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背景:胶质瘤的发生、发展不仅涉及细胞周期调控基因,也涉及到细胞侵袭、转移和细胞凋亡的调节基因。目的:用基因芯片技术分析不同分化程度的胶质瘤细胞增殖和凋亡相关的差异表达基因,为进一步研究胶质瘤细胞的增殖和凋亡机制奠定基础。设计:开放性实验。单位:解放军第三军医大学,西南医院病理学研究所,基础医学部神经生物学教研室,新桥医院药剂科。材料:人脑恶性胶质瘤细胞系CHG-5(WHO分级为Ⅱ级)由本实验室建立并保存培养;SHG-44(WHO分级为Ⅳ级),由苏州医学院附属二院脑肿瘤研究室提供,小牛血清由杭州四季青生物材料研究所生产并提供。实验中还采用了RPMI-1640培养基(Gibco)、Trizol试剂盒(Gibco-BRL)、RNAsecureTM溶液(Ambion,Austin,Texas)、光度适应计(Eppendorf,Hamburg,Germany)。基因芯片含9,984个人类cDNA片段,由香港城市大学准备和提供(UniGEMV2cloneset已知的基因和ESTs购自Incyte公司),SuperscriptⅡ反转录酶由Gibco-BRL公司提供,荧光染料Cy3&Cy5为AmershamPharmacia公司产品。方法:实验于2001/2003在重庆第三军医大学和香港城市大学完成。用Trizol试剂盒抽提总RNA,用SuperscriptⅡ反转录酶进行反转录聚合酶链反应,并用荧光染料Cy3&Cy5标记cDNA产物,然后进行芯片杂交,检测人胶质瘤细胞系CHG-5和SHG-44瘤细胞基因表达的差异,特别是与细胞增殖和凋亡相关基因表达的差异,并用Northernblot杂交来验证芯片结果。主要观察指标:不同分化程度的人胶质瘤细胞基因表达的差异;Northern杂交结果与相应基因芯片结果的比较。结果:①与CHG-5相比,SHG-44细胞中检测到了有120个基因表达明显上调和22个基因表达明显下调,这些差异表达基因的种类很多,其中凋亡类相关基因共6个,上调基因有3个,下调基因3个;细胞周期与增殖类相关基因共12个,上调基因有5个,下调基因7个。②芯片结果进一步得到Northernblot杂交结果的支持。结论:初步揭示了胶质瘤进展的差异表达基因,特别是与细胞增殖和凋亡相关的差异表达基因。
BACKGROUND: The development and progression of glioma involves not only the cell cycle regulatory genes but also the regulatory genes involved in cell invasion, metastasis and apoptosis. OBJECTIVE: To analyze differentially expressed genes related to proliferation and apoptosis of glioma cells with different differentiation by gene chip technology, which will lay the foundation for further study on the mechanism of glioma cell proliferation and apoptosis. Design: Open experiment. Unit: PLA Third Military Medical University, Southwest Hospital Pathology Institute, Department of Neurobiology, Department of Basic Medical Sciences, Xinqiao Hospital Pharmacy. MATERIALS: The human glioblastoma cell line CHG-5 (WHO grade Ⅱ) was established and maintained in our laboratory. SHG-44 (WHO grade Ⅳ) was obtained from the Brain Tumor Laboratory, Second Affiliated Hospital of Suzhou Medical College Provided, bovine serum produced by Hangzhou Sijiqing Biomaterials Institute and provided. RPMI-1640 medium (Gibco), Trizol kit (Gibco-BRL), RNAsecureTM solution (Ambion, Austin, Texas) and photometric fitter (Eppendorf, Hamburg, Germany) were also used in the experiment. The gene chip contains 9,984 human cDNA fragments, prepared and supplied by City University of Hong Kong (genes known as UniGEMV2cloneset and ESTs were purchased from Incyte), Superscript II reverse transcriptase was supplied by Gibco-BRL, and the fluorescent dyes Cy3 & Cy5 were from Amersham Pharmacia. Methods: The experiment was performed in Chongqing Third Military Medical University and City University of Hong Kong from 2001 to 2003. Total RNA was extracted with Trizol kit, reverse transcriptase polymerase chain reaction was performed with Superscript II reverse transcriptase, and the cDNA was labeled with fluorescent dyes Cy3 & Cy5. Then the hybridization was performed by chip to detect the expression of CHG-5 and SHG- 44 tumor cell gene expression differences, especially with the cell proliferation and apoptosis-related gene expression differences, and using Northern blot hybridization to verify the chip results. MAIN OUTCOME MEASURES: Differences in gene expression of human glioma cells with different degrees of differentiation; comparison of results of Northern hybridization with those of corresponding gene chips. Results: ① Compared with CHG-5, 120 genes were up-regulated and 22 genes were significantly down-regulated in SHG-44 cells. There are many kinds of differentially expressed genes, including 6 genes related to apoptosis, There are 3 up-regulated genes and 3 down-regulated genes. There are 12 genes related to cell cycle and proliferation, 5 up-regulated genes and 7 down-regulated genes. ② The chip results are further supported by Northern blot hybridization results. Conclusion: The differentially expressed genes of gliomas are revealed, especially the differentially expressed genes related to cell proliferation and apoptosis.