Investigation Of Gene Expression Profiles Of K562 Cells Treated By Total Saponin Of Panax Ginseng

Leukaemia is a malignant tumor of hematopoietic system,which can seriously destroy the health of human being.So far the treatment of leukaemia,which uses a great dose of combination chemotherapy,is not satisfactory because of the side effects such as the damage of immune system and harmfulness to hematopoietic system.The investigation of the mechanism of malignant tumor,especially for the mechanism of differentiation and retroconversion of leukaemia cells,is becoming one of the most interesting topic of the biomedicine.However the traditional Chinese medicine is getting more and more acceptance because of its benefits in reducing of side effects as well as extended effectiveness,etc.and it is becoming an important hope for cure of leukaemia that there are some useful medicine in traditional Chinese medicine which may either promote the normal haematogenesis or restrain the malignant generation of leukaemia cells.Panax Ginseng is an important herb of invigorating vital energy,which has been used in clinical practice for thousands of years.Total saponins panax ginseng(TSPG)is one of the active components of Panax Ginseng.Modern researches have proved that TSPG can be functioning effectively in nervous system, cardiovascular system and immune system.And TSPG have the effect of anti-infection,restraining apoptosis,expanding blood vessel,anti-aging,etc.It is also found that TSPG have an important effect in preventing and curing cancer. There are many kinds of mechanisms of anti-tumor,such as inducing the apoptosis of tumor cells,inducing cell differentiation,reversing the drag resistance of tumor cells,etc.The anti-tumor effects of TSPG are as follow.Ginsenoside Rh2(G-Rh2) contains an apoptotic inducing activity in SK-HEP-1 cells which functions via Bcl-2-insensitive activation of caspase-3 and involves the activation of cyclin A-Cdk2 by caspase 3-mediated cleavage of p21(WAF1/CIP1).Ginsenosides can induce the phenotypic and functional reverse transformation in cultured Morris hepatoma cells.Two saponin preparations,20(R)- and 20(S)-ginsenoside-Rg3, showed a significant inhibition of adhesion to fibronectin(FN)and laminin(LM)by B16-BL6 melanoma and they significantly inhibited the invasion of B16-BL6 cells into the reconstituted basement membrane(Matrigel)/FN,further more,they also have the anti-angiogenesis activity.However mechanisms of the anti-tumor effects of TSPG have not been fully discovered.In our study,we investigate the changes of gene expression of K562 cells treated by TSPG on molecular level using the gene expression microarray.By analyzing the optical signal of the probe,we try to find out the differentially expressed genes of K562 cells after treated by TSPG.Then we use a biological software to investigate the function of these genes,in order to provide some experimental evidence for clinical research and explore of this Chinese herb.There are two parts for this study:Part one:Use light microscope,flow cytometer,intra-cell haematoglobin benzidine staining and transmission electron microscope to examine the changes of K562 cells treated by TSPG.First,divide the K562 cells into two groups:control group is normal K562 cell;TSPG group is K562 cell treated by TSPG of different density.Then,observe the inhibitory action of hyperplasy of TSPG on k562 cells using the technique of 3,-4,5 dimethyliazol- 2,5 diphenyl tetrazolium bromide assay(MTT);observe the induction of differentiation of TSPG on k562 cells using the technique of intra-cell hemoglobin benzidine staining;detect the expression of surface antigen CD15 and CD235ab on the surface of the cells using the flow cytometer;and use light microscope and transmission electron microscope to observe the appearance of the cells.It was discovered that the degree of inhibition went deeper and deeper with the increase of density of TSPG using the MTT assay.After treated by 200μg/ml TSPG for 3 days,the amount of the haematoglobin in the K562 cells was greater than all other groups of cells and the expression of CD15 and CD235ab was significantly raised compared with control group(P＜0.01).It is demonstrated that 2001μg/ml TSPG can induce the K562 cells to differentiate to erythrocyte series and granulocyte series.It was observed under light microscope that the cells both in the two groups are round or orbicular-ovate,dispersing or clustering.The amount of cells in control group was more than that in TSPG group.And the diameters were between 3μm and 8μm in control group while they are between 2μm and 6μm in TSPG group.It was observed under transmission electron microscope that the cellular chromatospherites were becoming smaller and electron density increase in TSPG treated K562 cells.Further more,in the TSPG treated cells,the heterochromatin under caryotheca increased,membrane-coating granule of middling electron density could be found in endochylema and the shape of the cells looks more mature.It is evidenced that TSPG could inhibit the proliferation of K562 cells and induce the cells differentiate to erythrocyte series and granulocyte series.It suggests that TSPG may become a natural medicine which can both promote the normal haematogenesis and inhibit the proliferation of tumor cells such as leukemic cells.Part two:Apply microarray to detect the gene expression changes of K562 cells treated by TSPG.First,extract total RNA of control group and TSPG group, assess the quality of the RNA sample by 1%agarose gel electrophoresis.Second, use Low Input RNA Fluorescent Linear Amplification Kit to label RNA with Cy3 and Cy5:reverse transcript RNA to synthesize cDNA,label control group with Cy3 and TSPG group with Cy5,then synthesize fluorescently-labeled cRNA,and purify them.Then,hybridize the labeled cRNA with Agilent Human 1A 60mer Oligo Microarray,after the hybridization,wash slides by wash solutions one by one.Third, scan the microarray with Agilent scanner,collect and normalize the data with Feature Extraction.Then,use Panther to carry out biological function analysis.At last,use reverse transcription PCR to check the expression of ODZI,FOSL1, CCNE2 and LATS1.There are 362 differential expression genes in 22153 detection spots,20 genes are up-regulated,which related to cell proliferation and differentiation,signal transduction,developmental processes and et al.while 342 genes are down-regulated,which related to immune defence,protein biosynthesis,protein metabolism and modification,cell cycle and et al.Results of RT-PCR demonstrate that the expression of FOSL1,E2F2 and CCNE2 are down-regulated,the expression of ODZ1 is up-regulated,which are in accordance with the hybridization results.The gene expression changes of K562 treated by TSPG show as:differentiation promoting genes are up-graduated while proliferation promoting genes are down-regulated.The total tendency is promoting differentiation and inhibiting proliferation.Our study indicates that TSPG has the activity of promoting differentiation and inhibiting proliferation.Above all,our study used TSPG to treat K562 cells and applied MTT assay, light microscope,flow cytometer,intra-cell haematoglobin benzidine staining, transmission electron microscope and et al.to examine the changes of K562 cells. The study indicates that TSPG could inhibit K562 cells proliferate and induce differentiate.Then,we studied the gene expression profiling with microarray and by classifying the changed genes according their bio-functions,we investigate the anti-tumor mechanism of TSPG.The study provides experimental bases and idea of investigation for using TSPG at clinical practice in curing cacer.