AIM: To study the expression of hepatitis B virus core gene in Pichia pastoris and to obtain high-level expressed recombinant HBcAg with good immunoreactivity and high specificity. METHODS: HBV core gene was amplified by PCR from plasmid pHBV1 which contained HBV whole DNA sequence. The PCR product was cloned into pGEM-T vector by TA cloning strategy. After confirmed by DNA sequence analysis, the gene of interest was inserted into the yeast expression vector pPIC9. The recombinant plasmid pPIC9-cAg was constructed and transformed into GS115 by electroporation. The recombinant yeast GS115 was induced by 0.5% methanol. The expressed product was analysed by SDS-PAGE,Western blot and ELISA. RESULTS: The restriction analysis and DNA sequence analysis proved that HBV core gene had already been cloned to yeast expression plasmid pPIC9. The expressed HBcAg existed in SDS-PAGE. Good immunoreactivity and high specificity of the recombinant HBcAg have been proved by ELISA and Western blot. The titre of the recombinant HBcAg in the cell lysate was 1∶ 12 800 . CONCLUSION: The recombinant plasmid pPIC9-cAg was successfully constructed. The recombinant HBcAg with good immunoreactivity and high specificity was successfully expressed in Pichia pastoris expression system and can be applied to further developing HBcAb immunoassay. [

BACKGROUND: Bushen Huogu decoction can effectively prevent and treat osteoporosis, but the concrete mechanism of pharmacology is still not clear. 25-hydroxy vitamin D3 and 1, 25-dihydroxyvitamin D3 are important coupling factors, which can regulate bone resorption and formation.OBJECTIVE: To investigate the curative effects of Bushen Huogu decoction on the bone mineral density, bone biomechanics, and level of 25-hydroxy vitamin D3 and 1, 25-dihydroxyvitamin D3 in blood serum, liver and kidney in the ovariectomized osteoporotic rats.METHODS: Totally 108 healthy female Sprague-Dawley rats were randomly divided into model group, sham-operated group, andtreatment group. All rats had been ovariectomized to induce estrogen absence and further establish osteoporotic models, except those in sham-operated group. Treatment group of rats were intragastrically administrated with 2 mL Bushen Huogu decoction,twice a day.RESULTS AND CONCLUSION: Compared with model group, the bone mineral density in the rat femur was significantly increased in the treatment group (P < 0.05), the index of maximal stress and maximal loading of the femoral head were also increased (P <0.05). The concentration of 25-hydroxy vitamin D3 and 1, 25-dihydroxyvitamin D3 in blood serum, liver and kidney were significant higher in the treatment group than those in the model group, and the levels were similar with those in sham-operated group (P >0.05). In the early period of estrogenic hormone absence, the Chinese kidney-tonifying drugs could activate bone metabolism, raise bone mineral density and reinforce quality of bone through up-regulating expression of 25-hydroxy vitamin D3 and 1,25-dihydroxyvitamin D3.

Objective To establish a gene diagnosis assay for spinal muscular atrophy(SMA) in children. Method Analysis of the survival motor neuron (SMN) gene in 19 SMA patients and in 21 normal controls were performed by using polymerase chain reaction - fragment length polymorphism (PCR - RFLP) method. Result Deletion of exon 7and 8 in SMNt gene were found in all 19 SMA patients, while no such changes were found in normal controls. Conclusion The SMNt gene exon 7 and 8 examine can be applied to SMA gene diagnosis, and the PCR- RFLP method have higher sensitivity and particularity to the SMA diagnosis.

Objective To explore the differences in clinical features between the latent autoimmune diabetes in adults (LADA) and type 2 diabetes mellitus (T2DM) in the elderly.Methods Clinical features of 41 cases with LADA and 49 cases with T2DM were retrospectively analyzed and compared.Results (1) Body mass index (BMI),fasting and postprandial levels of cpeptide,triglyceride (TG) and serum albumin were lower in LADA patients than in T2DM patients.(2) The levels of fasting and postprandial blood glucose were higher in LADA patients than in T2DM patients.(3) There were no statistically significant differences in the disease course,blood pressure (BP),and the functions of liver and kidney between LADA and T2DM patients.Conclusions As compared with T2DM patients,LADA patients have higher blood glucose level,lower BMI and TG level,and poorer function of islet B cell.But there is no significant difference in the chronic complications between two groups.

