Objective To investigate the characteristics of HBV replication in HepG2.2. 15 cells treated with LAM alone or sequentially treated with LAM and IFN-α, and to further explore the different suppressive effect on HBV replication by LAM and IFN-α in vitro. Methods Untreated HepG2. 2. 15 cells were used as control group, HepG2. 2. 15 cells treated with 1 000 IU/ml of IFN-α alone for 10 d were served as IFN-α group. The HepG2.2. 15 cells treated with 0.2,1,5,20,100 μmol/L of LAM were used as LAM groups. HepG2. 2. 15 cells treated with 0. 04,0. 2,5,25,125,200 μmol/L of LAM for 7 d, then combined with 1 000 IU/ml of IFN-α for another 3 d. Afterwards, the cells were treated with 1 000 IU/ml of IFN-α only for another 10 d. These cells were served as LAM/IFN-α sequential treatment group. The ELISA was used for analyzing the secreted HBV antigens, while the Dot blot, Southern blot were applied for analyzing the extracellular HBV DNA and intracellular HBV replicative intermediate DNA in HepG2. 2. 15 cells of different treatment groups. Results The secreted HBsAg in the LAM group were 1. 77 ± 0. 22, 1.65 ±0.25, 1.95 ±0. 19, 1.34 ±0. 11, 1.07 ±0.05, respectively, and the secretion of HBeAg were 1.41 ±0. 13, 1.37 ± 0. 09, 1.63 ± 0. 07, 1.26 ± 0. 12, 1.05 ± 0. 09. The secreted HBsAg and HBeAg in control group were 3. 34 ± 0. 15 and 3.33 ± 0. 05. Statistical analysis showed that HBsAg and HBeAg secretion in the LAM group were significantly reduced by treatment with LAM. The t values of HBsAg were 10. 21,10.04, 9.94, 18.62, 24.86, and the t values of HBeAg were 23.87, 32.97, 34.22, 27.57, 38.35,respectively, all P values were ＜ 0.05. Dot blot, Northern blot hybridization analysis indicated that the extracellular HBV DNA and intracellular HBV replicative intermediate DNA could not be detected in the LAM group after cells treated by LAM for 10 days. When LAM was replaced with treatment of 1 000 IU/ml of IFN-α alone, it could not suppress the HBV replication effectively. Moreover, the intracellular HBV replicative intermediate DNA still existed in almost all groups, accompanied with the recovered expression of HBV antigens as well as extracellular HBV DNA, which suggested that the HBV particles restored replication again and secreted extracellular in HepG2. 2. 15 cells, although the sequential treatment lasted for 10 days.Conclusion The effect of viral suppression by LAM and IFN-α in vitro were different, which attributed to the different HBV replicative characteristcs in HepG2. 2. 15 cells.

BACKGROUND:Subcutaneous fat of human body is a rich reservoir of adipose derived stromal stem cells(ADSCs).ADSCs can proliferate rapidly when being cultured in vitro,and has the capacity of multi-directional differentiation.ADSCs attracted much attention in research of tissue engineered seed cells.OBJECTIVE:To isolate and culture stromal vascular fraction(SVF)cells from rabbit subcutaneous fat in vitro,and to testify whether it has multiple differentiation capacity.METHODS:SVF cells were isolated in vitro from rabbits,and cultured under standard condition.Cellular surface antigens CD44,CD45 and CD29 of passage 3 SVF cells were examined using flow cytometry.Passage 3 SVF calls were induced to differentiate into osteoblasts,chondrocytes,and lipocytes.Oil red staining was used to examine lipocyte induction.Alkaline phosphatase(ALP)staining,alizarin red staining and von Kossa staining were used to examine osteoblast induction.Type Ⅱ collagen immunohistochemical staining and type Ⅱ collagen mRNA RT-PCR were used to examine chondrocyte differentiation.RESULTS AND CONCLUSION:Primary SVF cells were multi-angular or short spindle-shaped.Passage 3 SVF calls were long spindle-shaped.Flow cytometry showed CD44+,CD29+,CD45.Oil red staining exhibited positive reaction in lipocyte induction group.ALP staining,alizarin red staining and Von kossa staining demonstrated positive reactions in osteoblast induction group.Type Ⅱ Collagen immunohistochemical staining and alcian blue staining have suggested positive reactions at 14 days of chondrogenic induction group.RT-PCR of type Ⅱ collagen mRNA test showed that the product band had strong signal at 14 days of chondrogenic induction group compared with that before induction.Above mentioned results have indicated that SVF cells isolated from rabbit subcutaneous fat have identical surface makers of stem cells,and have the ability to differentiate into lipocytes,ostsoblasts and chondrocytes in vitro by induction,and it could be concluded that the SVF cells were ADSCs.

