胃肠道间质瘤c-kit基因突变体的研究
刘晓红, 中国人民解放军第二军医大学长海医院胸心外科 上海市 200433
谢强, 白辰光, 冯菲, 马大烈, 中国人民解放军第二军医大学长海医院病理科上海市 200433
刘晓红, 女, 1976-09-04生, 山东济宁人, 汉族. 1999年济宁医学院本科毕业, 2002年第二军医大学硕士研究生毕业, 主要从事肿瘤的病理研究.
国家自然科学基金资助项目, No. 30070743
项目负责人: 马大烈, 200433, 上海市杨浦区长海路187号, 中国人民解放军第二军医大学长海医院病理科. daliema@yahoo.com.cn
电话: 021-25070660 传真: 021-25072135
收稿日期: 2004-09-24 接受日期: 2004-11-04
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: 2004-09-24 Accepted: 2004-11-24
Abstract
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.
Key Words:Mutant c-kit gene; Gastrointestinal stromal tumor; MTT assay; Flow cytometry
Liu XH, Xie Q, Feng F, Bai CG, Ma DL. Cell transformation effect of mutant c-kit gene in gastrointestinal stromal tumor. Shijie Huaren Xiaohua Zazhi 2005;13(3):321-324
摘要目的:研究突变型c-kit基因对细胞增生及细胞周期的影响从而揭示突变型c-kit基因对人体细胞的恶性转化作用.
方法: 将构建于pcDNA3的突变型c-kitcDNA和野生型c-kitcDNA重组质粒及空白pcDNA3质粒(空白对照)通过脂质体法稳定转染人胚胎肾细胞(human embryonic kidney cell，HEK)细胞.用Western blot方法检测c-kit蛋白的表达情况，观测转染细胞的生长曲线变化，用MTT法检测细胞增生活性的变化，并用流式细胞仪检测c-kit基因突变体对细胞周期的影响.
结果:与阴性对照、空白对照相比，实验组细胞生长速度明显增快，细胞增生活性明显增强，细胞周期检测显示:实验组和阳性对照组处于增生期细胞(S+G2-M)比例明显高于阴性对照组和空白对照组，各组处于增生期的细胞比例依次为:48.34%(实验组)、48.24%(阳性对照组)、42.03%(阴性对照组)、42.16%(空白对照组).
结论:c-kit基因突变可使人类细胞的增生活性明显增强，并可以使更多的细胞由静止期进入增生期，提示c-kit基因突变有可能是引起GIST的恶性转化的关键机制之一.
关键词:突变c-kit基因; 胃肠道间质瘤; 流式细胞术; MTT法
刘晓红, 谢强, 冯菲, 白辰光, 马大烈. 胃肠道间质瘤c-kit基因突变体的研究. 世界华人消化杂志 2005;13(3):321-324
(PDF) 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 Xho I，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 (PDF) 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).
图3 (PDF) 突变c-kit基因对HEK细胞生长速度的影响.
2.4 转染后各组细胞增生活性比较 将稳定转染pcDNA3，pcDNA3-KIT-W，pcDNA3-KIT-MW，pcDNA3-KIT-NW质粒HEK细胞吸光度检测结果，使用SPSS统计软件进行单向方差(One-way ANOVA)分析(表1).多重均数比较(LSD法)显示:转染pcDNA3与pcDNA3-KIT-W两组之间差异无统计学意义(P>0.05);转染pcDNA3-KIT-MW与pcDNA3-KIT-NW两组之间差异无统计学意义
(P>0.05).转染pcDNA3-KIT-MW与pcDNA3-KIT-NW组分别和pcDNA3与pcDNA3-KIT-W组有显著统计学意义(P<0.05)，以上结果显示转染含突变型c-kitDNA的HEK细胞比转染野生型c-kit及空白pcDNA3载体HEK细胞增生活性明显增强.
表1 各组细胞吸光度平均值检测单向方差分析结果
2.5 重组质粒对HEK细胞周期的影响 实验组、阳性对照组、阴性对照组和空白对照组细胞周期中位于增生期(S+G2-M)的细胞比例依次为48.3%、48.2%、42.0%、42.2%.与空白对照组和阴性对照组，转染含突变c-kit基因重组质粒的实验组和对照组处于增生期比例明显升高(表2).
