pSVL/GM-CSF真核表达质粒的构建及其表达活性观察

以真核瞬时表达质粒ｐＳＶＬ为载体构建ｐＳＶＬ／ＧＭ－ＣＳＦ真核表达质粒,用电穿孔法导入Ｃｏｓ－７细胞,以ＧＭ－ＣＳＦ依赖株ＴＦ１为靶细胞,检测其生物学活性;将不同剂量的ＰＳＶＬ／ＧＭ－ＣＳＦ质粒直接注射Ｂａ１ｂ／Ｃ小鼠股四头肌,动态观察小鼠血清中ＧＭ－ＣＳＦ的表达;将ｐＳＶＬ／ＧＭ－ＣＳＦ质粒导入Ｋ５６２细胞,观察ＧＭ－ＣＳＦ对瘤细胞生长状态的影响。结果表明：构建的ｐＳＶＬ／ＧＭ－ＣＳＦ真核表达质粒可在Ｃｏｓ－７细胞中有效表达ＧＭ－ＣＳＦ;小鼠骨骼肌直接注射ｐＳＶＬ／ＧＭ－ＣＳＦ（１００μｇ／及２００μｇ）,在血清中可检出ＧＭ－ＣＳＦ,在１０～１５ｄ达高峰（８Ｕ／ｍｌ）,为临床应用提供了实验基础;导入ＧＭ－ＣＳＦ基因的Ｋ５６２细胞可表达ＧＭ－ＣＳＦ,且经ＧＭ－ＣＳＦ基因修饰的Ｋ５６２细胞增殖状态明显优于对照组,提示对造血细胞肿瘤单用ＧＭ－ＣＳＦ应慎重,而与其它细胞因子联合应用可能是一个合理的方案。

CONSTRUCTION OF PSVL/GM-CSF EUKARYOTIC EXPRESSION PLASMID AND OBSERVE OF ITS BIOACTIVITY

The pSVL/GM-CSF eukaryotic ex-pression plasmid was constructed by insertion ofPCR amplified GM-CSF cDNA fragment into PSVLtransient expression vector. The construction wastransfered into COS-7 and K562 human erythroloeukamia cells by electroperation. The culturesupernatant was collected and GM-CSF activity wasassayed by using GM-CSF dependent cell line TF1.In order to explore the expression of pSVL/GMCSF in vivo, the mice quadriceps pretreated withbupricaine and the serum level of GM-CSF was assayed by bioassay as above. The results showed thatthe pSVL/GM-CSF plasmid was successfully constructed and GM-CSF was (下转第32页)(上接第16页) expressed in COS-7 cell at high level. The rats with direct PSVL/GM-CSF DNA injection showed a siginificant expression of GM-CSFand the peak serum level in 200 μg of plasmid DNArat group was higher than that in 100 μg of plasmidDNA rat group, although there was no significantdifference. K562 cell transfected with pSVL/GMCSF secret bioactive GM-CSF in the culture supernatant, which was lower that that in COS-7 cells.More interestedly, K562 cells modified by pSVL/GM-CSF grow more rapidly than its parent cells. Itseems that GM-CSF could stimulate the proliferation of K562 cells, this indicates that clinical applicatoin of GM-CSF in leukmia therapy might be verycautious.