小鼠造血干细胞因子基因克隆及在 C H O 细胞中的表达

目的： 克隆造血干细胞因子（ Ｓ Ｃ Ｆ）并在 Ｃ Ｈ Ｏ 细胞中表达。方法： 采用 Ｐ Ｃ Ｒ 方法从ｐ Ｒ Ｃ／ Ｃ Ｍ Ｖ（含 Ｓ Ｃ Ｆｃ Ｄ Ｎ Ａ）中钓取 Ｓ Ｃ Ｆ ｃ Ｄ Ｎ Ａ 膜外段活性区，克隆入真核表达载体ｐｃ Ｄ Ｎ Ａ３，转染入 Ｃ Ｈ Ｏ 细胞；用 Ｒ Ｔ－ Ｐ Ｃ Ｒ 方法检测 ｍ Ｒ Ｎ Ａ 水平表达及通过协同刺激骨髓细胞集落形成来检测转染细胞上清的生物活性。结果：转染 Ｓ Ｃ Ｆｃ Ｄ Ｎ Ａ 的 Ｃ Ｈ Ｏ 细胞可表达 Ｓ Ｃ Ｆｍ Ｒ Ｎ Ａ，其细胞培养上清可协同 Ｇ Ｍ － Ｃ Ｓ Ｆ刺激骨髓细胞形成集落数比 Ｇ Ｍ － Ｃ Ｓ Ｆ 单独作用高 ２ 倍，转染外源基因对细胞周期没有明显影响。结论：转染 Ｓ Ｃ Ｆｃ Ｄ Ｎ Ａ 在 Ｃ Ｈ Ｏ 细胞中表达，转染细胞上清协同 Ｇ Ｍ － Ｃ Ｓ Ｆ 刺激骨髓细胞集落的形成，而本身对集落的形成没有影响。

Cloning of murine stem cell factor and it's expression in CHO cells

Objective: To study the expression of murine stem cell factor(SCF) in CHO cells. Methods: Murine SCF cDNA encoding extra cellular domain was isolated using PCR from plasmid pRC/CMV(containing SCF gene),and then recombined into the expression vector pcDNA3, and transferred into CHO cells. Semi-quantitative RT-PCR was used to examine the expression on mRNA level . And the effects of supernatant of transfected CHO cells were investigated alone or in coordinate with GM-CSF by colony formation test of human bone marrow cells in semisolid culture.Results: SCF mRNA was expressed in transfected CHO cells. The supernatant of transfected CHO cells could increase the number of CFU-GM, synergizing with GM-CSF. SCF gene has no effect on the cycle of transferred cell.Conclusion: The supernatant of CHO cells transfected with vector containing mSCF gene can stimulate the colony formation of human bone marrow cells in combination with GM-CSF.