BACKGROUND: Small intestinal submucosa (SIS) has good compatibility with cells and tissues, and has good degradabUity. It is an ideal scaffold for tissue engineering. Inducing adipose derived mesenchymal stem cells (ADSCs) seeded on SIS can construct target tissues, which has the potential to be used in clinical treatment.OBJECTIVE: To prepare decellularized porcina SIS matrix, and testify its biocompatibility with rabbit ADSCs cultured in vitro. METHODS: SIS was processed by enzyme digestion-hypertonic saline decellularization, lyophilized at low temperature, and sterilized by gamma radiation. Paraffin sections were used to observe the effect of decellularization of SIS, and the surface structures of SIS were observed by scanning electron microscope (SEM). Rabbit ADSCs were isolated and cultured, and passage 3 ADSCs were seeded onto one side or both sides of SIS. After one weak of co-culture, the cell-scaffold composites were observed.RESULTS AND CONCLUSION: SIS was white and semi-transparent film. Paraffin sections showed no cells on SIS matrix; electron microscopy showed loose weave structure of serosal surface and dense packing structure of mucosal surface. After one week of co-cultivation, plenty of ADSCs were observed on the surface of SIS. In ADSCs seeded onto one side of SIS group, a large number of cells grew on the superior surface, and few even no cells were observed on inferior surface of SIS. When ADSCs were seeded onto both sides of SIS, cells adhered to SIS in paraffin sections. Results show that enzyme digestion-hypartonic saline decellulariation can decellularize SIS completely, and SIS can support ADSCs growth.

Objective To study the relationship between serum Treg cells content and superoxide dismutase(SOD) level with the severity and clinical prognosis in the patients with preeclampsia.Methods Forty cases of preeclampsia in our hospital from September 2015 to September 2016 were selected as the research subjects divided into the mild preeclampsia group(25 cases) and severe preeclampsia groups(15 cases) according to the disease severity,and 20 healthy pregnant women served as the control group.The peripheral blood Treg cells and serum SOD level were compared between the two groups.The relationship between Treg cell content and serum SOD level with the disease severity and prognosis in the patients with preeclampsia was investigated.Results The systolic and diastolic blood pressure in the severe preeclampsia group were(145.3±10.6)mm Hg and(102.3±7.8)mm Hg,the occurrence rates of premature delivery,placental abruption and fetal distress were 33.3%,20.0% and 13.3% respectively,BMI before pregnancy was 25.4±1.3,which were significantly higher than those in the mild preeclampsia group and control group,while the mild preeclampsia group was higher than the control group.The Treg cell content and SOD level in the severe preeclampsia group were (2.3±0.7)×106/L and(2.6±0.7)μg/mL,which were significantly lower than those in the mild preeclampsia group and control group and which in the mild preeclampsia group were (3.4±0.6)×106/L and(4.3±0.9)μg/mL,and significantly lower than those in the control group,the differences were statistically significant(P<0.05).The Logistic regression analysis results indicated that BMI index(P=0.018),Treg cell content(P=0.024) and SOD level(P=0.029) were the independent risk factors for poor pregnancy outcome.Conclusion The peripheral blood Treg cells content and serum SOD level are closely related to poor pregnancy outcome in the patients with preeclampsia.

Objective:To optimize the water extraction process of traditional Chinese medicine ( TMC) Qubai granule. Methods:The orthogonal test was used to study four influencing factors including water amount, soaking time, extraction time and extraction times with dry extract yielding rate and the content of ferulic acid as the evaluation indices. Results:The optimum extraction process was as follows:A2 B1 C2 D2 , namely adding 10-fold amount of water, without soaking in advance, extracting twice with 2 h for each time. Con-clusion:The process is simple, stable and reproducible, which provides basis for the industrial production.

OBJECTIVE:To observe the effects of adding chitosan in the hydrotubation on increasing conception rates.METHODS:184tubal infertility cases were divided into2groups at random,the95cases in the treatment group were injected with0.3%chitosan solution10ml after the routine hydrotubation;89cases in the control group were only given the conven-tional hydrotubation.RESULTS:The conception rates for the treatment group and the control group were40.7%and24.4%respectively within6months after hydrotubation(P

