Objective To explore the possibility of differentiation of canine bone marrow-derived mesenchymal stem cells(BMSCs) into smooth muscle cells(SMCs) and the potential of using these SMCs as cell sources for engineering of blood vessel construction.Methods Canine BMSCs were isolated by density gradient centrifugation and cultivated in DMEM supplemented with PDGF-BB and vitamin C(VIT-C).The phenotypic characteristics of BMSCs were identified by morphological observation,?-SMA and SMMHC in SMCs at passage 4-6 were analyzed by immunofluorescent staining,and the positive rates of SMCs at passage 5,6 were detected by flow cytometry.Results The BMSCs cultivated in the conditioned medium for SMCs showed SMC-like morphology:they displayed spindle-shape in morphology and were positive for ?-SMA but negative for SMMHC.42 d and 33 d were needed to obtain 107-108 seeding cells without or with PDGF-BB/VIT-C in culture medium respectively.With the subculture,the percentage of ?-SMA positive cells increased from 57.8% at passage 5 to 66.8% at passage 6,suggesting under aforementioned cultured conditions,more BMSCs turned into SMCs.Conclusion BMSCs can be differentiated into SMCs under appropriate culture conditions,suggesting the potentiality of using these SMCs as cell sources for tissue engineering of blood vessel construction.

Objective To evaluate the performance of self‐established angiotensin converting enzyme (ACE) detection system . Methods The performance evaluation of ACE reagent kit produced by the Jiuqiang Company including precision ,linearity ,correla‐tion with the reference reagent and reportable range were conducted by using the automatic biochemical analyzer according to the re‐quirements of the EP5‐A2 and EP9‐A documentation in the Clinical and Laboratory Standards Institute (CLSI) .Results Intra‐as‐say CV of the system were 6 .87% ,2 .39% and inter‐assay CV were 6 .09% ,1 .81% ,respectively .During the day CV were 8 .00 %and 2 .8% respectively ,which were less than those provided by the manufacturer (<10% );the lenearity result was R2 =0 .99 .The correlation coefficient (r) of the system comparing with the reference reagent was 0 .990 56 ,moreover the average bias was 8 .55% , showing good correlation ;the repotable range was 9 .0U/L‐600U/L .Conclusion Self‐established ACE detection system can meet the requirements for clinical application .

This study was aimed to establish a rapid detection method for timosaponin BⅡ in Anemarrhenae Rhizoma in order to determine its concentration quickly,conveniently and efficiently.The concentration of timosaponin BⅡ in A.Rhizomadetected by HPLC in the Chinese Pharmacopeia was used as the actual measured value.The near-infrared spectroscopy (NIRS) was used to collect the spectrogram of A.Rhizomasamples.The partial least squares (PLS) of TQ Analyst 8.0 were used in the data analysis.Through the pretreatment,wavelength range and principal component number selection,the actual measured value and NIRS information were associated for the establishment of the optimal quantitative analysis model of timosaponin BⅡ.The results showed that the correlation coefficients (R2),root-mean-square error of calibration (RMSEC),root-mean-square error of prediction (RMSEP),root-mean-square error of cross-validation (RMSECV) and the performance index (PI) of the established model were 0.975 15,0.094 2,0.080 0,0.369 20,and 91.0,respectively.It was concluded that the established quantitative analysis model by NIRS with HPLC was able to determine the concentration of timosaponin BⅡ in A.Rhizomaquickly and accurately.

Objective To measure the expression of indoleamine 2, 3-dioxygenase(IDO)in condy-loma acuminatum (CA) lesions, and to evaluate its ability to locally metabolize tryptophan. Methods Immunohistochemical study was performed to observe the protein expression of IDO in skin lesions of patients with CA, and count the number of IDO-positive cells. Immunofluorescence assay was conducted to estimate the relationship between IDO-positive cells and dendritic cells. Epidermal cells and keratinocytes were isolated from warts of 30 patients with CA and prepuces of 11 healthy controls respectively, and both in vitro incubated with tryptophan solution for 4 hours. Then, high-performance liquid chromatography (HPLC)was performed to detect the level of tryptophan metabolite, kynurenine, in the culture supernatant of the above cells, which could reflect the ability of epidermal cells to metabolize tryptophan. Results Rare IDO-positive cells were found in the normal skin, but a lot of IDO-positive cells gathered in the epidermis of the wart tissues. The IDO-positive cell/total cell ratio was significantly higher in the wart tissues than in the normal skin(48.3%± 15.4%vs. 5.2%± 2.4%, P<0.05). The fluorescence signals of IDO-positive cells and CD1a-positive Langerhans cells were not overlapped with each other, suggesting that IDO-positive cells were derived from epidermal cells of the wart tissues. Compared with the keratinocytes from the healthy skin, the epidermal cells from warts had a stronger ability to metabolize tryptophan in vitro. Conclusion A large number of IDO-positive cells exist in CA warts, and may be involved in occurrence of CA.

