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Objective@#To compare two Taq-man Real-time PCR methods for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in serum or liver tissue.@*Methods@#Two sets of primers and probes (common Taq-Man probe and MGB Taq-Man probe) were synthesized according to the reference papers, and the sensitivity and specificity of the two methods were compared using prepared plasmid as standard curve, and HBV DNA samples were exlracted from serum and liver tissue samples of hepatitis B patients. The samples were tested with both methods separately before or after the digestion with a Plasmid-Safe ATP-dependent Dnase (PSAD).@*Results@#Both of these two kinds of detection methods had a good linear relationship with the prepared plasmid as standard curve (R2 0.989 or 0.976 respectively, CV were within 4% ), and obtained good specificity when the HBV DNA samples were tested before or after digestion with PSAD. The common Taq-Man probe had lower Ct value than MGB probe when the samples in the same concentration.@*Conclusions@#Both methods can be used for HBV cccDNA detection. The common Taq-Man probe has slightly higher sensitivity than MGB probe, while the MGB probe has lower background than the common Taq-Man probe in our test. One can select the appropriate probe according to the need.
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Objective To discuss the protective effect of creatine phosphate (CP)on isolated rat liver against cold preservation.Methods Isolated perfused rat liver model under simple cold preservation was established.The liver of the control group was perfused with pure University of Wisconsin (UW)solution. With UW solution as the base fluid,the liver of the low-dose group was perfused with 1 g/100 ml CP in UW solution;the liver of the middle-dose group was perfused with 2 g/100 ml CP in UW solution;the liver of the high-dose group was perfused with 3 g/100 ml CP in UW solution.The livers of each group were cold preserved in the corresponding perfusion fluid at 4 ℃.The content of alanine aminotransferase (ALT)and lactate dehydrogenase (LDH)in preservation solution in infrahepatic vena cava were determined.The content of malondialdehyde (MDA)and activity of myeloperoxidase (MPO)in liver tissues were detected.The apoptosis index (AI)of liver cells in liver tissues and positive expression rate of NF-κB in liver tissues were observed. Pathologic changes of liver tissues were observed under optical microscope.Results At 12 h after the cold preservation,the content of ALT and LDH in the rat livers of low-,middle-and high-dose groups were lower than those of the control group (all in P <0.05).At 18 h after the cold preservation,the content of MDA and MPO in the liver tissues of low-,middle-and high-dose groups were lower than those of the control group (all in P <0.05).At 12 h and 18 h after the cold preservation,AI and positive expression rate of NF-κB in liver cells in the rat livers of low-,middle-and high-dose groups were lower than those of the control group (all in P<0.05).At 24 h after the cold preservation,the content of ALT and MDA in preservation solution of the high-dose group was obviously higher than that of the control group as well as the low-and middle-dose groups (all in P <0.05).The results of pathological examination indicated that the injuries to the livers of the high-,middle-and low-dose groups were obviously lighter than that of the control group.There was no obvious difference among each dose group.Conclusions CP in UW solution may well protect the isolated rat liver against cold preservation,which is better than pure UW solution.
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Objective To detect the changes of CD4 + CD25 + CD127low/-regulatory T (Treg) cells in peripheral blood in patients with portal hypertension and hepatitis B virus infection before and after splenectomy,and to study the effects of splenectomy on the immune function of patients with portal hypertension.Methods The clinical data of 20 patients with portal hypertension,hepatitis B virus infection and hypersplenism who were admitted to the Third Hospital of Hebei Medical University from May 2012 to May 2013 were retrospectively analyzed.The dynamic levels of CD4 + CD25 + CD127low/ Treg cells in the peripheral blood of the 20 patients (portal hypertension group) at 1 day before splenectomy and at postoperative week 1,month 1 and month 3 were detected by flow cytometry,and the dynamic levels of CD4 + CD25 + CD127low/ Treg cells in the peripheral blood of 10healthy individuals (control group) from the same hospital were also detected by flow cytometry.The effects of changes of Treg cells on the immune function were analyzed.All data were analyzed using the t test or repeated measures analysis of variance.Results The proportions of CD4 + CD25 + CD127low/-Treg cells before operation were 5.1％ ± 3.5％ in the portal hypertension group and 1.4％ ± 0.2％ in the control group,with significant difference between the 2 groups (t =2.573,P ＜ 0.05).The proportions of CD4 + CD25 + CD127low/ Treg cells in the portal hypertension group at postoperative week 1,month 1 and month 3 were 9.2％ ±2.7％,5.6％ ± 1.7％and 2.5％± 2.1％,respectively.There was significant difference in the proportion of CD4+ CD25 + CD127low/-Treg cells between postoperative week 1 and that before operation (F =9.814,P ＜ 0.05),while there was no significant difference in the proportion of CD4 + CD25 + CD127low/-Treg cells between postoperative month 3 and that before operation (F =2.364,P ＞ 0.05).Conclusion The proportion of Treg cells increases in a short period after splenectomy,and then it decreases as time passed by,which indicates that splenectomy has slight influence on the immune system from the perspective of Treg cells.
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Objective:To investigate the renal injury of portal hypertensive rat after one hour of occlusion of portal vein and inferior vena cava.Methods:Healthy male Wistar rats were taken randomly as normal control、portal hypertensive control and trial group.The recoverable portal hypertensive model was induced firstly.Three weeks later ,15 model rats were taken randomly as portal hypertensive control group,others had another operation and were divided randomly into 0,6h,12h,24h,48h,72h,7d group according to different reperfusion time after 1 hour of occlusion of portal vein and inferior vena cava.At the corresponding time points after reperfusion,the examinations below were done:serum ALT,TBIL,BUN,Cr concentrations;mor-phological changes of liver and kidney,the ultrastructure of renal tissue.Results:Serum and Cr in trail group reached their peak value 12~24 hours after reperfusion,then decreased gradually,and returned to normal 72 hours after reperfusion.The main injury of kidney was located in proximal tubular epithelial cell ,it peaked at 12 hours and 24 hours after reperfusion,the sporadic karyopyknosis and karyorrhexis could be seen,but the basement membrane preserved well.48 hours later,the restoration could be seen.7 days later it restored obviously.Conclusion:There are obvious injury in tubular epithelial cell in the portal hypertensive rat after 1 hour of occlusion of portal vein and inferior vena cava.But the injury of kidney is reversible.