Objective To detect nucleos(t)ide-resistance mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) isolated from hepatocytes of patients with chronic HBV infection and to analyze the correlation between the mutations found in cccDNA and relaxed circular DNA (rcDNA). MethodsForty patients with chronic HBV infection were investigated. Preoperation serum samples and non-tumor liver tissues were collected.Intrahepatic HBV cccDNA and rcDNA were selectively extracted by co-precipitation of sodium dodecyl sulphate-protein and QIAamp DNA Mini Kit, and further purification with plasmid-safe ATP-dependent DNase (PSAD).Thereafter,cccDNA were amplified by selective polymerase chain reaction (PCR) or nested PCR using the primers spanning both the two gaps in HBV genome and covering the common mutations associated with nucleoside analogues resistance (rt169- rt250).Intrahepatic HBV rcDNA and pre-operation serum HBV rcDNA were also extracted and then amplified by PCR.The PCR products were then purified and sequenced.Results Among the 40 patients,intrahepatic HBV cccDNA were detected in 31 patients,and HBV rcDNA were detected in liver samples of 35 patients and pre-operation serum samples of 21 patients. The PCR products amplified from these samples were all successfully sequenced.rtM204Ⅰ mutation was detected in intracellular HBV cccDNA,rcDNA and serum HBV rcDNA in 2 patients.Both rtM204Ⅰ and rtQ215H were detected in intrahepatic HBV cccDNA and rcDNA in 2 patients,while no corresponding mutation was observed in serum HBV rcDNA of these two patients.Three variants including rtM204V,rtM204V and rtV173L-rtL180M-rtM204V were detected in serum HBV rcDNA in 3 patients,while corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA of these patients.Condsions The results suggest that antiviral nucleos(t) ide resistance mutations can be found in HBV cccDNA in chronic hepatitis B patients. The dominant resistant mutation found in intrahepatic HBV cccDNA/rcDNA may be different from that in serum HBV rcDNA.

Objective To investigate the dynamic changes of osteopontin (OPN) in the guinea pig model of cholesterol gallstone disease.Methods Forty-four guinea pigs were randomly assigned to cholesterol gallstone group and control group.The animals were sequentially killed on Day 7,14,28,42,56 and 70 after operation.The expressions of OPN mRNA in gallbladder and liver tissues were detected by real-time PCR.The changes of OPN,mucin,α1-acid glycoprotein (AAP) and apolipoprotein A1 (APOA1) in bile and blood plasma were detected by ELISA kits.Results The expression of OPN mRNA in gallbladder and liver tissues increased gradually and reached the peak in the 6th week,and then decreased.The increased expression of OPN in bile in the gallstone group started from the 1st week and reached the peak in the 6th week (P ＜ 0.05),which gradually decreased to the baseline in the 10th week (P ＞ 0.05).The expressions of OPN in bile and blood demonstrated similar trends,while the peak time in blood samples was much earlier (4th week).The changes of APOA1 in bile and blood were similar to OPN,although there was no advanced peak value in blood.The levels of mucin and AAP in bile and blood increased after operation,and were kept at high level throughout the study.Conclusions OPN is involved in the whole process of cholesterol gallstone formation,which may be associated with other nucleation-active factors.

In order to investigate the effects of hyperbaric oxygen (HBO) on the proliferation and differentiation of human osteoblasts isolated from alveolar bone under various pressure and exposure time, osteoblasts from human alveolar bone were seeded in 24 well plates at a cell density of 2 500 cells per well. There are four treatment groups which were 2.4ATA for 90 min, 2.4ATA for 30 min, 1.5ATA for 90 min and 1.5ATA for 30 min. Osteoblasts culture were treated one time everyday for up to 10 days in a temperature and humidity controlled custom-made seven-litre hyperbaric unit. Control samples were incubated in a standard humidified incubator at 37℃ containing 5% CO2 and 95% atmospheric air. Proliferation of osteoblasts were evaluated by WST-1 assay before and 16 h after HBO on day 1,2,3,4,6,8,10. The cytotoxic effect of HBO on osteoblasts was assessed by a toxicology assay kit. For differentiation study, osteoblasts were seeded in 96 well plates at a cell density of 10 000 cells per well. After 3 days normal culture, medium was changed to osteogenic medium. Subsequently, cultures were exposed daily to HBO of 2.4ATA for 90 min and 1.5ATA for 90 min up to 13 days. Mineralization was evaluated by calcium deposition assay, alkaline phosphatase (ALP) activity and von Kossa staining. To assess the effect of pressure on cell proliferation and differentiation, hyperbaric air treatment was observed in this study. It showed that HBO treatment promotes proliferation of osteoblast in the presence of 10% foetal calf serum (FCS). No significant change in extracellular LDH activity before and after HBO treatment. The study of differentiation demonstrated that HBO enhanced differentiation associated with increased bone nodule formation, calcium deposition and alkaline phosphatase activity. These result suggests that HBO treatment significantly stimulated osteogentic differentiation, which implies a potential application of HBO in bone tissue engineering.

