BACKGROUND:Tissue engineering requires a lot of seed cells. Osteoblasts have become important seed cells in bone tissue engineering. However, it is difficult to culture the osteoblasts, and cellnumber, purity, proliferation and differentiation activity are different obtained by different culture methods. OBJECTIVE:To identify and compare three common primary osteoblat culture methods, and to explore a method for the primary culture of osteoblasts which is easy to operate, economical and effective, in order to provide basis for the further experimental research. METHODS:Calvarias were dissected from newborn Sprague Dawley rats in 72 hours, and osteoblasts were isolated with col agenase digestion method, sequential digestion method and bone tissue method respectively. The morphological observation and cytochemical staining were performed, the growth curve of the cells was drawn with cellCounting Kit-8 method, and the rate of living osteobalsts was counted with trypan blue staining. RESULTS AND CONCLUSION:The proliferation of the insolated and cultured osteoblasts was wel with typical characteristics of osteoblasts, cytochemical staining results were positive. Compared with the sequential col agenase digestion method, the col agenase digestion method presented higher production of osteoblasts and higher cellsurvival rate (P<0.05), and the col agenase digestion method was easier than the sequential col agenase digestion method and cost less than sequential col agenase digestion method. Bone tissue method was the easiest method with less damage to cells, but bone tissue method presented lower production of osteoblasts and cost much more time, which cannot be used in large-scale osteoblast culture. The col agenase digestion method is a simple, efficient and ideal method for isolation and culture of primary osteoblasts.

Objective To investigate the current situation of occupational burnout of doctors in west China and to explore the relationships among burnout,social support and work-family conflicts.Methods 611 doctors in west China were assessed by Chinese Maslach burnout inventory( CMBI),social support rating scale(SSRS) and work-interference-with-family and family-interference-with-work questionnaire.Results 14.1％ of the doctors in west China got high scores on emotional exhaustion(EE),49.4％ of them high scores on depersonalization(DP) and 33.1％ high scores on reduced personal accomplishment (RPA) ; 27.3％ experienced mild burnout,43.7％ moderate burnout and 3.3％ severe burnout.Doctors who had worked for 5 ～ 10 years and those for 11 ～ 20 years experienced greater EE.Those doctors with bachelor or master degree and intermediate or senior titles got highest level of depersonalization.Doctors in second-class hospitals got highest scores of depersonalization,and those who worked in first-class hospital experienced the highest sense of achievement.Logistic regression analysis showed that social support was a protective factor for burnout,but the conflict between work and family was a risk factor.Conclusion The status of occupational burnout among the doctors in west China is in a grave condition,the balance between family and work,and social supporting is crucial for doctors to resist the occupational burnout.

Objective To study the effects of perinatal exposure to bisphenol A(BPA) on the expression of estrogen receptor ? and ? mRNA in the brain of F1 male offsprings. Methods Pregnant SD rats were given BPA at 2, 20, 100 mg/kg bw per day respectively from eleventh day of gestation throughout the whole lactation by gastric gavage until their pups were weaned on postnatal day 21, F1 male pups from each group were killed on postnatal day 21 respectively, the brain was removed for detecting the expression of estrogen receptor ? and ? mRNA by RT-PCR. Results BPA treatment caused a up-regulation of ER? mRNA and ER? mRNA relative expression in the brain,especially in the middle-dose and low-dose groups it was so obvious. Conclusion Perinatal exposure to BPA can cause a change of ER? mRNA and ER? mRNA expression in the brain of the male offspring,which may be a part of the mechanism of BPA induced brain development damage.

Objective To investigate telomerase activity in patients with psoriasis. Methods The telomerase activity in the skin lesions of patients was detected by TRAP (Telomeric repeat amplification protocol)——ELISA which is based upon PCR amplification of the initial telomerase product and detected by ELISA. Results Telomerase activity was detected in the lesions of psoriatic patients, but the level of it was lower in comparison with that in tissues of malignant tumor and K562 cell line. Conclusion These findings support that the increase of telomerase activity may be linked to the pathogenesis of psoriasis and that the telomerase activity can be detected not only in malignant tumors but also in nonmalignant skin diseases.

A matrix solid phase dispersion extraction_dispersed liquid phase microextraction_gas chromatography_mass spectrometric method has been developed for the determination of three pyrethroids ( tetramethrin, permethrin and deltamethrin) in soil. The optimal conditions for the analysis were as follows. About 0. 5 g soil and 1. 5 g C18 solid phase extraction powder were grinding for 5 min. The mixture was eluted with 10 mL of acetone. The eluent was concentrated to 0. 4 mL, and then mixed with 20 μL of tetrachloromethane and 5. 0 mL of ultrapure water to form a homogeneous cloudy solution. The emulsion was broken by centrifugation. About 1 μL of sediment was injected and analyzed directly by GC_MS. Good linearities for three pyrethroids were ranged from 5 to 200 μg/kg (r2≥0. 9989), and recoveries at three spiked levels were ranged from 86 . 5% to 108 . 0% with RSDs less than 7 . 8%. The LODs of three pyrethroids were 1. 00-1. 48 μg/kg. This method can meet the determination of trace pyrethroids in soil.

