The Establishment Of The Gene Expression Profile And Cloning The Genes Associated With Differentiation Inducing Of Human Glioma Cells.*

I Objective] New comprehension and deeper understanding of the disease process have been acquired since molecular biology was utilized in neurosurgery. The various molecular treatment strategies were also improved. At the beginning of the new century, when the HGP will be accomplished soon, what to do after HGP is a serious question for life scientists to consider. Malignant neoplasm is still a big challenge in life science, so the genomics and gene informatics of neoplasm should be a hot topic for a long time. Many potent techniques have greatly improved the efficiency of gene analysis. Although in an undifferentiating status, malignant glioma cells still have differentiation potential and can be induced by some certain reagents. This phenomenon has been a new target of glioma treatment strategy. In this paper, we will screen out the most effective and practical inducing reagent, then induce the glioma cells with it, for the purpose of establishing the expression changed genes profile in the ultra-early period or late period, clone some associated genes, inquire into the mechanism of glioma differentiation, lay the foundation for molecular diagnosis and therapy of glioma. [Methods] 1. Establish human glioma cell strains and culture normal astrocytes. Under phase-contract microscope, single glioma cell was siphoned with a capillary peptite, cultured with nutrient cells, later transferred V *Gz.~ted by National Nature Science Foundation. (No. 39870862) into culture bottles. 2. Screen the differentiation inducing agents. Test cell proliferation by MTT method, study cell morphology by conventional Giemsa or I-LE stain. Test CFE by double layer soft agar method. Study cell cycle dynamics by flow cytometry. Measure GFAP expression by in-ununoflurecent test. Determine cell mobility by scratch test. 3. Establish gene expression profile of glioma differentiation by cDNA array. Prepare the PCR product(pick the clones, shake over night, extract the plasmids, PCR), spot them on.nylon membrane by a robot, extract total RNA of induced tumor cells, isolate mRNA, labeled with Q -33P-dATP, hybridize with the array membrane, collect the signal by phosphorus screen and a special scanner, prove the results by multi-dot blot. 4. Clone glioma differentiation associated genes by RAP-DD-PCR. Extract total RNA from cells, reverse transcribe and PCR with a same random sequence primer, label with isotope~ PAGE, autography. Harvest the differential bands, amplify again, SSCP purify. Prove the results by dot-blot, northern blot. Clone the reproducible genes, sequence, analyze with inforniatics tools. [Results] 1. The characteristics of 3 glioma cell strains, and culture of human normal astrocytes. 25 cell clones were got after 7days, which were named SHG-44- 1 to SHG-44-25. Among them the 9th, l7~, 15æ•€ were further studied, which represented relatively low, middle, high differentiation degree, in biology character such as morphology, cytometry, GFAP expression, cell mobility, in vivo behavior. The 9æ•€ cells showed more malignant. VI 2...