Objective To investigate the nursing methods of transferring peduncle island skin flap in foot. Methods Nursing about 19 patients who have undergone the transferring of peduncle island skin flaps because of soft tissue injury in foot. The preoperative nursing included skin nursing, individual psycho-behavior guidance, room allocation. Postoperative nursing included placement of limbs correctly, nursing about the transferring district flaps, observation and nursing the vescular crisis, correct using anticoagulant and functional exercises of ankle-joint. Results There were 18 flaps survived completely, 1 flaps in distal end had partly necrosis, but flap had recovered well afterⅡ-period skin grafting. Conclusion Overall elaborate clinical nursing can guarantee the survival of the graft peduncle island skin flaps in foot.

ObjectiveTo investigate and analyze the health education needs of TCM rehabilitation after hepatobiliary surgery,and the application effect of health education. Methods105 cases of hepatobiliary surgery patients were chosen.Homemade questionnaires with TCM rehabilitation related knowledge needs were used in the investigation.And then the health education of TCM rehabilitation was given to the patients.After these patients accepted the education and were rehabilitated,these patients were again taken TCM and rehabilitation of health education needs for investigation.The changes of the needs of the patients before and after education for the rehabilitation of health education on TCM were compared.The evaluation of the recovery effect for patients with TCM rehabilitation of health education in the liver and gallbladder surgery were taken for investigation. Results105 cases of patients with TCM and rehabilitation health knowledge education before and after the survey results showed that,the urgent need on the knowledge of TCM health rehabilitation rate after hepatobiliary surgery in patients increased significantly compared with before education.In the evaluation of the recovery effect of patients for TCM rehabilitation of health education in theliver and gallbladder surgery,the rate of thinking it effective in promoting postoperative recovery was 91.43％. ConclusionsHepatobiliary surgery patients were lack of knowledge of postoperative recovery of health knowledge of TCM and rehabilitation.By education and practical application,the patients understand the importance of health education on their postoperative rehabilitation,so TCM rehabilitation of health education has an important clinical significance in hepatobiliary surgery patients.

Objective To evaluate liver biopsy for the diagnosis in liver transplant patients with suspected acute rejection. Methods From Oct. 2004 to Apr. 2005, liver biopsies were performed 53 times in 39 transplant cases. Results Based on Banff schema for grading liver allograft rejection, laboratory abnormalities and result of treatment, acute rejection was diagnosed on 16 episodes, preservation injury in 12, bile duct strictures in 9, drug-induced injury in 11, chronic rejection in 3 and acute hepatic failure in 2. Conclusions Hepatocyte ballooning with necrosis features preservation injury. Drug-induced injury commonly has a combination of hepatocyte denaturalization with mild portal inflammation. Histologic features of early bile duct strictures in liver biopsy show prominent bile ductular proliferation and the canalicular cholestasis with mild hepatocyte damage which help to exclude acute rejection.

Objective This study aims to investigate the sex difference in the hippocampus and parahippocampus in patients with bipolar disorder. Methods We acquired T1－weighted structural MRI from 133 bipolar type I patients (60 males) and 144 normal controls (81 males). The General Linear Model was used to examine the relationship between sex and brain volumes of the hippocampus and parahippocampus, with age and intracranial volume as covariates. Results Patients showed significantly smaller volumes of the bilateral hippocampus and parahippocampus (P＜0.01). There were sex－by－diagnosis interactions in the left parahippocampus gyrus (F=6.534, P=0.044). Male patients had significant smaller volumes of the left parahippocampus gyrus compared to the male normal controls (P＜0.001) whereas the volumes were not significantly different between female patients and female normal controls (P＞0.05). Conclusion The results suggest sex difference in the left parahippocampus gyrus volume in patients with bipolar type I disorder, which deserves further investigation in the future bipolar imaging researches.

Objective To examine the heritability of neurocognitive functions in bipolar I disorder(BD-I)families and BD-associated cognitive endophenotypes. Methods Seventy-nine nuclear families consisting of euthymic BD-I probands and their healthy parents were recruited. Cognitive functions including attention, working memory, processing speed and executive function were evaluated by 7 classic neurocognitive tests, and the heritability of neuroconitive functions in these families was estimated using parent-offspring regression indexes of quantitative traits.Furthermore,the heritable cognitions were compared between 79 BD probands and 140 normal controls. Results After adjusted by age and education,mistake numbers of Trail Making Test A(TMT-A),total score and completed mission numbers of Tower of Hanoi (TOH) were significantly heritable (P<0.05). The comparison of these heritable cognitions between patients and normal controls showed that TOH total score and TOH completed mission numbers were significantly impaired in the patient group (P<0.05). Conclusion Processing speed and executive function are probably heritable in BD nuclear families. Executive function impairments may be disease-related which could be candidate endophenotypes for bipolar disorder.