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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 construct three eukaryotic expression vectors of TPO-pEGFP-N1, IL-6-pcDNA3.1 (+) and IL-11-pcDNA3.1 (-) and observe their expression in co-transfected human bone marrow stromal cells Express the situation. Methods Three eukaryotic expression vectors of TPO-pEGFP-N1, IL-6-pcDNA3.1 (+) and IL-11-pcDNA3.1 (-) were constructed by gene recombination technique. The transient expression, protein content and expression of TPO, IL-6 and IL-11 were observed in human bone marrow stromal cells cultured in vitro for 24 h, and compared with the untransfected and empty vector transfected groups. Results 1) The correctness of the three eukaryotic expression vectors of TPO-pEGFP-N1, IL-6-pcDNA3.1 (+) and IL-11-pcDNA3.1 (-) could be verified by digestion and sequencing. 2) 24 h after transfection, pEGFP-N1 expression was observed under fluorescence microscope and 25% of cells showed green fluorescence. Immunocytochemical staining showed that IL-6 and IL-11 could be expressed in cells after transfection. 3) After 48 h of transfection, the protein expression levels of TPO, IL-6 and IL-11 in bone marrow stromal cells were detected by Western blot. Conclusion The three genes of TPO, IL-6 and IL-11 can be co-transfected into human bone marrow stromal cells and efficiently expressed in the cells.