OBJECTIVE To investigate the staphylococcal cassette chromosome mec(SCCmec) genotype characteristics and antibiotic-resistant genes in meticillin-resistant Staphylococcus aureus(MRSA) isolated from Lianyungang.METHODS The SCCmec of clinically isolated MRSA strains were genotyped with a novel multiplex PCR strategy reported by Zhangetal.Antibiotic-resistant genes of aac(6′)/aph(2″),aph(3′)Ⅲ,tetM,erm,TEM,and ant(4′,4″) were analyzed by traditional PCR.RESULTS The isolates were almost SCCmec Ⅲ positve,only one isolate couldn′t be typed.The positive rates of aac(6′)/aph(2″),aph(3′)Ⅲ,tetM,and erm were 98%,46%,72% and 86%,respectively.TEM and ant(4′,4″) tested were all negative.CONCLUSIONS Almost all genotypes of MRSA prevailing in Lianyungang carry the SCCmec Ⅲ gene.There are high positive percentages of antibiotic-resistant genes of aac(6′)/aph(2″),aph(3′)Ⅲ,tetM and erm in the isolates.The novel multiplex PCR strategy recommended by Zhang et al can be applied into genotyping study of MRSA SCCmec effectively.

Aim To construct and express the eukary-otic expression vector of double-stranded RNA-depend-ent protein kinase (PKR)fusion green fluorescent and analyse its antiviral activity of HBV in vitro.Methods The PKR gene was cloned into an empty expression vector pEGFP-N1 using molecular clone technology. After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pEGFP-PKR that was subsequently transfect-ed into HepG2.2.15 cells using LipofectamineTM2000. The expression level of PKR in HepG2.2.15 cells was confirmed by using fluorescent microscopy. Mean-while,HBV DNA and HBsAg/HBeAg were detected by real-time PCR and electrochemiluminescence meth-od,respectively.Results Both restriction enyme di-gestion and sequencing assays showed that the recombi-nant vector pEGFP-PKR was successfully constructed in our study.Fluorescent microscopy observation indi-cated that the fusion protein pEGFP-PKR expressed ef-ficiently in HepG2.2.15 cells.Moreover,compared with the empty vector group,the expression of HBV antigen in supernatants was significantly decreased (P<0.05 ).However,the extracellular HBV DNA ex-pression was not inhibited significantly.Conclusion In vitro,PKR proteion has certain antiviral activity of HBV.

Objective To make a comparison between the plain radiograph, CT, and MR findings of gouty arthritis and to analyse the relationship between clinical data and imaging findings. Methods Fifty-four joints of 33 patients with a confirmed diagnosis of gouty arthritis were included in this study. In the morning, the blood uric acid level of patients was tested before meal. In the afternoon, their clinical data were recorded and joints were examinated by plain radiography, CT, and MRI. The imaging findings were evaluated by tophi, bone erosion, soft tissue swelling, hydrarthrosis, synovial thickness, and bone oedema. The data was analyzed by Chi-square test, indepentdent-samples t test, and logistic regression. Results The Chi-square test was utilized to evaluate number of joints with tophi(CT>plain radiography, MR>plain radiography, P<0. 01), soft tissue swelling (MR>CT>plain radiography, P<0. 01), hydrarthrosis(MR>CT, P<0. 01). In addition, 35 joints had bone oedema and 50 joints had synovial thickness. The course of disease(tophi positive group vs tophi negative group, P<0. 01) was analyzed by indepentdent-samples t test. The tophi's causative agents including bone erosion and course of disease were analysed by logistic regression(P<0. 01). Conclusions MRI is superior to CT and plain radiography on the early diagnosis of gout. Tophi and bone erosion may not affect the blood uric acid level. With the progression of disease, the probability of tophi formation increases. The relationship between the formation of tophi and bone erosion may be interpromotied.

Objective To evaluation the method of rapid detection of Methicillin-resistant Stphylococcus aureus (MRSA) ST239 clones with multiplex PCR assay and investigation of the epidemic status of MRSA blood stream infections in Hefei area.Methods Antibiotic susceptibility testing were applied to MRSA isolates from bloodstream infection,rapid screening and confirmation of MRSA ST239 clones by using multiplex PCR,Multilocus Sequence typing (MLST) and Staphyloccoccal Cassette Chromosome mec(SCCmec) typing.Results 51 of 106 clinic isolates Staphylococcus aureus were identified as MRSA,accounting for 48.1％.The resistance rate of MRSA to erythromycin,aminoglycosides and quinolone were significantly higher than Methicillin Sensitive Staphylococcus aureus (MSSA).Both MRSA and MSSA had a high sensitivity to cotrimoxazole,the sensivity rates were 86.3％ and 94.5％,respectively; 47 of 51 isolates of MRSA that detected by MRSA ST239 rapid screening were ST239 clones.Randomly selected 20 positive screening stains were confirmed as MRSA-ST239-SCCmec Ⅲ by MLST and SCCmectyping.Conclusions In Hefei area,nearly half of MRSA bloodstream infections in clinical isolates are MRSA-ST239-SCCmeclⅢ type and serious multidrug-resistance.The rapid detection of ST239 clones by multiplex PCR is a reliable and effective method for large-scale screening in laboratory.