ABSTRACT
Objective To establish an absolute reverse transcription fluorescence quantitative PCR method (RT-qPCR) that can accurately quantify the circRNA hsa_circ_0004123, and verify the copy number of hsa_circ_0004123 in the leukemia cell line. Methods Total RNA was extracted from HMy2.CIR cell line, and reverse transcribed to cDNA. Using cDNA as template, a 236 bp characteristic segment containing the back-spliced junction of hsa_circ_0004123 transcript was cloned and inserted into the pGM-T vector, and then the recombinant plasmid was sequenced. Regarding the gradient diluted recombinant plasmid as standard substance, the standard curve of hsa_circ_0004123 was established by SYBR fluorescent staining RT-qPCR, and then the copy number of hsa_circ_0004123 in leukemia cell lines (HMy2.CIR, Jurkat, Raji, K562) were verified. Results The absolute RT-qPCR method was established that can accurately quantify the copy number of hsa_circ_0004123 with wide linear range (9.63×103–9.63×109) copies/μl, high sensitivity (4528±516) copies/μl, intra-batch coefficient of variation (CV) of 0.30%-1.86%, inter-batch CV of 1.40%-2.30%, and a single peak dissolution curve. The copy number of hsa_circ_0004123 in leukemia cell lines HMy2.CIR, Jurkat, Raji and K562 was detected respectively. The expression of hsa_circ_0004123 was significantly different (P<0.001) in the 4 kinds of leukemia cell lines. Conclusion The absolute RT-qPCR method used to detect the copy number of hsa_circ_0004123 has a wide linear range, high sensitivity and specificity, and good repeatability, and lays a technical foundation for accurate quantification of hsa_circ_0004123 in leukemia.
ABSTRACT
Objective To learn the effect of indomethacin (IND)of different concentrations on the expression of lysozyme protein induced by SiO2 in rat alveolar macrophages (AM).Methods Pure AM was prepared with the method of bronchoalveolar lavage in rats.In the model group,the silica dust poisoning model was replicated by adding SiO2 dust suspension (50 μg /ml).In the intervention group,following the adding of SiO2 dust suspension,the IND of 1 0 -5 ,1 0 -4 , and 1 0 -3 g/ml were added respectively.In the control group,the same volume of PBS was given.After 8 h,1 6 h,the cell morphology was observed.Results Compared with the model group,the AM cells in the intervention group were relatively complete,and that there was a concentration dependent trend.The expression of LSZ protein in AMof the model group was significantly higher than that of the control group (P<0. 05 ),while the expression of LSZ protein in the intervention group decreased compared with that of the model group.After incubation with IND and SiO2 ,the expression of LSZ protein in the intervention group decreased compared with that of the model group.Conclusion IND can inhibit the increased expression of LSZ protein in AMcaused by the stimulation of SiO2dust,and can reduce the damage of SiO2on the membrane of alveolar macrophage and thus has the protective effect on the AM cell membrane.