未知基因KH的真核表达载体构建及其对细胞增殖的影响

目的 构建未知基因KH的开放阅读框架(KH-ORF)的真核表达体系,初步探讨未知基因KH对细胞增殖的影响。方法 采用DNA重组技术构建KH-ORF的表达载体pCI-neo-KH-ORF,通过脂质体法转染COS-7细胞和K562细胞,RT-PCR检测目的基因的表达。采用流式细胞术检测：KH-ORF对COS-7细胞和K562细胞周期的影响,四甲基偶氮唑蓝(MTT)法检测KH-ORF对COS-7细胞增殖速度的影响,生长曲线与乳酸脱氢酶(LDH)活性测定检测KH-ORF对K562细胞增殖速度的影响。结果 (1)KH-ORF在COS-7细胞与K562细胞中获得表达。(2)KH-ORF对COS-7细胞的细胞周期与增殖速度无明显影响。(3)流式细胞术检测显示,KH-ORF使K562／pCi-KH-ORF细胞周期中S期细胞增多;生长曲线显示,KH-ORF促进K562／pCI-KH-ORF细胞增殖;LDH活性测定结果表明,K562／pCI-KH-ORF细胞LDH比活性上升,高于对照组细胞。结论 KH基因可能是一个与K562细胞增殖有关的白血病未知基因。

Construction of eukaryotic expression vector of unknown KH gene and its effect on cell proliferation

Objective To construct an eukaryotic expression vector of open reading frame of unknown KH gene (KH ORF), and investigate its effect on cell proliferation. Methods The pCI neo KH ORF expression vector was constructed by DNA recombinant technique and was introduced into COS 7 cells and K562 cells by lipofectactin mediated DNA transfection. Expression of KH ORF mRNA was detected by RT PCR. The effect of KH ORF on cell cycle of COS 7 cells and K562 cells was evaluated by flow cytometry (FCM). Effect on cell proliferation of COS 7 cells was tested by MTT assay and that on K562 cells was analyzed by growth curves and LDH activity measurement. Results (1)KH ORF mRNA was expressed both in COS 7 cells and K562 cells. (2)The cell cycle and cell proliferation of COS 7 cells were unaffected significantly. (3)The proportion of cells in S phase was increased in pCI neo KH ORF transfected K562 cells; and growth curves and LDH activity indicated enhanced cell proliferation. Conclusion KH gene may be a leukemia gene related to proliferation of K562 cells.