GM-CSF基因修飾的卵巢癌細胞NuTu-19/GM-CSF的構建

【摘要】  　　目的 構建能分泌表達粒細胞-單核細胞集落刺激因子(GM-CSF)的卵巢癌細胞NuTu-19/GM-CSF。方法 將T-h GM-CSF重組質粒轉化入感受態(tài)大腸桿菌中,篩選出耐藥性單克隆菌落送測序。利用PCR技術擴增出GM-CSF目的基因片段，將PCR產(chǎn)物連入pGEM-T easy載體，轉化入大腸桿菌中擴增，用SDS堿裂解法提取Teasy-GM-CSF重組質粒，用限制性內切酶酶切Teasy-GM-CSF重組載體，連入經(jīng)相同酶酶切的pLXSN反轉錄病毒載體中。用磷酸鈣沉淀法轉染PA317包裝細胞系進行病毒包裝，用含有病毒的上清液感染卵巢癌NuTu-19細胞，經(jīng)G418加壓篩選感染成功的NuTu-19/GM-CSF細胞，用免疫細胞化學和ELISA法檢測NuTu-19/GM-CSF分泌表達GM-CSF蛋白的情況。結果 T-h GM-CSF重組質粒測序結果與GenBank Accession#：NM_000758基因序列對比后確定為人類GM-CSF基因編碼序列；PCR產(chǎn)物酶切電泳在Mark 400-500 bp中有一條帶，符合GM-CSF基因序列長度；重組Teasy-GM-CSF質粒用EcoRⅠ、BamHⅠ限制性內切酶酶切后，電泳鑒定在400-500 bp之間有一目的條帶，重組反轉錄病毒質粒pLGM-CSFSN酶切電泳有目的條帶；G418加壓篩選感染后的NuTu-19細胞，14 d后慢慢形成單克隆繼續(xù)生長；免疫細胞化學檢測到感染成功的NuTu-19細胞膜表面有明顯的棕褐色顆粒沉淀，ELISA法檢測細胞培養(yǎng)上清液中GM-CSF的質量濃度為150 pg/mL。結論 成功構建了能分泌表達人GM-CSF蛋白的卵巢癌細胞NuTu-19/GM-CSF，為以后研究卵巢癌免疫治療提供必備、有效、可靠的工具基礎。

【關鍵詞】  NuTu-19細胞 粒細胞單核細胞集落刺激因子 卵巢腫瘤 免疫治療

　　ABSTRACT: Objective  To construct NuTu-19/GM-CSF ovarian cancer ce學術論文發(fā)表 lls which can secrete and express granulocyte-macrophage colony-stimulating factor (GM-CSF). Methods  The T-h GM-CSF recombinant plasmid was transformed into competent Top109 Bacillus colis and the positive colonies were cultivated in soft agar medium. The T-h GM-CSF recombinant plasmid was used as models to amplify GM-CSF gene fragment by PCR. The certain fragment was linked with the pGEM-T easy vector and transformed into Top109 bacillus coil. SDS alkaline lysis was used to get T easy-GM-CSF recombinant plasmid with pLXSN retrovirus vector cut by the same enzymes. The pseudotype virus was packed after transfecting PA317 cells using calcium phosphate precipitation. The supernate fluid which contained virus to infect NuTu-19 cells was saved. The positive cells were harvested through pressurizing selected by G418. The GM-CSF protein secreted by NuTu-19/GM-CSF cells was tested by immunocytochemistry and ELISA. Results  T-h-GM-CSF included the human GM-CSF gene fragment which contained 435 bp and signal peptide coding region, compared with the sequence of Genebank Accession#: NM-000758. Production of PCR was between 400 bp and 500 bp in the agarose electrophoresis, and had the same length with hGM-CSF gene. There was an electrophoretic strap between 400 bp and 500 bp and the sequence of the cutting fragment was completely correct after cutting T easy-GM-CSF recombinant plasmid by EcoRⅠ and BamHⅠ enzymes. It showed that the amplification of　the GM-CSF gene fragment and the connection with T easy vector were successful. There was still the same strap between 400 bp and 500 bp in the agarose electrophoresis of pLGM-CSFSN after being cut by EcoRⅠ and BamHⅠ enzymes. The infected NuTu-19/GM-CSF cells cultivated in medium containing G418 continued to grow in the forms of monoclone 14 days later. There were some visible granules on the surface of the cellular membrane. The amount of GM-CSF in suspent liquid of cells culture was 150 pg/mL tested by ELISA. Conclusion  The construction of NuTu-19/GM-CSF which can secret and express GM-CSF protein is successful, and offers effective and necessary basis for future study on ovarian cancer immunotherapy.

　　KEY WORDS: NuTu-19 cell; granulocyte-macrophage colony-stimalating factor (GM-CSF); ovarian cancer; immunotherapy

　　卵巢癌是女性生殖系統(tǒng)常見的惡性腫瘤之一,其主要特點是隱蔽性高，臨床不易被發(fā)現(xiàn)，易發(fā)生轉移。近20年來國際婦產(chǎn)聯(lián)盟(FIGO)提出Ⅲ-Ⅳ期卵巢癌患者5年生存率一直徘徊在15%-30%，病死率高居婦科腫瘤首位，其根本原因在于腫瘤的復發(fā)和轉移。在過去的20多年內，免疫治療是繼手術、放療、化療外的又一大腫瘤輔助療法。我們利用分子生物學技術構建