Objective To investigate the expressions of thymidylate synthase (TS) and adenosine triphosphate (ATP)-binding cassette superfamily G member 2 (ABCG2) in advanced gastric cancer (GC) and to explore their correlation with clinical pathological features.Methods A total of 80surgical specimens of advanced gastric cancer patients were collected.The expressions of TS and ABCG2 in gastric cancer tissues and adjacent normal gastric tissues were detected by immunohistochemical method.The expression of P-glycoprotein in gastric cancer tissues was also examined.The correlations between TS,ABCG2 and clinical pathological features and P-glycoprotein were analyzed.Chi-square test was performed for two groups comparison and Kruskal-Wallis H test were used for multi-groups comparison.Results The positive rates of both TS and ABCG2 in gastric cancer tissues [85.0％ (68/80) and 90.0％ (72/80)] were higher than those of adjacent normal gastric tissues [62.5 ％ (50/80) and 78.7 ％ (63/80)],the differences were statistically significant (x2 =11.466 and 16.463,P=0.009 and 0.001).There were close correlation between the expression of TS,ABCG2 and tumor TNM stage,differentiation status,invasion depth (TS:x2 =30.686,61.470 and 40.545 ; ABCG2:x2=48.192,63.150 and 47.512; all P＜0.01).The later the tumor staged,the worse the cells differentiated and the deeper the tumor invaded,the higher level they expressed.Both TS and ABCG2expressions in gastric cancer tissues were correlated with the expression level of P glycoprotein (x2 =43.977and 29.509,both P＜0.01).Conclusion TS and ABCG2 may be potential indexes to predict the degree of malignancy,progression,drug resistance and prognosis in gastric cancer.

Objective To investigate the inhibition of pathogenic viral gene expression in HPV6bE6-positive cell line by specific siRNA, which might have great potential for clinical use. Methods B16 cells were transfected with recombinant plasmid pcDNA3.1(+)-GFP/HPV6bE6, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. Four specific siRNAs, none of which shares homology with exons of known human genes, were designed and synthesized to target HPV6bE6 mRNA. Quantitative real-time PCR was performed to measure the inhibition rates of target gene expression by comparing HPV6bE6 mRNA concentrations before siRNA transfection with those after transfection. The inhibition rates of target gene expression with different siRNA concentrations of 0.2 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L, 150 nmol/L and with different treatment time at 24 h, 48 h, 72 h and 96 h after transfection were measured respectively. Results More than 90% reduction of HPV6bE6 mRNA was observed following treatment with HPV6bE6-siRNA, and HPV-negative cells were apparently unaffected by HPV6bE6-siRNA. The decrease of HPV6bE6 mRNA was maximal at 24 h after siRNA treatment and sustained for at least 4 days. The minimal level of siRNA to efficiently silence the homogeneous target gene HPV6bE6 was 1 nmol/L. Conclusion HPV6bE6-siRNA can efficiently and specifically silence target genes and may be developed as a potential therapeutic approach for HPV infection.

Objective To study the value of MP-DNA in the early diagnosis of pneumonia by analyzing the result of MP-DNA in bronchoalveolar lavage fluid(BALF) and MP-IgM antibodies in serum.Methods 103 hospitalized children were detected for MP-DNA in BALF(positive:DNA copies ＞ 102/ml) and MP-IgM antibodies in serum.Results The MP-DNA positive in BALF and the MP-IgM positive in serum in children with pneumoina were significantly higher than those in children with foreign body in bronchus (x2 =13.00,4.11,all P ＜ 0.05).33.01％ of 103 children was MP-DNA positive,19.43％ of 103 children was MP-IgM positive.The MP-DNA positive in BALF was significantly higher than the MP-IgM positive in serum(x2 =4.92,P ＜0.05).Conclusion The samples of MP-DNA in BALF were collected more difficulty than the samples of MP-IgM in serum,but the detection of MP-DNA can avoid the false negative,which may be of value in the diagnosis and therapy of MPP.

Objective To understand the HIV infection status and epidemic characteristics among blood donors in Hefei area to provide the basis for recruiting the blood donors and establishing the blood detection scheme.Methods Two kinds of serological re-agent were adopted for screening the blood donors.HRV RNA in the detection samples of the nucleic acid test system and positive serological samples were simultaneously sent to the municipal Center for Disease Control for conducting the confirmation detection from 2012.Results 495 279 blood donors were performed the serological screening,44 cases were confirmed HIV positive with the detection positive rate of 0.9;112 940 samples were performed the NAT detection,20 cases were confirmed HIV positive,all were the serological dual reagent positive samples.Conclusion The HIV infection among unpaid blood donors in Hefei area shows the increasing trend year by year.Effective measures must be taken to ensure the safety of blood.

Objective To evaluate the effect of calcitriol combined with losartan on diabetic nephropathy in grade Ⅲ and early Ⅳ.Methods 47 patients with diabetic nephropathy were enrolled.Patients were randomly assigned to receive losartan or both losartan and calcitriol according to randomized table for 6 months.At baseline time and after 6 months,the 24-hour urinary protein excretion,estimated glomerular filtration rate (eGFR),serum creatinine(SCr),blood pressure,fasting blood-glucose,serum calcium,serum phosphorus,pulse wave velocity(PWV) and ankle brachial index(ABI) were measured.Results The urinary protein excretion showed that there was significant decrease in the mix-treated group[(824.81 ± 307.84) g/24h vs (390.75 ± 173.51) g/24h,t =10.51,P ＜ 0.01] and the control group [(860.64 ± 313.89) g/24h vs (676.16 ± 297.71)g/24h,t =6.91,P ＜ 0.01].Furthermore,the mix-treated group had the lower proteinuria compared the group given losartan only(t =2.56,P =0.015).No significant differences were observed decrease in estimated eGFR and change in serum calcium,serum phosphorus,PWV and ABI between the two groups.Conclusion Addition of calcitriol to a renin-angiotensin system inhibitor resulted in a safe decrease in proteinuria in patients with diabetic nephropathy.