Targeted Apollon Gene Eukaryotic Expression Vector Combined With Ligustrazine On The Proliferation,Apoptosis And Drug Sensitivity Of K562 Cells

ObjectiveScreening effective targeted Apollon small interfering RNA (small interference, siRNA) sequence and build pGPHI-GFP-Neo-Apollon eukaryotic vector,discusses siRNA Apollon gene silence combined TMP on chronic myelogenous leukemia cell line K562 proliferation, apoptosis,and the change of the sensitivity to chemotherapy drugs.Methods1.Synthesize design of the Apollon siRNA sequences transfection MCF-7 cell of breast cancer,using RT-PCR cellular immune fluorescent confocal laser technology,determined by MTT selected silent efficient Apollon siRNA sequences,build pGPHI-GFP-Neo-Apollon specificity eukaryotic expression vector.2.PGPHI-GFP-Neo-Apollon carrier stable transfection K562 cell,RT-PCR detection Apollon mRNA expression level;Cellular immune fluorescent confocal laser technology analysis Apollon protein expression of the amount of change;Determined by MTT and FCM method respectively detect Apollon siRNA alone and with TMP K562 cells to chemotherapy drugs after VCR and DNR sensitivity and apoptosis rate of change.Results1.In the design of small interfering RNA synthesis siRNA2 Apollon gene expression is the most significant inhibition,Apollon mRNA inhibition rate (inhibitory rate,IR)(67.308±7.686)%,Apollon protein expression is (14.97±2.08)%,cell proliferation inhibition rate was (73.361±2.118)%.Enzyme identification and sequencing displays pGPHI/GFP/Neo-Apollon eukaryotic expression vector to construct a success.2.PGPHI-GFP-Neo-Apollon carrier can stable expression in K562 cell,its Apollon mRNA and protein expression of the relative amount (27.622±0.057)%, respectively (15.96+1.71)%, IR for cell proliferation inhibition rate=(63.1±6.3)%,promote cell apoptosis rate was (16.513±1.060)%,compared with cells in the control group was statistically significant difference (P<0.05).3.PGPHI-GFP-Neo-Apollon carrier stable transfection K562 cells and its sensitivity to the VCR,DNR increased, the apoptosis rate,respectively (57.200± 0.066)%,(50.861±1.893)%,half inhibitory concentration (half inhibitory concentration,IC50) cells significantly lower than the control group (P<0.05);Steady turn add Chinese traditional medicine after the TMP cell proliferation inhibition rate IR (80.1 +6.5)%,and RNA interference,TMP+RNA interference group comparative difference was statistically significant (P<0.05);Stable transfection cells with different concentrations of TMP may VCR was reduced and DNR chemotherapy drug IC50 alue concentration of K562 cells,and the concentration dependence of TMP concentration to 320 (including g/mL of VCR and DNR reverse ratio were 3.477,2.885,and IC50 alue concentration of cells in the control group and the negative control group differences have statistical significance (P<0.05).Conclusions1.Select targeted Apollon the expression of gene sequence can effectively silence Apollon siRNA2,inhibit breast cancer MCF-7 cell proliferation,apoptosis significantly increased.2.Successful build pGPHI-GFP-Neo-Apollon carrier and stable expression in K562 cell,restructuring the carrier can effectively silence Apollon mRNA and protein expression,inhibit the proliferation of K562 cells and promote its apoptosis.3.Stable expression pGPHI-GFP-Neo-Apollon carrier of K562 cells increased sensitivity of VCR,DNR,cell apoptosis rate increased; Stable transfection cells combined Chinese medicine TMP,VCR,significantly lower IC50 of DNR, obviously promote apoptosis, targeted silence Apollon gene combined traditional Chinese medicine is expected to become a kind of new train of thought of leukemia treatment.