ABSTRACT
Objective To observe the changes in help T cell 1/2 cytokines and transcription factors in murine cytomegalovirus (MCMV) infected newborn mice and to determine the effectiveness of intervention with thymopentin.Methods One hundred and twenty newborn BALB/c mice were randomized into a blank group,model group and thymopentin group,with 40 mice in each group.Mice in the model and thymopentin group received an intraperitoneal injection of MCMV suspension within 4-6 hours after birth at the dosage of 20 μ L to and tissue culture infections dose of 10431 U/0.1 ml,establish a systemic infection model,and the same volume of normal saline was injected into mice in the control group.Mice in the thymopentin group also received thymopentin 0.3 mg/(kg · d).On Day 3,5,7,10 and 14,8 mice in each group were sacrificed and their splenic tissues were harvested.Interferon (IFN) γ and interleukin (IL)-10 expression in the supernatant of a splenic lymphocyte culture was assessed by enzyme-linked immunosorbent assay.T-bet/GATA-3 mRNA in splenic tissues were analyzed using reverse transcription-polymerase chain reaction.One way ANOVA,including LSD and Dunnett T3 methods,was used for statistical analysis.Results (1) IFN-γ expression in the model and thymopentin groups peaked on Day 3,and was higher than that in the control group [(280.73 ± 14.88),(286.03 ± 15.44) and (149.42±5.43) pg/ml,respectively,F=183.532,P=0.000].Expression in the thymopentin group decreased after Day 3,and increased by Day 7.At Day 14,the IFN-γ expression level in the thymopentin group was higher than that in the control and model groups [(252.33±8.33),(149.07±7.05) and (148.57±4.53) pg/ml,respectively,F=385.487,P=0.000].(2) IL-10 expression in the model group gradually increased.By Day 14,the expression became obviously higher than that in the control and thymopentin groups [(71.19± 1.50),(36.67 ± 2.55) and (40.01 ± 1.28) pg/ml,respectively,F=523.670,P=0.000].IL-10 expression in the thymopentin group increased after Day 3 and decreased by Day 7.On Day 14,the expression in the thymopentin group was lower than that in the model group,but higher than that in the control group.(3) T-bet mRNA expression was obviously increased in the model and thymopentin groups.On Day 3,the expression was higher than that in the control group (relative value of gray-scale:0.74±0.02,0.71 ±0.04 and 0.30±0.01,respectively,F=741.630,P=0.000).By Day 3,the expression in the thymopentin group decreased,and gradually recovered on Day 5,and on Day 14 it was higher than that in the control and model groups (relative value of gray-scale:0.45 ± 0.01,0.30±0.01 and 0.30±0.01,respectively,F=257.571,P=0.000).(4) GATA-3 mRNA expression in the model and thymopentin groups increased on Day 3,and was higher than that in the control group (relative value of gray scale:0.48±0.02,0.53±0.01 and 0.33±0.01,respectively,F=345.167,P=0.000).On Day 14,the expression in the model group was higher than that in the thymopentin group,which was higher than that in the control group (relative value of gray-scale:0.99 ± 0.02,0.55 ± 0.02 and 0.34 ± 0.01,respectively,F=1 767.505,P=0.000).Conclusions A Th1/Th2 shift may be induced in MCMV infected neonatal mice,which manifests as a state of predominant Th2 response.Thymopentin can ameliorate this situation.
ABSTRACT
Objective To establish a screening system of efficient small interfering RNA (siRNA) target sites directed against truncated region of human cytomegalovirus (HCMV) UL54 gene (UL54S) with siRNA expression vectors. Methods Two small hairpin RNA (shRNA) expression vectors targeting truncated region of HCMV UL54 gene were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with the fusion protein expression vectors pUL54S-enhanced green fluorescent protein (EGFP). The levels of mRNA and EGFP were evaluated by means of reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and flow cytometry so as to assess the inhibitory efficiency of siRNA. Analysis of variance was applied to analyze the variance of total fluorescence intensity to screen out the efficient target sites of siRNA.Results shRNA expression vector psiUL54-2 and fusion protein expression vector pUL54S-EGFP were cotransfected into AD293 cells. The EGFP expression level in pUL54S-EGFP/psiUL54-2 cotransfected group was lower than that in pUL54S-EGFP/pAVU6 +27 cotransfected group after 48 h of transfection. Gel analysis showed that the mRNA relative level of UL54S was 19.6 after 48 h of psiUL54-2/pUL54S-EGFP cotransfection, which was significantly lower than those in pUL54S-EGFP/psiUL54-1 group (96.6) and control group (100.0). Cotransfection of psiUL54-1/pUL54S-EGFP for 48h didn't show any effects on the expression of fusion protein UL54S-EGFP (P>0. 05).While psiUL54-1/pUL54S-EGFP cotransfection inhibited the expression of fusion protein UL54S-EGFP(19.43×104±2.29×104vs27.89×104±5.50×104, P<0.01).Conclusion Thescreening system of efficient siRNA targeting truncated region of HCMV UL54S is established successfully. The 1532th-1550th nucleotide acids of UL54S coding sequence are efficient siRNA target sites.