抗白介素-4受体单抗与眼镜蛇毒细胞毒素构建的免疫毒素靶向治疗胰腺癌的实验研究

发布时间：2018-04-14 08:43

本文选题：靶向治疗 + 胰腺癌　；参考：《福建医科大学》2008年博士论文

【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。
[Abstract]:One purpose of the study:
In order to explore a new immunotoxin for the treatment of pancreatic cancer, we constructed the immunotoxin MAIL4R-CTX by using SPDP chemical coupling method. We used the cobra venom cytotoxin (CTX) and anti IL-4R monoclonal antibody (MAIL4R) to observe whether it had the function of killing pancreatic cancer cells.
Two materials and methods
1. isolation and purification of cytotoxin from cobra venom. First, SP-Sephadex C-25 ion exchange column chromatography was used, then Superdex 75 gel filtration and Phenyl- Sepharose High Performance hydrophobic chromatography were used to purify CTX. from Zhoushan cobra venom in two steps.
2. immunotoxin preparation: application of SPDP coupling method at room temperature, the CTX and SPDP reaction of CTX-PDP; then MAIL4R reacts with SPDP to form MAIL4R-PDP, the latter under the effect of DTT reduced to MAIL4R-SH; CTX-PDP and MAIL4R-SH finally fully react at room temperature for at least 24 hours after the formation of anti interleukin 4- receptor monoclonal antibody cell toxin immunotoxin (MAIL4R-CTX).
3. the composition of immuno toxin was detected by SDS- polyacrylamide gel electrophoresis, bi-directional immuno diffusion and immuno spot test.
4. immunohistochemical staining was used to detect the expression of IL-4R in pancreatic cancer tissue. The expression of IL-4R in human pancreatic cancer cell line PANC-1 and BxPC-3 was detected by immunocytochemical staining.
5. the binding ability of immuno toxin MAIL4R-CTX to high expression IL-4R cells BxPC-3, PANC-1 and non expression of IL-4R human lung adenocarcinoma cell H1299 was detected by DAB method.
6. the effect of CTX, MAIL4R and MAIL4R-CTX on the culture of pancreatic cancer cells and lung cancer cells in vitro was measured by MTT method.
Three results
1. SP-Sephadex C-25 column chromatography of crude venom of cobra venom separated 14 protein peaks, and XIII was identified as cytotoxin. Then the single symmetric protein peak was obtained by Superdex 75 gel filtration and Phenyl-Sepharose HP hydrophobic chromatography, and its molecular weight was 77.381kDa.: it was named CTX..
The first peak of 2.CTX and SPDP reaction by Superdex30 gel column after the first peak is CTX-PDP; MAIL4R and SPDP reaction by Superdex30 gel column to obtain the MAIL4R-PDP; the first peak of MAIL4R-PDP reduction by DTT followed by gel column to obtain the MAIL4R-SH; finally the CTX-PDP and MAIL4R-SH excess reactants by Superdex30 gel filtration to obtain 2 protein peaks, the first peak is the immunotoxin MAIL4R-CTX.
3.SDS- polyacrylamide gel electrophoresis, bi-directional immuno diffusion and immuno spot test showed that the immune toxin contained both CTX and MAIL4R.
4. immunohistochemical staining showed that in pancreatic cancer cells, the cytoplasm showed Brown particles uniformly colored, and normal pancreatic tissue cells were negative staining. Immunohistochemistry showed in pancreatic cancer PANC-1, BxPC-3 cells, cytoplasm showed Brown particles and coloring, and H1299 cells were negative staining.
5. BXPC-3 and PANC-1 cells with high expression of IL-4R were stained with DAB, while H1299 without IL-4R cells was not stained.
6. using the method of MTT was observed at 1.2 ug/ml MAIL4R-CTX, which inhibit PANC-1 and BxPC-3 cell growth at the concentration of 18.75ug/ml 4h PANC-1 and BxPC-3 cells were 86.4% and 95.2% were killed, only 26.8% of H1299 cells; CTX of PANC-1, BxPC-3 and H1299 cells were significantly inhibited at the concentration of 8ug/ml inhibition of the three cell lines were 88.5%, 87.2% and 90%, but there was no significant difference between the three; MAIL4R for PANC-1, BxPC-3 and H1299 cells had no obvious inhibitory effect.
Four conclusion
1., using SP-Sephadex C-25 cation exchange column chromatography, Superdex 75 gel filtration and Phenyl-Sepharose HP hydrophobic chromatography to separate and purify three times, we can get low toxicity and high efficiency Naja naja toxin cytotoxin (CTX) pure product.
2. the anti interleukins -4 receptor monoclonal antibody (MAIL4R) and CTX can be successfully constructed by SPDP method to form immuno toxin MAIL4R-CTX.
3., the expression of IL-4R was higher in pancreatic cancer and pancreatic cancer cells. The expression of IL-4R. immunotoxin MAIL4R-CTX in normal pancreas tissues had a selective killing effect on pancreatic cancer cells with high expression of IL-4R.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R735.9

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