3种血小板生成的关键因子共转染人骨髓基质细胞的研究  

Three kind of platelet growth factors in hBMSC by gene transfection

作　　者：林放[1] 王甜甜[1] 王世春[1] 孟强[1] 尹智平[1] 陆华[1] 蒋天伦[1] 赵树铭[1] 

机构地区：[1]第三军医大学西南医院输血科,重庆400038

出　　处：《中国输血杂志》2012年第3期207-212,共6页Chinese Journal of Blood Transfusion

基　　金：重庆市科委科技攻关项目(CSTC:2011AC5039)

摘　　要：目的构建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)3个真核表达载体,并观察其在共转染的人骨髓基质细胞中的表达情况。方法应用基因重组技术,构建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)3个真核表达载体,在脂质体介导下将其导入体外培养的人骨髓基质细胞内,24 h后观察TPO、IL-6、IL-11瞬时表达情况、蛋白含量及表达,并与未转染组、空载体转染组做对比。结果 1)酶切及测序结果均可证实TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)3个真核表达载体的正确性。2)细胞转染24 h后,荧光显微镜下可观察到pEGFP-N1的表达,25%细胞发出绿色荧光;免疫细胞化学染色检测出IL-6及IL-11转染后能在细胞内表达。3)转染48 h后,Western blot检测出骨髓基质细胞内TPO、IL-6及IL-11的蛋白表达量。结论 TPO、IL-6、IL-11这3个基因可共转染人骨髓基质细胞,并在细胞内有效表达。Objective To establish throe eukaryotie expression vectors TPO-pEGFP-NI, IL-6-peDNA3.1 （ ＋ ） and IL-11-pcDNA3.1 （ - ）, and then observe their expressing situation in bone marrow stromal cells of eo-transfection peo- ple. Methods use recombinant DNA technology to establish throe eukaryotic expression vectors TPO-pEGFP-N1, IL-6- peDNA3.1 （ ＋ ） and IL-11-peDNA3. 1 （ - ）, under the mediate of Liposome to import into human bone marrow stromal cells, which are in vitro eulturo; 24h later, observe the transient expression, protein content and expression of TPO, IL-6 and IL-11, and compared them with non-transfeetion group and transfeetion group of no load. Results 1 ） the results of di- gestion and sequencing both can prove the eukaryotie expression vectors TPO-pEGFP-N1, IL-6-peDNA3. 1 （ ＋ ） and IL-11- peDNA3. 1 （ - ）. 2）24h after the transfection of cells, the expression of pEGFP-N1 can be observed under fluorescence mi- croscope, 25% of the cells will give out green fluorescence; the chemical dyeing test of immune ceils find out that IL-6 and IL-11 can be expressed in cells after transfeetion. 3 ）After 48h, Western blot detects the protein expression of TPO, IL-6 and IL-11 in bone marrow stmmal cells. Conclusion The three genes, TPO, IL-6 and IL-11 can be eo-transfeeted hBMSC and effectively expressed in cells.

关 键 词：真核表达载体 血小板相关因子 TPO IL-6 IL-11 骨髓基质细胞 共转染 

分 类 号：R446[医药卫生—诊断学] R457[医药卫生—临床医学]