AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time...AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.展开更多
MicroRNA (miR)-125b has been shown to play a potential role in the development of glioma stem cells.However,the relationship between miRNA and glioma stem cells is still elusive.This study was designed to elucidate th...MicroRNA (miR)-125b has been shown to play a potential role in the development of glioma stem cells.However,the relationship between miRNA and glioma stem cells is still elusive.This study was designed to elucidate this potential relationship.We established a highly invasive glioma stem cell and progenitor (GSCP) cell line SU3.SU3 cell suspensions were injected into nude mice brains in situ,and the invasiveness of graft tumors was analyzed using hematoxylin and eosin staining as well as immunohistochemistry.Real-time polymerase chain reaction (PCR) was used to measure the expression levels of miR-125b in SU3 and other cells.In vitro,SU3 cells expressed CD133 and nestin as well as differentiation markers glial fibrillary acidic protein (GFAP) and β-tubulin III,which were consistent with the characteristics of glioma stem cells.Scratch assays indicated that the migration ability of SU3 cells was stronger than that of U251 stem cells (U251s).In vivo,SU3 cells invaded into each part of the mouse brain from the caudate nucleus in a diffuse pattern and highly expressed invasive and proliferative cell markers matrix metalloprotease 2 (MMP2),MMP9,and Ki-67.Real-time PCR results revealed that the levels of miR-125b and MMP9 were significantly higher in SU3 and SU2,also a highly invasive GSCP cell line we established before,than in U251s.High expression of miR-125b both in newly established GSCPs,SU3,and long-term cultured GSCPs,SU2 suggests that miR-125b exhibits oncogene-like behavior.This behavior should be considered in further studies of miR-125b in cancer stem cells.Furthermore,MMP9,which plays a role in cancer stem cell invasion,may be a target gene of miR-125b.展开更多
基金SuppoSed by CRCG grant from the University of Hong KongCERG grant from University Grant Council of Hong Kong Research Fund from Science and Technology Commission of Shanghai,China
文摘AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.
基金funded by grants from the Natural Science Foundation of China(NO.81172400,81000963)the Natural Science Foundation of Jiangsu Province(NO.BK2011341)the Natural Science Foundation of Suzhou(NO.SYS201063,SYS201161)
文摘MicroRNA (miR)-125b has been shown to play a potential role in the development of glioma stem cells.However,the relationship between miRNA and glioma stem cells is still elusive.This study was designed to elucidate this potential relationship.We established a highly invasive glioma stem cell and progenitor (GSCP) cell line SU3.SU3 cell suspensions were injected into nude mice brains in situ,and the invasiveness of graft tumors was analyzed using hematoxylin and eosin staining as well as immunohistochemistry.Real-time polymerase chain reaction (PCR) was used to measure the expression levels of miR-125b in SU3 and other cells.In vitro,SU3 cells expressed CD133 and nestin as well as differentiation markers glial fibrillary acidic protein (GFAP) and β-tubulin III,which were consistent with the characteristics of glioma stem cells.Scratch assays indicated that the migration ability of SU3 cells was stronger than that of U251 stem cells (U251s).In vivo,SU3 cells invaded into each part of the mouse brain from the caudate nucleus in a diffuse pattern and highly expressed invasive and proliferative cell markers matrix metalloprotease 2 (MMP2),MMP9,and Ki-67.Real-time PCR results revealed that the levels of miR-125b and MMP9 were significantly higher in SU3 and SU2,also a highly invasive GSCP cell line we established before,than in U251s.High expression of miR-125b both in newly established GSCPs,SU3,and long-term cultured GSCPs,SU2 suggests that miR-125b exhibits oncogene-like behavior.This behavior should be considered in further studies of miR-125b in cancer stem cells.Furthermore,MMP9,which plays a role in cancer stem cell invasion,may be a target gene of miR-125b.