OBJECTIVETo investigate the effects of advanced glycosylation end products (AGEs) modified bovine serum albumin (AGE-BSA) on the expression of reactive oxygen species (ROS) and monocyte chemoattractant protein-1 (MCP-1) in human renal mesangial cells (HRMCs).

METHODSHRMCs were cultured in vitro with medium containing different doses of AGE-BSA or BSA (50,100, 200, 400 mg/L) for 48 hours, or with AGE-BSA (200 mg/L) for different times (12, 24, 48, 72 h). Immunocytochemistry assay was used to estimate the protein level of RAGE. The ROS in cells were measured by flow cytometry and the mRNA expression of MCP-1 were analyzed by semi-quantiative reverse transcription-polymerase chain reaction (RT-PCR) after treatment with AGE-BSA or BSA.

RESULTSThe protein level of RAGE was upregulated in the HRMCs with AGE-BSA. The expression of ROS and MCP-1 significantly enhanced by incubation of AGE-BSA in a time- and dose-dependent manner. The effects of AGE-BSA-induced up-regulation of ROS and MCP-1 level was significantly blocked by neutralizing antibodies to RAGE, while the expression of ROS and MCP-1 stood nearly unchanged after cultured with huamn IgG.

CONCLUSIONThe expression of ROS and MCP-1 in HRMCs is induced by AGE-BSA through RAGE, which may have potential effects in the pathgenic mechanism of diabetic nephropathy.

Cells, Cultured ; Chemokine CCL2 ; metabolism ; Glycation End Products, Advanced ; pharmacology ; Humans ; Mesangial Cells ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Serum Albumin, Bovine ; pharmacology

BACKGROUND: Although overuse-induced patella-patellar tendon junction injury is a common disease, its pathogenesis and healing mechanism remain unclear.OBJECTIVE: To establish an overuse-induced injury animal model by a repetitive traction at the patellar tendon insertion,and to explore its pathogenesis and healing mechanism based on histomorphology and growth factor expression levels.METHODS: Twenty-eight New Zealand rabbits were randomly divided into four groups: the rabbit left and right posterior limbs without any intervention as controls (n=4); cyclic loading training targeting the left posterior limb was done in model,healing and training groups (n=8 per group) for 4 weeks. Afterwards, the rabbits in the model group were killed immediately, rabbits in the healing group relaxed for another 4 weeks and then were killed, while rabbits in the training group were subjected to 4-week low-intensity training. The histological changes and growth factor expression levels during patella-patellar tendon junction injury and healing after load training were observed.RESULTS AND CONCLUSION: Compared with the control group, in the model group, the cells and collagenous fibers arranged in disorder, and the tidemark disappeared. The healing and training groups healed incompletely, but showed better histological changes than the model group. The thickness of fibrocartilage and basic fibroblast growth factor level in the control group were significantly lower than those in the other three groups (P < 0.05). The cell density and vascular endothelial growth factor level in the model group were significantly higher than those in the control and healing groups (P < 0.05). These results show that the animal model of overuse-induced patella-patellar tendon junction injury cannot return to normal after 4 weeks of rest, but the low-intensity training is conductive to healing quality.

Objective To investigate the clinical feature,diagnosis,treatment and prognosis of endometriosis of the bladder.Methods A retrospective study was conducted to review the clinical data of 10 patients with bladder endometriosis.Patient's age ranged from 30 to 48 years (with mean age of 38 years).Eight cases were admitted to hospital with urinary tract irritating symptoms during the menstrual period and 6 cases with hematuria; 2 patients without any symptoms were found through examination.The course of disease was 1-36 months (with mean of18 months).Ultrasound shows with low echo,single,wide base and no significant blood flow mass whose boundaries are less clear within the bladder wall.CT reveals soft-mass protruding into the bladder.Results Eight of the 10 patients were undergone partial cystectomy.And 2 cases was treated with transurethral resection.All cases were pathologically confirmed to be bladder endometriosis.Recurrence and ectopic lesion had not be found during follow-up period from 10 to 72 months (with mean of 30 ± 5.6 mon).Conclusions Endometriosis is a common disease in females in their reproductive years,but thebladder endometriosis is rare.The initial diagnosis needs to be made combining with imaging studies.It is confirmed by cystoscopy and pathological biopsy.Surgery is the option for the treatment of bladder endometriosis.