表2 各转染组细胞周期检测结果
3 讨论许多学者证实exon11 Condon550-560是GIST中c-kit基因突变热点，该区域的突变均为“获得性功能突变”，他有可能是可能成为判断GIST恶性变的独立预后指标[2，10-21].由于发现GIST和c-kit原癌基因之间的关系使理解GIST发病机制迈出了关键的一步，该基因经过转录、翻译成c-kit蛋白，深入细胞膜作为酪氨酸的跨膜受体.目前关于生理状态下c-kit癌基因相关的信号传导途径已有报道[22-28]，主要表现为c-kit蛋白与其配体结合可以使两个相邻的Kit受体分子出现二聚体化，导致蛋白分子结构发生改变，使得c-kit受体酪氨酸激酶区相互作用，引起对方的酪氨酸残基磷酸化，被磷酸化的酪氨酸残基可结合信号传导通路中的下游底物，或者通过磷酸化作用于胞内其他蛋白底物，从而激活相应的信号传导通路.我们对82例GIST研究中发现41.5%的GIST存在c-kit基因突变，而且突变主要发生在恶性GIST，其中一例GIST在突变热点区域之外检测到c-kit蛋白肽链第579位密码子插入4个氨基酸:Leu-Pro-Tyr-Asp(亮氨酸-脯氨酸-酪氨酸-天冬氨酸).本课题将深入了解该突变体是否也存在类似的获得性功能突变及其在GIST发生、发展过程中的作用机制，我们用分子生物学手段分别从细胞水平，蛋白水平了解该突变体所引起的c-kit蛋白生物活性的改变.
HEK细胞系虽含有c-kit原癌基因，但蛋白表达率极低，Western blot免疫印迹方法不能检测出[29-30]，本研究根据所
检测出的突变位点以及文献所报道的codon559-560缺失性突变人工突变野生型c-kitcDNA，同时设置阴性对照，空白对照，转染HEK肾细胞.Western blot免疫印迹方法检测四种质粒在HEK细胞的表达，结果显示转染pcDNA3空白对照组未显示条带，而其余各组在相同位置均显示一淡兰色条带，这说明除空白对照组外，其余各组均有c-kit蛋白表达，从而证实各重组质粒已在HEK细胞中稳定表达.
随后，观察C-Kit基因突变体的cDNA对HEK增生活性和细胞周期的影响，结果显示:转染pcDNA3、pcDNA3-KIT-W、pcDNA3-KIT-NW和pcDNA3-KIT-MW质粒各组HEK细胞的细胞周期中位于增生期(S+G2-M)的细胞比例依次为42.2%、42.0%、48.3%、48.2%;MTT检测表明以上各组细胞吸光度均值依次为0.1 947±3.537´10-3;0.1830±6.034´10-3;0.1340±2.353´10-3;0.1 274±3.537´10-3.SPSS单向方差分析结果显示实验组和阳性对照组之间无统计学意义(P>0.05);而空白对照组和阴性对照组之间无统计学意义(P>0.05).实验组和阳性对照组分别与空白对照组和阴性对照组之间有统计学意义(P<0.05)，实验组和阳性对照组与空白对照组和阴性对照组相比，前二者吸光度明显高于后二者.以上结果表明稳定转染c-kit突变体与转染空白pcDNA3载体及转染野生型c-kitcDNA组的HEK细胞系相比，细胞生长速度加快，增生活性更强，处于增生期的细胞比例明显较高.该结果提示，GIST中的c-kit基因突变可使人类细胞的增生活性明显增强，并可以使更多的细胞进入细胞周期中的增生期;而野生型c-kit基因表达对细胞功能状态无明显影响，这说明突变型c-kit可影响人类正常细胞周期调节机制，促进更多的细胞由静止期进入增生期，从而对GIST的发生、发展起推动作用.
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编辑 潘伯荣 审读 张海宁, 百拇医药( 刘晓红, 谢 强, 冯 菲, 白辰光, 马大烈)
谢强, 白辰光, 冯菲, 马大烈, 中国人民解放军第二军医大学长海医院病理科上海市 200433
刘晓红, 女, 1976-09-04生, 山东济宁人, 汉族. 1999年济宁医学院本科毕业, 2002年第二军医大学硕士研究生毕业, 主要从事肿瘤的病理研究.
