發(fā)布時(shí)間：2018-04-14 08:43

  本文選題：靶向治療 + 胰腺癌��； 參考：《福建醫(yī)科大學(xué)》2008年博士論文

【摘要】： 一研究目的: 為探索一種新的免疫毒素治療胰腺癌的方法,通過SPDP化學(xué)偶聯(lián)法,我們將眼鏡蛇毒細(xì)胞毒素(CTX)與抗IL-4R單克隆抗體(MAIL4R)構(gòu)建成免疫毒素MAIL4R-CTX,觀察其是否具有導(dǎo)向性殺傷胰腺癌細(xì)胞的作用。 二材料與方法 1.眼鏡蛇毒細(xì)胞毒素的分離純化:先采用SP-Sephadex C-25離子交換柱層析、再用Superdex 75凝膠過濾及Phenyl- Sepharose High Performance疏水層析兩步從舟山眼鏡蛇粗毒中精細(xì)純化分離CTX。 2.免疫毒素的制備:應(yīng)用SPDP偶聯(lián)法,在室溫下,先將CTX與SPDP反應(yīng)生成CTX-PDP;再將MAIL4R與SPDP反應(yīng)生成MAIL4R-PDP,后者在DTT的作用下還原為MAIL4R-SH;最后CTX-PDP與MAIL4R-SH在室溫下充分反應(yīng)至少24小時(shí)后,生成抗白介素4-受體單抗-細(xì)胞毒素免疫毒素(MAIL4R-CTX)。 3.采用SDS-聚丙烯酰胺凝膠電泳、雙向免疫擴(kuò)散和免疫斑點(diǎn)試驗(yàn)檢測免疫毒素的組成。 4.采用免疫組織化學(xué)染色檢測胰腺癌組織中IL-4R的表達(dá)。采用免疫細(xì)胞化學(xué)染色檢測體外培養(yǎng)人胰腺癌細(xì)胞株P(guān)ANC-1、BxPC-3中IL-4R的表達(dá)。 5.采用DAB法檢測免疫毒素MAIL4R-CTX對高表達(dá)IL-4R細(xì)胞BxPC-3、PANC-1和不表達(dá)IL-4R人肺腺癌細(xì)胞H1299的結(jié)合能力。 6.采用MTT法分別測定CTX、MAIL4R及MAIL4R-CTX對體外培養(yǎng)胰腺癌細(xì)胞和肺癌細(xì)胞的作用。 三結(jié)果 1.眼鏡蛇毒粗毒的SP-Sephadex C-25柱層析分離共得14個(gè)蛋白峰;組分XIII鑒定為細(xì)胞毒素,再經(jīng)Superdex 75凝膠過濾和Phenyl-Sepharose HP疏水層析獲得單一對稱蛋白峰,測其分子量為:77.381kDa。將其命名為CTX。 2.CTX與SPDP反應(yīng)物經(jīng)Superdex30凝膠柱后獲得的第一峰為CTX-PDP;MAIL4R與SPDP反應(yīng)物經(jīng)Superdex30凝膠柱獲得的第一峰為MAIL4R-PDP;MAIL4R-PDP經(jīng)DTT還原后再經(jīng)凝膠柱獲得的第一峰為MAIL4R-SH;最后將過量的CTX-PDP與MAIL4R-SH反應(yīng)物經(jīng)Superdex30凝膠過濾,獲得2個(gè)蛋白峰,第一峰即為免疫毒素MAIL4R-CTX。 3.SDS-聚丙烯酰胺凝膠電泳、雙向免疫擴(kuò)散和免疫斑點(diǎn)試驗(yàn)顯示該免疫毒素分子中既含有CTX也含有MAIL4R。 4.免疫組織化學(xué)染色顯示在胰腺癌細(xì)胞中,細(xì)胞質(zhì)呈現(xiàn)均勻著色的棕黃色顆粒,而正常胰腺組織細(xì)胞均為陰性著色。免疫細(xì)胞化學(xué)顯示在胰腺癌PANC-1、BxPC-3細(xì)胞中,細(xì)胞質(zhì)呈現(xiàn)棕黃色顆粒著色,而H1299細(xì)胞為陰性著色。 5.高表達(dá)IL-4R的BXPC-3和PANC-1細(xì)胞DAB染色為棕色,而不表達(dá)IL-4R細(xì)胞H1299則未被染色。 6.采用MTT法觀察到,MAIL4R-CTX在1.2 ug/ml時(shí)即明顯抑制PANC-1、BxPC-3細(xì)胞的生長,在濃度為18.75ug/ml作用4h時(shí)PANC-1和BxPC-3細(xì)胞分別有86.4%和95.2%被殺傷,H1299細(xì)胞僅為26.8%;CTX對PANC-1,BxPC-3和H1299細(xì)胞均有明顯抑制作用,在濃度為8ug/ml時(shí)對三株細(xì)胞的抑制率分別達(dá)到88.5%,87.2%和90%,但三者無明顯差別;MAIL4R對PANC-1、BxPC-3和H1299細(xì)胞均無明顯抑制作用。 四結(jié)論 1.采用SP-Sephadex C-25陽離子交換柱層析、Superdex 75凝膠過濾及Phenyl-Sepharose HP疏水層析三步分離純化可得到低毒高效的眼鏡蛇毒細(xì)胞毒素(CTX)純品。 2.以SPDP法可以成功地將抗白介素-4受體單克隆抗體(MAIL4R)與CTX構(gòu)建成免疫毒素MAIL4R-CTX。 3.胰腺癌組織和胰腺癌細(xì)胞高表達(dá)IL-4R,正常胰腺組織不表達(dá)IL-4R。免疫毒素MAIL4R-CTX對高表達(dá)IL-4R的胰腺癌細(xì)胞具有選擇性殺傷作用。
[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.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392;R735.9

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