稳定表达基质细胞衍生因子1α的内皮细胞系构建及其与单核细胞的粘附作用

Stable Expression Stromal Cell-Derived Factor -1 Alpha Endothelial Cells Line Establishment and it’s Adhesion Activity with Monocyte

目的构建稳定表达基质细胞衍生因子1α的ECV304细胞系,研究基质细胞衍生因子1α在单核细胞/内皮细胞粘附中的作用。方法将大鼠基质细胞衍生因子1α基因聚合酶链反应产物及质粒载体pcDNA3.1用EcoRⅠ进行酶切,去磷酸化,置于连接反应体系中进行连接,将连接产物氯化钙法转化DH5α菌。氨苄青霉素筛选并扩增阳性菌落,经酶切鉴定插入方向正确后,再用G418筛选、逆转录聚合酶链反应检测基质细胞衍生因子1α浓度,建立稳定转染ECV304细胞系。在6孔板上种植转染基质细胞衍生因子1α基因的ECV304细胞,加THP-1细胞37℃孵育30min,磷酸缓冲液轻洗3遍,去除未粘附细胞,倒置显微镜下计数上、下、左、右、中5个视野,取其平均值得到每个视野粘附单核细胞数。结果经逆转录聚合酶链反应检测证实,稳定表达基质细胞衍生因子1α的ECV304细胞系构建成功,细胞计数表明,转染ECV304细胞系粘附THP-1细胞数是转染空质粒的十几倍,CXCR4单抗基质细胞衍生因子1α多抗均显著减少其粘附力(P<0.01)。结论内源性基质细胞衍生因子1α能促进ECV304细胞与单核细胞的粘附。

Aim To study the effect of stromal cell derived factor-1 alpha （SDF-1α） on adherence of ECV304/THP-1. Methods Rat SDF-1α gene and pcDNA3.1 were both cut with EcoRⅠ, dephosphorylated, and connected in connecting buffer. Then, the connecting product was transformed to DH5α by calcium chloride, scanned by ampicillin, and cut by enzyme for the right inserting identification, and sub-selected by G418, SDF-1α expression of ECV304 was measured by reverse transcription polymerase chain reaction （RT-PCR） to get a stabilized SDF-1α expressional cells line. Trans-gene ECV304 was seeded in a six-pore plate. Then, THP-1 was added and incubated for 30 minutes, washed with PBS about 3 times, to remove unattached cells. Cells number was counted under microscope from 5 visual field -up, under, left, right and center. Results Stable SDF-1α expression cells line was obtained which was identified by RT-PCR （about 360 bp）, and the adherence ability of trans-gene ECV304 was ten times and more than control, and CXCR4 mono-antibody obviously inhibited this adherence ability （P〈0.01）. Conclusion Endogenous SDF-1α improves adhesion of ECV304/THP-1.