Identification of differentially expressed genes related to cell proliferation and apoptosis in gliomas to different differentiating degree / 中国组织工程研究

ABSTRACT

BACKGROUND: Occurrence and development of the glioma are not only related to regulative gene of cell cycle, but also dealt with adjustive genes of cell invasion, cell metastasis and apoptosis.OBJECTIVE: To identify the differentially expressed genes related to cell proliferation and apoptosis in glioma cell of different differentiating degree with cDNA microarray, and provide basic data for further research of mechanisms of cell proliferation and apoptosis in human gliomas.DESIGN: Opening-up experiment.SETTING: Institute of Pathology, Southwest Hospital and Department of Neurobiology, Basic Medicine Faculty as well as Department of Pharmacy,Xinqiao Hospital, Third Military Medical University of Chinese PLA.MATERIALS: Human malignant glioma cell line CHG-5 ( Ⅱ grading according to the WHO standard) was constructed, kept and cultured in this experiment. SHG-44 (Ⅳ grade according to the WHO standard) was provided by Institute of Brain Tumor, Second Hospital Affiliated to Suzhou Medical College. Serum of calf was produced and provided by Hangzhou Sijiqing Biomaterial Institute. In experiment RPMI-1640 medium (Gibco),Trizol test kit (Gibco-BRL), RNAsecureTM solutions (Ambion, Austin,Texas), biophotometer (Eppendorf, Hamburg, Germany) were also applied.Gene chip contained 9 984 human cDNA segment, which was prepared and provided by City University of Hongkong (UniGEMV2 cloneset known gene and ESTs were purchased from Incyte Company), Superscript Ⅱ reverse transcriptase was provided by Gibco-BRL Company. Fluorochrome Cy3 & Cy5 was the products of Amersham Pharmacia Company.METHODS: The experiment was performed at Chongqing Third Military Medical University of Chinese PLA and City University of Hongkong between 2001 and 2003. Total RNA was extracted from Trizol test kit. Reverse transcription polymerase chain reaction (RT-PCR) was conducted in Superscript Ⅱ reverse transcriptase, and cDNA product was marked with fluorochrome Cy3 & CyS. Followed by chip hybridizationto detect the difference of gene expressions between human glioma cell line CHG-5 and SHG-44 tumor cell, especially the difference of related genic expression between cell proliferation and apoptosis, and the chip result was verified with Northern blot hybridization.MAIN OUTCOME MEASURES: The difference of gene expression of human glioma cell in different differentiating degree; Comparison of result between Northern blot and related gene chip.RESULTS: ①Compared with CHG-5, 120 gene expressions were detected obvious up-regulation and 22 gene expressions were significant down regulation in SHG-44 cells, and the variety of these differentially expressed genes was many, in which apoptosis related genes were 6, including 3 up-regulation genes and 3 down-regulation genes; Cell cycle and proliferating related genes were 12, including 5 up-regulation genes and 7 down-regulation genes. ②Chip result was supported by Northern blot result further.CONCLUSION: The differentially expressed genes of glioma are revealed primarily, especially the differentially expressed genes related with cell proliferation and apoptosis.