Objective To evaluate clinical significance of two real-time fluorescence quantitative PCR kits for quantitative detection of HBV DNA and detection performance at different viral load levels.Methods A series of calibrators with different concentrations(1×106,5×105,1×105,5×104,1×104,5×103,1×103,5×102,1×102,1×101 kIU/L) were prepared with AB-type sera using the second generation WHO international standard (NIBSC code:97/750). HBV viral load in the sera of 78 patients,30 healthy blood donors and 10 calibrators were detected by real-time fluorescence quantitative PCR HBV DNA test kit from PIJI Bio-Technical Development Company Ltd (PG kit) and Cobas AmpliPrep/Cobas TaqMan HBV test kit. The correlation of the two methods was evaluated, and the performance of the two kits different viral load levels was evaluated. The false negative rate was analyzed. Negative control, low positive control and high positive control were included in every batch. Results Both two kits showed the correct results for the 10 specimens from the WHO international standards. The lowest detection limit of HBV DNA for Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit were 2.00 (kIU/L, lg) and 3.00 (kIU/L,lg) ,respectively. There was linear correlation between the results from Roche Cobas AmpliPrep/Cobas TaqMan HBV test and PG kit ( R2=0.938 7, P < 0.01 ), the upper limit of Roche kit had coincided with theoretical value. The samples with HBV DNA level above the upper limit of detection were diluted and retested to obtain the precise result. The result form Roche Cobas AmpliPrep/Cabas TaqMan HBV test [(8.35±0.20) kIU/L, lg] was higher than that from PG kit [(7.73±0.42 ) kIU/L, lg] (t=3. 776, P <0.05) . The detection of 108 serum samples showed that the level of HBV DNA detected by Roche Cobas AmpliPrep/Cobas TaqMan HBV test [(5.88±1.64) kIU/L, lg] was higher than that by PG kit [(5.25±1.55 kIU/L,lg] (t=12. 297 ,P <0.01 ). The correlation coefficients were high in samples with high HBV viral load[HBV DNA(>5.00 and≤7.00) kIU/L,Ig,R2=0. 779 7, P <0.01 ;HBV DNA( >7.00 ands≤9.00) kIU/L,lg,R2=0.603 7, P <0.01]. The correlation coefficient was low in samples with low HBV viral load[HBV DNA ( > 3.00 and≤5.00) kIU/L, lg, R2=0. 417 3, P <0.01 )]. When HBV DNA ( >3.00 and≤4.00) kIU/L,lg,the false negative rate was 33.3% (5/15). When HBV DNA ( > 1.08and≤3.00) kIU/L,lg,none of positive samples was detected with PG kit. Conclusions PG kit is not as good as Cobas AmpliPrep/Cobas TaqMan HBV test . The linear correlation between the results from the two kits is good. The correlation between the results detected with PG kit and Cobas AmpliPrep/Cobas TaqMan HBV test is higher in the high viral load groups than in the low viral load group. It is suggested that PG kit had a narrower linear range.

