| The human erythroleukemia K562 cell is a low-differentiation blast cell line with the potentiality of differentiation towards multi-direction. Matrine, one of the components in traditional Chinese herb Sophora flavescens, has the pharmacological effect of anti-tumor. Our previous study showed that a definite concentration of matrine could inhibit proliferation and induce differentiation in K562 cells in vitro. But the direction of differentiation and the molecular mechanism involved are poorly understood. To elucidate the mechanism, present study investigated the changes of cell morphology, cell surface marker-cluster of differentiation (SmCD) and the expression of oncongenes and immediate difference genes treated with matrine at various concentrations and for different time. Focus of the study was to screen some unknown genes involved in differentiation and to understand the function of unknown gene by studying it's expression product.In the first part of our studies, we observed the effects of matrine on proliferation by trypan blue exclusion and LDH measurement. The effect of matrine on inhibiting proliferation of K562 cells was observed at 0.2mg/ml concentration after the 3rd day. Meanwhile, we found the K562 cells had the morphological tendency of differentiation observed by Wright's staining under optical-microscopes, and the apoptosis was found on the 9th day. The results indicated that 0.2mg/ml matrine had the effects of inhibiting proliferation and inducing differentiation .In the second part of our studies, the SmCD, such as CD34, CD33, CD14, CD15, CD20, CD27, CD41, HIR2 on K562 cells treated with 0.2mg/ml matrine for 6 days were measured by flow cytometry. The results showed that 0.2mg/ml matrine could induce the differentiation of K562 cells towards erythrocyte and granulocyte. The results were in accordance with the morphology changes.The differentiation of tumor cells is regulated by several kinds of factors, and oncogenes is one of the regulation factors. In the third part, the expression of some proto-oncogenes of K562 cells treated with 0.2mg/ml matrine were measured by RT-PCR assay. The results showed that the changes of c-myc, c-jun and HNF-1αmRNA were dramatic, their relative levels of expression were dropped, and levels of H-ras and p21 mRNA were increased at the early stage (3h). It is suggested that the effect of inducing differentiation of matrine on K562 cells was regulated at the gene level by a network including multiple factors and pathways, and DNA replication, transcription and signal transduction might be involed.In the fourth part, for the purpose of exploring the molecular mechanisms of K562 cells proliferation suppression and differentiation induced by matrine, and discovering unknown genes, a sensitive reverse transcript differential display PCR (DDRT-PCR) was used to screen some important genes with low-level expression in the earlier phase of matrine induced differentiation. After cloning and sequencing of different cDNA by DDRT-PCR, the results were identified through public database of biological information. The results showed that the mRNA levels of several genes coding proteins and proteinase was changed, which may be associated with cell proliferation and differentiation. In addition, we cloned an unknown cDNA fragment (GenBank ID BM077298, dbEST ID 10245338, http://www.ncbi.mlm.nih.gov/GenBank/index.html ,we called KH gene). Then RT-PCR and Northern blot were performed respectively to learn the distribution of unknown cDNA expression in 11 cell lines and 12 human tissues. It was shown that the unknown cDNA distributed mainly in human brain and leukemia cells. Although rapid amplification of cDNA ends (RACE) was used to extend the unknown cDNA fragment to full length ,but we failed to get it. So we cloned, subcloned and reconstructed expressive vector of the open reading frame (ORF) on the unknown cDNA fragment to study the function of the expressive product. A 24kD peptide was obtained in BL21(DE3)plysS by SDS-PAGE. Meanwhile, COS-7 cell...
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