国家自然科学基金资助项目, No. 30070743
项目负责人: 马大烈, 200433, 上海市杨浦区长海路187号, 中国人民解放军第二军医大学长海医院病理科. daliema@yahoo.com.cn
电话: 021-25070660 传真: 021-25072135
收稿日期: 2004-09-24 接受日期: 2004-11-04
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: 2004-09-24 Accepted: 2004-11-24
Abstract
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.
Key Words:Mutant c-kit gene; Gastrointestinal stromal tumor; MTT assay; Flow cytometry
Liu XH, Xie Q, Feng F, Bai CG, Ma DL. Cell transformation effect of mutant c-kit gene in gastrointestinal stromal tumor. Shijie Huaren Xiaohua Zazhi 2005;13(3):321-324
摘要目的:研究突变型c-kit基因对细胞增生及细胞周期的影响从而揭示突变型c-kit基因对人体细胞的恶性转化作用.
方法: 将构建于pcDNA3的突变型c-kitcDNA和野生型c-kitcDNA重组质粒及空白pcDNA3质粒(空白对照)通过脂质体法稳定转染人胚胎肾细胞(human embryonic kidney cell，HEK)细胞.用Western blot方法检测c-kit蛋白的表达情况，观测转染细胞的生长曲线变化，用MTT法检测细胞增生活性的变化，并用流式细胞仪检测c-kit基因突变体对细胞周期的影响.
结果:与阴性对照、空白对照相比，实验组细胞生长速度明显增快，细胞增生活性明显增强，细胞周期检测显示:实验组和阳性对照组处于增生期细胞(S+G2-M)比例明显高于阴性对照组和空白对照组，各组处于增生期的细胞比例依次为:48.34%(实验组)、48.24%(阳性对照组)、42.03%(阴性对照组)、42.16%(空白对照组).
结论:c-kit基因突变可使人类细胞的增生活性明显增强，并可以使更多的细胞由静止期进入增生期，提示c-kit基因突变有可能是引起GIST的恶性转化的关键机制之一.
关键词:突变c-kit基因; 胃肠道间质瘤; 流式细胞术; MTT法
刘晓红, 谢强, 冯菲, 白辰光, 马大烈. 胃肠道间质瘤c-kit基因突变体的研究. 世界华人消化杂志 2005;13(3):321-324
(PDF) 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 Xho I，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 (PDF) 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).
图3 (PDF) 突变c-kit基因对HEK细胞生长速度的影响.
2.4 转染后各组细胞增生活性比较 将稳定转染pcDNA3，pcDNA3-KIT-W，pcDNA3-KIT-MW，pcDNA3-KIT-NW质粒HEK细胞吸光度检测结果，使用SPSS统计软件进行单向方差(One-way ANOVA)分析(表1).多重均数比较(LSD法)显示:转染pcDNA3与pcDNA3-KIT-W两组之间差异无统计学意义(P>0.05);转染pcDNA3-KIT-MW与pcDNA3-KIT-NW两组之间差异无统计学意义
(P>0.05).转染pcDNA3-KIT-MW与pcDNA3-KIT-NW组分别和pcDNA3与pcDNA3-KIT-W组有显著统计学意义(P<0.05)，以上结果显示转染含突变型c-kitDNA的HEK细胞比转染野生型c-kit及空白pcDNA3载体HEK细胞增生活性明显增强.
表1 各组细胞吸光度平均值检测单向方差分析结果
| HEK cell | pcDNA3 | pcDNA3-KIT-NW | pcDNA3-KIT-MW | pcDNA3-KIT-W |
| 平均值 | 0.1 274 | 0.1 830 | 0.1 947 | 0.1 340 |
| 标准误 | 3.537´10-3 | 3.888´10-3 | 6.034´10-3 | 2.353´10-3 |
2.5 重组质粒对HEK细胞周期的影响 实验组、阳性对照组、阴性对照组和空白对照组细胞周期中位于增生期(S+G2-M)的细胞比例依次为48.3%、48.2%、42.0%、42.2%.与空白对照组和阴性对照组，转染含突变c-kit基因重组质粒的实验组和对照组处于增生期比例明显升高(表2).
