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Cell transformation effect of mutant c-kit gene in gastrointestinal stromal tumor
Xiao-Hong Liu, Qiang Xie, Chen-Guang Bai, Fei Feng, Da-Lie Ma
Xiao-Hong Liu, Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Qiang Xie, Fei Feng, Chen-Guang Bai, Da-Lie Ma, Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Supported by: National Natural Science Foundation of China, No. 30070743.
Correspondence to: Da-Lie Ma, Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. Daliema@yahoo.com.cn
Received: September 24, 2004 Revised: October 22, 2004 Accepted: November 4, 2004 Published online: February 1, 2005
AIM: To investigate the implication in tumorigenesis of a novel c-kit gene mutant, which was identified recently in gastrointestinal stromal tumor (GIST), by examining its effect on cell proliferation and cell cycle.
METHODS: Recombinant plasmids, which contained mutant or wile-type c-kit gene, were stably transfected into human embryonic kidney (HEK) cells. The expression of c-kit protein was detected by Western blot. The proliferation and cell cycle of the transfected cells were detected by MTT colorimetic assay and flow cytometry, respectively.
RESULTS: In comparison with the cells transfected with wild-type c-kit cDNA and empty pcDNA3 vector, the proliferation of the cells transfected with mutant c-kit cDNA was increased significantly. The percentages of cells in proliferation phase (S+G2+M) were 48.34%, 48.24%, 42.03% and 42.16% in test, positive control, negative control and empty control group, respectively.
CONCLUSION: The mutant c-kit gene can promote human cell proliferation, which may play an important role in the malignant transformation of GIST.
Citation: Liu XH, Xie Q, Bai CG, Feng F, Ma DL. Cell transformation effect of mutant c-kit gene in gastrointestinal stromal tumor. Shijie Huaren Xiaohua Zazhi 2005; 13(3): 321-324
图1 c-kit cDNA和pcDNA3载体重组质粒双酶切鉴定.
M: l-EcoT14 DNA Marker, 1 pcDNA3 vector disgested by XhoI, 2 pcDNA3-KIT-W disgested by Xho I/BamH I, 3 pcDNA3-KIT-MW disgested by Xho I/BamH I, 4 pcDNA3- KIT-NW disgested by Xho I/BamH I.
2.2 c-kit蛋白在HEK细胞中的表达
Western blot免疫印迹结果显示转染pcDNA3的HEK工程细胞c-kit蛋白表达为阴性, 转染重组质粒pcDNA3-KIT-W, pcDNA3-KIT-MW, pcDNA3- KIT-NW在HEK工程细胞均有c-kit蛋白表达(图2).
图2 Western blot 免疫印迹结果.
1-2: pcDNA3-KIT-W; 3-4: pcDNA3-KIT-MW; 5-6: pcDNA3-KIT-NW; 7-8: pcDNA3.
2.3 重组质粒对HEK细胞生长的影响
细胞生长曲线(图3)显示稳定转染质粒pcDNA3和pcDNA3-KIT-W组HEK工程细胞在4 d出现快速增长期, 转染pcDNA3-KIT-MW和pcDNA3-KIT-NW HEK工程细胞3 d出现快速增长期; 导入突变型c-kit基因组生长速度显著变快. 将转染各质粒的HEK工程细胞生长曲线拟合为直线并求其直线回归方程: pcDNA3空载体: Y = -17.73+9.93X(P<0.05); pcDNA3-KIT-W: Y = -18.34+10.44X(P<0.05); pcDNA3-KIT-MW: Y = -17.7+11.73X(P<0.05); pcDNA3-KIT-NW: Y = -19.0+12.51X(P<0.05).
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