ABSTRACT
Objective@#To observe the integration frequency of aadA2 resistance cassette at attI site of the integron under different concentration of streptomycin. @*Methods@#Class 1 integron with known gene sequence was cloned into plasmid pACYC184 to produce recombinant plasmid pACIDA, meanwhile the integrase gene was cloned into plasmid pET28a to construct recombinant plasmid pETINT. These two recombinant plasmids were consecutively transformed into E. coli BL(DE3). These transformed bacteria was cultured in the LB medium at 37 ℃ overnight with addition of different concentration of streptomycin. The copy number of total integrons and the copy number of integrated aadA2 at attI site of integrons were determined by using real-time PCR. and the integration frequency is the result of the former divided by the latter. @*Results@#The resulting frequencies were (1.97±0.24)×10-3, (3.23±1.77)×10-3, (3.27±0.67)×10-3, 0.45±0.13 and 1.32±0.11, with respective streptomycin concentrations of 0, 20, 30, 40 and 50 μg/ml. The background frequency of integration without integrase overexpression was less than (1.75±0.33)×10-7. @*Conclusion@#These findings indicate that antibiotic concentration significantly increase recombination frequency of aadA2 resistance cassette at attI site of the integron, catalyzed by integron integrase.(Chin J Lab Med, 2018, 41: 531-535)
ABSTRACT
Objective To investigate the relationship of PI3K/Akt signaling pathway with the activation of hepatic stellate cells (HSC) and its role in radiation-induced hepatic fibrosis.Methods HSC was treated with 6 MV X-ray irradiation (IR) together with the inhibitor of PI3K/Akt signaling pathway.The cells were divided into inhibitor group,10 Gy IR group,10 Gy + inhibitor group,20 Gy IR group,an 20 Gy + inhibitor group and blank control group.Then cell apoptosis rate was detected,the expression of transforming growth factor β1 (TGF-β1) in cell supernatant and the mRNA expressions of α-smooth muscle actin (α-SMA) and phosphorylation protein kinase B (p-Akt) were measured.Results Compared with the control group,the apoptosis rate of 10 and 20 Gy IR group increased with irradiation dose (t =8.43,11.63,P ＜0.05) but they were reduced by the inhibitor of PI3K/Akt (t =8.09,4.88,P ＜0.05).The expressions of TGF-β1,α-SMA,and p-Akt also increased with irradiation dose (t =6.91,7.80,9.28,P＜0.05) but they were declined by this inhibitor for both 10 Gy IR (t =6.17,15.11,10.34,P＜0.05) and 20 Gy IR (t =10.04,6.85,23.84,P＜0.05).Conclusions X-ray irradiation could activate HSC through PI3K/Akt signaling pathway,which may further result in hepatic fibrosis.