| [Objective] : The research on the molecular mechanism of induced apoptosis of leukemia CEM cellsin vitro of diffusa aqueous extract and Inhibition of it on multidrug resistance in leukemia cell line CEM/VCR ?[Methods]: 1?The mechanisms of Hedyotis diffusa willd induce apoptosis in vitro effect to leukemia CEM cells.With different concentrations of Hedyotis diffusa willd water extract acts on the logarithmic growth phase CEM cells,MTT assay drugs 24,48,72 h of cell growth,measuring OD values,calculated for different concentrations of Hedyotis diffusa willd CEM cell growth inhibition rate and then select three differernt concentrations,and to design appropriate control group,under an inverted microscope culture flask cell morphology,then smear,Giemsa staining were observed in oil microscope changes in cell structure;Observed under an inverted microscope cell morphology,then smear Giemsa staining changes in cell structure in the oil microscope;Select three corresponding concentration according to MTT assay results and make them acting on 1×107 number of CEM cells,the control group to accede to an equal amount of distilled water,after 24,48,72 h culture,collecte the all cells,the expression of bcl-2,bax,P53 gene was detected by reverse transcription PCR(RT-PCR).2.Study of Hedyotis diffusa inhibitory effect on multidrug resistance in leukemiacell line CEM/VCR With different concentrations of Hedyotis diffusa willd water extract act in CEM and CEM/VCR cells,MTT assay of drug action in different time periods,different drug concentrations on the two kind of cell inhibitory effect,by comparing the inhibitory rate and IC50,to determine whether CEM/VCR cell multidrug resistance cell line resistant to Oldenlandia diffusa;Different concentrations of the drugs and the role of time the cells under an inverted microscope to observe and record changes in cell morphology,and then smear,changes were observed in the cellular structure of the oil microscope.Logarithmic growth phase of the CEM/VCR cell suspension,according to the MTT colorimetric assay concentration selected three role,the role of magnitude CEM/VCR cells at 3×106,without adding the same amount of drug control group distilled water,after 24,48,72 h culture,according to kit instructions apoptotic DNA agarose gel electrophoresis to detect DNA apoptosis.MTT colorimetric assay based on the selected concentration of three effectsacting on the 3×106 projects at the logarithmic phase of CEM/VCR cells and the control group an equal amount of distilled water,after 24,48,72 h culture,according to DNA withered death kit instructions by agarose gel electrophoresis to detect DNA apoptosis.[Results]: 1.MTT test results Diffusa water extract were able to inhibit the growth of leukemia CEM and CEM / VCR cells.In the same drug concentration,the growth inhibition strength with increasing time of growing drug action;Action at the same time,CEM,CEM/VCR rate of cell growth inhibition increases with increasing dose.Drugs on CEM,CEM/VCR cell growth inhibition dose intensity and duration of action of drugs with dependence.2.Changes in cell morphology Observed under an inverted microscope for not adding the number of drug interventiongrown CEM,CEM/VCR cell growth-intensive,cells rounded shape,uniform size,the cells plump and bright,the refractive index is good;medication experimental group CEM and CEM/VCR cells were found reducing the number of cells of differentdegrees of reduced growth density,uneven size,reduced size,cell debris,and poorrefractive index change,and the degree of change with the increase of drug concentration and drug effect increased with increasing time.Where under Oil microscope were observed not add drug intervention grown CEM,CEM/VCR cells rounded shape,and a large,nuclear membrane and membrane integrity;medication experimental group CEM,CEM/VCR cells were found varying degrees of deformation of cells,nuclear shrinkage,chromatin clumping,cell membrane damage and otherchanges,and the degree of change with time and medication drug concentrations were positively correlated.3.RT-PCR assay CEM cells bcl-2,bax,P53 gene expression.It was found,Bcl-2 gene expression is inversely proportional to the amount of Hedyotis diffusa willd dosage,medication group bcl-2 expression was significantly lower than the control group;and with prolonged duration of action of drugs,expression decreased.drug concentration and treatment time was positively correlated with the expression of bax gene,Expression and drug concentration and treatment time was positively correlated with bax gene,expression levels higher in the treatment group,the control group did not express basically,medication group,the duration of action of drugs at the same level,the higher the concentration of drug bax expression more;For the same concentration,the longer the drug,the more bax gene expression.The expression of P53 gene is similar to bax gene,and the expression level of the drug concentration and time of action was a positive correlation between the P53 gene that controls almost no expression,the cells promote the expression of drug action,the higher the drug concentration and the longer duration of action,the more expression of it.4.DNA agarose gel electrophoresis to detect cell apoptosis Diffusa water extract induced apoptosis in leukemic CEM cells in the experimental group were apparent ladder and strip brightness time extension with drug or drug concentration increases significantly;the control group only one DNA macromolecules bands,a more complete description of the cells,no apoptosis occurred.[Conclusions]: 1.Hedyotis diffusa can effectively inhibit leukemia CEM and CEM/VCR cells,and the inhibition of cell growth rate and the drug concentration and action time of the drug were positively correlated,induce cell apoptosis may be one of the mechanism of inhibition;2.Hedyotis diffusa willd induce the apoptosis of CEM cells and the molecular mechanism may be associated with the down-regulation of bcl-2 gene expression,up regulation of Bax,P53 gene expression. |
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