Objective This study reviews the 198 consecutive mitral valve operations for elderly patients(＞ 65 y) with ischemic mitral insufficiency performed at Anzhen Hospital between January 2000 and june 2016.The results for mitral valve reconstruction are compared with those for biological mitral valve replacement.Methods From January 2000 to June 2016,198 elderly patients with ischemic mitral insufficiency underwent mitral valve reconstruction (n =150) or biological mitral valve replacement(n =48).All of them coronary artery bypass gafting was performed at the same time.Preoperative clinical characteristics,procedural characteristics,major and minor complications after surgery,preoperative and postoperative left ventricular ejection fraction (LVEF) by echocardiography,and outcome (survivor or death,mitral regurgitation,NYHA degree) were assessed.Results There was no significant difference between the two groups in the rate of mortality during hospitalization and early postoperative cardiac function.The proportion of severe MR in 1,3,and 5 years after MVP were 5.1％ 、6.3％、7.7％ respectively.There was no MVR due to the recurrent moderate-to-severe reflux.There were 2 SBE but no reflux appeared after MVR.There was no significant difference in cardiac function and mortality between the two groups within 5 years.Conclusion There is no significant difference between CABG + MVP and CABG + MVR(BV) in cardiac function and mortality in the treatment of elderly patients with IMR.,There was a possibility of MR recurrence after CABG + MVP.There was no MR after CABG + MVR(BV),but the risk of SBE is higher than MVP.We can select operation mode personalized according to the type of mitral valve disease and cardiac function.

Objective To study the relationship between angiogenesis and pathology and prognosis of cholangiocarcinoma. Methods Immunohistochemistry was used to determine micro-vessel density (MVD) and the expression of vascular endothelial growth factor(VEGF) and its receptor Flk-1/KDR in 50 cholangiocarcinoma cases. Results were compared with pathological and follow-up parameters. Results MVD in cholangiocarcinoma tissues and para-tumor tissues (34.04 ? 11.08, 32.80 ?9.28) were higher than normal bile duct tissues ( 11. 67 ? 4. 64) ( P

BACKGROUND:Patella-patellar tendon junction (PPTJ) injuries are a common disease in athletes and vulnerable to re-injury. Post-injury training is a double-edged sword. OBJECTIVE:To examine the effect of post-injury resting days on the quality of healing in PPTJ in view of histomorphology, expression levels of growth factor and collagen, and muscle tension. METHODS:Forty New Zealand white rabbits were randomly divided into control, and five post-injury training groups representing different time to restart training after acute injury:the rabbits restarted training at 24 (PIT24), 48 (PIT48), 72 (PIT72), 96 (PIT96) and 120 (PIT120) hours, respectively. All post-injury training groups received acupuncture at the PPTJ and then underwent 4-week low-intensity training. Afterwards, all rabbits were killed to take specimens for hematoxylin-eosin, safranin O and immuohistochemical staining. The muscle tension and hardness were detected using Myoton-3. The thickness of fibrous cartilage zones, cell density, as well as the expression levels of vascular endothelial growth factor and collagen were measured on Metamorph software. RESULTS AND CONCLUSION:Compared with the control group, in the PIT groups, the collagens arranged in disorder, and decreased cell density, increased mucopolysaccharide andrising tidephenomenon appeared. The fibrocartilage in the PIT24 and PIT120 groups was significantly thicker than that in the control group (P<0.05). The ratio of collagen type Ⅰ/Ⅲ was significantly decreased in the PIT24 and PIT48 groups compared with the control group (P<0.05), and the highest in the PIT72 group. The expression level of vascular endothelial growth factor in the PIT48, PIT72 and PIT96 groups was significantly higher than that in the control group (P<0.05). The muscle tension was negatively correlated with the collagen ratio (r=-0.538;P<0.01) and chondrocyte density (r=-0.570;P<0.01), while muscle hardness was positively correlated with the expression level of vascular endothelial growth factor (r=0.613;P<0.01). Our results suggest that different resting time makes significant differences in the PPTJ healing and the 72-hour resting is the best choice. Moreover, muscular properties are related to the cel density, collagen synthesis and growth factor expression, which can be used as a non-invasive indicator to guide post-injury training and prevent recurrence.