Objective:To explore the expression of peptidyl prolyl cis-trans isomerase (Pin1) activity in peripheral blood of patients with rheumatoid arthritis (RA) and the value and correlation of T helper cell 17/regulatory cells (Th17/Treg) cells, and to analyze the effect and influence of all-transretinoic acid (ATRA) on it.Methods:① Comparing the difference of Pin1 expression and absolute counts of Th17 and Treg between RA patients before and after treatment and healthy control group, Kruskal-Wallis rank sum test was used for analysis. ② To analyze the correlation between the expression of Pin1 and its general data, activity indicators [such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and disease activity score 28 joints (DAS28) scores], Th17, Treg and some cytokines in RA patients, and to use Pearson and Spearman correlation tests. ③ To analyze the difference of Pin1 expression and Th17/Treg in peripheral blood of RA patients treated with low-dose all-trans retinoic acid (10 mg twice a week) and traditional immunosuppressants such as hydroxychloroquine for 3 months respectively. Mann-Whitney U test was used for comparison between the two groups, and the difference was statistically significant with ( P<0.05). Results:① The activity of Pin1 in peripheral blood of the newly treated group of RA was [13.62(9.16, 19.42)] higher than that of the healthy control group [8.97(7.62, 11.45)]( Z=42.82 , P<0.05), and Th17 was [18.28(12.76, 24.08)] higher than that of the healthy control group [6.04(4.96, 4.96)]( Z=48.83 , P<0.05). Treg [11.06(5.31, 21.87). It was lower than that of healthy control group [40.41(24.33, 48.52)]( Z=42.21 , P<0.05). ② the activity of Pin1 in peripheral blood of RA patients was positively correlated with CRP, the number of involved joints, DAS28 score, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) ( r=0.396, P<0.05; r=0.683, P<0.05; r=0.466, P<0.05; r=0.315, P<0.05; r=0.416, P<0.05). ③ Compared with the newly treated RA group, the activity of Pin1 [6.94(5.96, 8.77), Z=42.82 , P<0.05] and Th17 7.38 decreased [7.38(3.85, 11.21), Z=48.83 , P<0.05], while Treg [40.41 (17.77, 33.47)] increased ( Z=42.21 , P<0.05). ④ Compared with the traditional medicine group, Treg [28.9(21.73, 37.36)] was higher in the retinoic acid group, and the difference was statistically significant ( Z=-2.683 , P<0.05). The activity of Pin1 was [6.23(5.58, 8.75)], but there was no statistical significance ( Z=-1.622 , P=0.104). Conclusion:Pin1 in peripheral blood of RA patients is over-expressed. Th17 is increased and Treg is decreased. ATRA combined with other traditional drugs can reduce Pin1 activity, promote Treg growth and improve disease activity of RApatients to a certain extent.

Objective To determine the distribution of HCV genotypes in patients with chronic hepatitis C,study the distribution of genotype in different gender and the relationship between genotypes and serum HCV-RNA levels.Methods Two hundred and six cases of HCV RNA positive patients(all with relevant clinical data) receiving pegylated interferon therapy were collected from May to December 2010.HCV RNA was detected in 206 hepatitis C patients from 40 hospitals in China by Roche Cobas AmpliPrep/Cobas TaqMan HBV test,and genotype was determined by Abbott RealTime HCV G enotype Ⅱ .The distribution of genotypes in the gender was analyzed by chi-square test analysis.The relationship between genotypes and serum HCV RNA levels was detected by single factor analysis and two independent sample t test analysis.Results There were seven different subtypes of HCV in 206 samples,including genotype 1,7 cases(3.4% ,7/206); genotype 1a,2 cases(1.0%,2/206); genotype 1b,123 cases (59.7 %,123/206); genotype 2,32 cases(15.5 %,32/206); genotype 3,27 cases(13.1%,27/206); genotype 6,6 cases(2.9% ,6/206) ;genotype 1/6,5 cases(2.4% ,5/206) ;genotype 2/4,1 cases(0.5%,1/206).There was no significant difference between HCV genotype and gender in 132 cases with genotype 1 and 65 cases with non-genotype 1(genotype 2,3,6) (x2 = 0.000,P ＞ 0.05).There was significant association between quantity of HCV RNA and genotype in 188 patients with HCV(F = 3.371,P＜ 0.01).The 197 patients with HCV single genotype were divided into five groups in terms of region(East,South,West,North and Center).There was no significant difference between HCV genotype 1 and non-genotype 1 in the five groups(x2 = 5.840,P ＞ 0.05).Conclusions It is suggested that HCV 1 b is the most prevalent type in China,followed by HCV 2.There is no significant difference between HCV genotype and gender.The levels of HCV RNA with genotype 1b are significantly higher than those with genotype 3.The levels of HCV RNA with genotype 2 are significantly higher than those with genotype 3.The levels of HCV RNA with genotype 6 are significantly higher than those with genotype 3.

Objective To study the proliferation inhibition effect by silencing PLCε gene expression with RNA interference in BIU-87 cells. Methods The specific short hairpin RNA recombinant plasmids were constructed by gene clone technology.The expression level of PLCε protein and mRNA were detected by Western blot and RT-PCR respectively after transfected recombinant plasmids into BIU-87 cells.The influence on proliferation was check by MTT.The changes of proliferating cell nuclear antigen(PCNA)were analyzed by immunocytochemical method,and the distribution of cell cycle was analyzed using flow cytometry. Results After transfected with the specific recombinant plasmids,PCNA expression was decreased 33.08%,and the analysis of cell cycle indicated that cells of G0/G1 phase were increased comparision with(40.75±2.30)%and(40.00±1.76)0A,and its G2/M phase cells(8.16±0.51)%were decreased strikingly compared with group control(31.20±1.76)%and group NP(35.94±1.58)%.Cells were blocked at G0/G1 phase,the cell proliferation was inhibited obviously. Conclusion PLCε may play an important role in proliferation of bladder cancer cells,which could be a potential target of biological treatment on bladder cancer in the future.