H1299则未被染色。6.采用MTT法观察到,MAIL4R-CTX在1.2 ug/ml时即明显抑制PANC-1、BxPC-3细胞的生长,在浓度为18.75ug/ml作用4h时PANC-1和BxPC-3细胞分别有86.4%和95.2%被杀伤,H1299细胞仅为26.8%;CTX对PANC-1,BxPC-3和H1299细胞均有明显抑制作用,在浓度为8ug/ml时对三株细胞的抑制率分别达到88.5%,87.2%和90%,但三者无明显差别;MAIL4R对PANC-1、BxPC-3和H1299细胞均无明显抑制作用。四结论1.采用SP-Sephadex C-25阳离子交换柱层析、Superdex 75凝胶过滤及Phenyl-Sepharose HP疏水层析三步分离纯化可得到低毒高效的眼镜蛇毒细胞毒素(CTX)纯品。2.以SPDP法可以成功地将抗白介素-4受体单克隆抗体(MAIL4R)与CTX构建成免疫毒素MAIL4R-CTX。3.胰腺癌组织和胰腺癌细胞高表达IL-4R,正常胰腺组织不表达IL-4R。免疫毒素MAIL4R-CTX对高表达IL-4R的胰腺癌细胞具有选择性杀伤作用。更多还原

【Abstract】 1. ObjectiveTo explore an effective new approach that against pancreatic cancer with a novel immunotoxin ,we purify a effective Cobra venom cytotoxin(CTX) from cobra (Naja Naja atra)venom. Then CTX was selected as a "warheads" and anti-IL4-R monoclonal antibody (MAIL4R) as a carrier . With heterotypic bifunctional coupling agent SPDP method, MAIL4R and CTX was connected together and the immunotoxin MAIL4R-CTX was obtained. The selective killing effects on pancreatic carcinoma cells which has high expression of IL-4R was observed and compared so as to provide experimental data for new treatment method of pancreatic cancer.2. Materials and methods1. Isolation and Purification of CTX: In this study, the crude venom of cobra venom(Naja atra) was first isolated on SP-Sephadex C-25 cation-exchange column, The effect of tenth～thirteenth protein peaks to the rat isolated heart perfuse preparations and the rat isolated phrenic nerve-diaphragm preparations then be observed; protein peak XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each; SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains be used to assess the purity of peak XIII and its mol. wt of them were calculated.2. Immunotoxin MAIL4R-CTX preparation: In room temperature, the cytotoxin of Naja naja atra reacted with SPDP to form CTX-PDP. Then we made the anti-interleukin4-receptor monoclonal antibody and SPDP have the same reaction to form anti-IL-4-receptor monoclonal antibody-PDP, and it was reduced to monoclonal antibody-SH by DTT. In the end, the monoclonal antibody-SH was thoroughly mixed with CTX-PDP in room temperature for at least 24 hours to produce a new type immunotoxin MAIL4R-CTX. 3. Identified the composition of immunotoxin MAIL4R-CTX by the means of SDS-PAGE polyacrylamide gel electrophoresis and double immunodiffusion and dotimmunobindingassay.4. Immunohistochemical technique was used to detect the expression of IL-4R in normal and pancreatic cancer specimens and immunocytochemical technique was used to detect the expression of IL-4R in human pancreatic cancer cell lines (PANC-1 and BxPC-3) and human NSCLC cells.5. Binding ability of the conjugate to BxPC-3 and PANC-1 cells with highly expressed IL-4R and H1299 cells without IL-4R expression was measured by DAB method.8. Using MTT method, Cytotoxic activity of AIL-4R ,CTX and CTX-AIL-4R were tested in PANC-1,BXPC-3 and H1299 cell lines in vitro respectively.3. Results1. Isolation and Purification of CTX: The crude venom of cobra venom(Naja atra) was isolated on SP-Sephadex C-25 cation-exchange column and fractionated into 14 protein peaks; The protein peak XIII could produce the rat isolated Langendorffs preparations to decrease their contracture heights, ultimately leading to complete cardiac arrest. Peak XIII were further purified by Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography once each and a single protein peak be obtained. It migrated as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by Coomassie brilliant blue stains which showed that it was homogeneous with one band. Its mol. wt was calculated to be 7.381 kDa orderly by Image Master VDS. The results showed that the protein peak XIII was a CTX and then we named it CTX in this paper.2. Gel filtration chromatography of immunotoxin: The reaction products of CTX and SPDP were loaded on Superdex30 gel column and three major protein peaks were obtained, in which the first was CTX-PDP with large molecular weight. The reaction products of MAIL4R and SPDP were loaded on Superdex30 gel column, and 2 peaks were obtained, in which the first peak was MAIL4R-PDP; After reduction of MAIL4R-PDP by DTT, two main peaks were obtained after filtration on the column, in which the first peak was MAIL4R-SH. Finally, filtration of excessive reaction products of CTX-PDP and MAIL4R-SH were carried out on Superdex30 gel column with two protein peaks obtained, in which the first peak was the immunotoxin MAIL4R-CTX, and the second peak was unbound CTX-PDP.3. In reduced SDS-PAGE polyacrylamide gel electrophoresis, MAIL4R-CTX showed two zones of minor molecular weight protein. Then antivenomous serum made immunoprecipitation among MAIL4R-CTX, CTX, CTX-PDP and peakⅡof MAIL4R-CTX, and eventually the first three samples formed the lines of sedimentation. It is found in Dotimmunobindingassay of the immunotoxin that, immunotoxin MAIL4R-CTX not only had strong antigen-antibody response with anti-cobra venom antibody, but also could directly react with goat anti-mouse antibody, suggesting that MAIL4R-CTX contained both the cobra venom cytotoxin and mouse antibody (ie MAIL4R)4. Normal pancreas tissue samples did not stain or showed weak positive staining for IL-4R. In contrast, all 26 pancreatic cancer specimens overexpress IL-4R; Both human pancreatic cancer cell lines BxPC-3and PANC-1 were stained brown, while human lung cancer cells H1299 who did not express IL-4R were not stained.5. BxPC-3 and PANC-1 cells have high expression of IL-4R were stained brown and H1299 cells that do not express IL-4R were not stained in DAB method.6. By MTT method, we observed that MAIL4R-CTX significantly inhibited growth of BxPC-3, PANC-1 cells that have high expression of IL-4R at the concentration of 1.2 ug / ml, while for H1299 cells with no or low expression of IL-4R, inhibitory effect appeared when the concentration was more than 5 ug / ml. At the concentration of 18.75 ug / ml, the cell killing rate of MAIL4R-CTX on PANC-1 and BxPC-3 cells was 86.4% and 95.2% respectively, while it was only 26.8% on H1299 cells; CTX had rapid killing effects on PANC-1, BxPC-3 and H1299 cells. After 4h’s action,the inhibition rate of CTX in the concentration of 8 ug / ml on three cell strains was 88.5%, 87.2% and 90% respectively; Monoclonal antibody of interleukin-4 receptor had no obvious killing effect on the above cells. 4. Conclusions1. A purified CTX isoforms from the Naja atra venom was obtained by SP-Sephadex C-25 cation-exchange chromatography, Superdex 75 gel filtration and phenyl-Sepharose HP hydrophobic interaction chromatography step by step.2. With heterotypic bifunctional coupling agent SPDP method, MAIL4R and CTX can be successful connected together to be a novel immunotoxin MAIL4R-CTX.3. IL-4R overexpress in pancreatic cancer specimens and cells but not in normal pancreas. Immunotoxin MAIL4R-CTX had selective killing effect on pancreatic cancer cells which have high expression of IL-4R.更多还原