表2 各转染组细胞周期检测结果
| HEK cell | G0-G1期 | S期 | G2-M期 | S+G2-M期 |
| 空白对照组 | 57.0% | 10.4% | 31.8% | 42.2% |
| 阴性对照组 | 58.0% | 9.6% | 32.4% | 42.0% |
| 阳性对照组 | 51.6% | 3.7% | 44.5% | 48.2% |
| 实验组 | 50.8% | 6.3% | 42.0% | 48.3% |
3 讨论许多学者证实exon11 Condon550-560是GIST中c-kit基因突变热点，该区域的突变均为“获得性功能突变”，他有可能是可能成为判断GIST恶性变的独立预后指标[2，10-21].由于发现GIST和c-kit原癌基因之间的关系使理解GIST发病机制迈出了关键的一步，该基因经过转录、翻译成c-kit蛋白，深入细胞膜作为酪氨酸的跨膜受体.目前关于生理状态下c-kit癌基因相关的信号传导途径已有报道[22-28]，主要表现为c-kit蛋白与其配体结合可以使两个相邻的Kit受体分子出现二聚体化，导致蛋白分子结构发生改变，使得c-kit受体酪氨酸激酶区相互作用，引起对方的酪氨酸残基磷酸化，被磷酸化的酪氨酸残基可结合信号传导通路中的下游底物，或者通过磷酸化作用于胞内其他蛋白底物，从而激活相应的信号传导通路.我们对82例GIST研究中发现41.5%的GIST存在c-kit基因突变，而且突变主要发生在恶性GIST，其中一例GIST在突变热点区域之外检测到c-kit蛋白肽链第579位密码子插入4个氨基酸:Leu-Pro-Tyr-Asp(亮氨酸-脯氨酸-酪氨酸-天冬氨酸).本课题将深入了解该突变体是否也存在类似的获得性功能突变及其在GIST发生、发展过程中的作用机制，我们用分子生物学手段分别从细胞水平，蛋白水平了解该突变体所引起的c-kit蛋白生物活性的改变.
HEK细胞系虽含有c-kit原癌基因，但蛋白表达率极低，Western blot免疫印迹方法不能检测出[29-30]，本研究根据所
检测出的突变位点以及文献所报道的codon559-560缺失性突变人工突变野生型c-kitcDNA，同时设置阴性对照，空白对照，转染HEK肾细胞.Western blot免疫印迹方法检测四种质粒在HEK细胞的表达，结果显示转染pcDNA3空白对照组未显示条带，而其余各组在相同位置均显示一淡兰色条带，这说明除空白对照组外，其余各组均有c-kit蛋白表达，从而证实各重组质粒已在HEK细胞中稳定表达.
随后，观察C-Kit基因突变体的cDNA对HEK增生活性和细胞周期的影响，结果显示:转染pcDNA3、pcDNA3-KIT-W、pcDNA3-KIT-NW和pcDNA3-KIT-MW质粒各组HEK细胞的细胞周期中位于增生期(S+G2-M)的细胞比例依次为42.2%、42.0%、48.3%、48.2%;MTT检测表明以上各组细胞吸光度均值依次为0.1 947±3.537´10-3;0.1830±6.034´10-3;0.1340±2.353´10-3;0.1 274±3.537´10-3.SPSS单向方差分析结果显示实验组和阳性对照组之间无统计学意义(P>0.05);而空白对照组和阴性对照组之间无统计学意义(P>0.05).实验组和阳性对照组分别与空白对照组和阴性对照组之间有统计学意义(P<0.05)，实验组和阳性对照组与空白对照组和阴性对照组相比，前二者吸光度明显高于后二者.以上结果表明稳定转染c-kit突变体与转染空白pcDNA3载体及转染野生型c-kitcDNA组的HEK细胞系相比，细胞生长速度加快，增生活性更强，处于增生期的细胞比例明显较高.该结果提示，GIST中的c-kit基因突变可使人类细胞的增生活性明显增强，并可以使更多的细胞进入细胞周期中的增生期;而野生型c-kit基因表达对细胞功能状态无明显影响，这说明突变型c-kit可影响人类正常细胞周期调节机制，促进更多的细胞由静止期进入增生期，从而对GIST的发生、发展起推动作用.
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编辑 潘伯荣 审读 张海宁, 百拇医药( 刘晓红, 谢 强, 冯 菲, 白辰光, 马大烈)
