Objective To establish a method for the determination of indigo content in Qingxin Sushitie by HPLC. Methods At 30℃, HPLC was performed to determine indigo on ODS column(4.6?250 mm), the mobile phase was composed of methanol-acetonitrile-0.1mol/L ammonium acetate(60: 4: 36), flow rate was 1.0 mL/min; injection volume was 20 ?L and detection wavelength at 280 nm. Results The linearity of indigo was good within 0.44～6.60 ?g/mL (r=0.9991), the average recovery was 103.6% and RSD=1.11%. Conclusion This method is simple, sensitive and accurate, and can be used for quality control of Qingxin Sushitie.

Objective To establish a HPLC method for determining the content of paeoniflorin in Dahuang Zhechong Tablets. Methods Stationary phase was C18 column (4.6 mm?250 mm, 5 ?m), mobile phase was acetonitrice-0.1% phosphoric (14∶86), detection wavelength was 230 nm, flow rate was 1.0 mL/min and column temperature was 30 ℃. Results There was good linear relationship between the concentration of paeoniflorin and area integral value in the range of 0.10～0.90 ?g, r =0.999 7. The average recovery was 99.98% with RSD=0.81% (n =6). Conclusion This method is simple, accurate, with good reproducibility and high precision, and can be used to control the quality of Dahuang Zhechong Tablets effectively.

Objective To analyze the drug resistance status of mycobacterium tuberculosis in patients with double immunization of human immunodeficiency virus (HIV) and tuberculosis (TB) by phage bioassay (PhaB),and to optimize the control strategy.Methods One hundred and twelve cases of HIV/TB infected patients.in Chongqing Ninth People's Hospital were treated with PhaB method,and the drug susceptibility testing results were compared with 208 cases of simple pulmonary tuberculosis patients.Results The anti-tuberculosis drug resistance rate of HIV/TB patients was lower than that of simple pulmonary tuberculosis patients.The resistance rates of 5 common anti-tuberculosis drugs in HIV/TB patients were 7.14％ of isoniazid (INH),7.14％ of pyrazinamide (PZA),5.36 ％ of rifampicin(RFP) streptomycin(SM),and 4.46 ％ of ethambutol (EMB),compared with simple pulmonary tuberculosis(resistance rates of RFP were 17.31％,IN H 13.46 ％,PZA 11.54 ％,EMB 10.58 ％,SM 9.62 ％),RFP resistance rate of HIV/TB infected patients was lower(P＜0.05).There was no significant difference between two groups in the other four anti-tuberculosis drug(P＞0.05).The coincidence rate with the absolute concentration method were INH 96.4％,RFP 98.2％,PZA 96.4％,EMB 93.8％ and SM 96.4％,respectively.Conclusion The resistance rate of mycobacterium tuberculosis to RFP in patients with HIV/TB infection in this region is lower than that in patients with common pulmonary tuberculosis,which is related to the good medication compliance of these patients.PhaB has the characteristic of fast,simple,without special equipment,it can be used as a rapid screening of mycobacterium tuberculosis drug resistance method.

Objective To evaluate the value of quantitatively analyzing tissue perfusion parameters of contrast-enhanced ultrasound in the evaluation of antiangiogenic therapeutics effect. Methods Kun-min mouse were subcutaneously implanted with H22 cells. Ten mouse were treated with thalidomide (200 mg/kg once daily) by intraperitoneal injection over 7 days,starting at day 2 post-implantation. Ten control mouse were treated with an equivalent volume of 0.5 CMC. Contrast-enhanced gray-scale ultrasound was performed on day 8 after bolus injection of SonoVue (0.1 ml) and the imaging was recorded on cine.Regions of interest within tumour were analyzed off-line with the software of SonoLiver to determine the area under the curve (AUC), maximum intensity (Imax), perfusion index (PI), mean transit time (mTT),time to peak (TTP) and quality of fit (QOF). Immediately after imaging, minces were euthanized and tumour tissue removed for fixation in a 10% formalin solution. Microvascular density (MVD) was measured after anti-CD34 staining. Results The body weight and the tumor volume of treated tumors were significant different (lower) than that of control tumors (P ＜0.05). Treatment with thalidomide resulted into a significant decrease in AUC,PI and Imax in comparison with control tumors ( P ＜0.05). There were no significant difference in mTT and TTP between control and treated tumours ( P ＞0.05). Treated tumours were associated with a significantly lower MVD as compared with control tumours( P ＜0.05). ConclusionsQuantitatively analyzing tissue perfusion parameters of contrast enhanced ultrasound shows promise for monitoring tumor response to antiangiogenic therapy.

Objective:To investigate the effect of miR-204 on the proliferation and differentiation of osteoblasts in osteoporosis mice by Wnt signaling pathway and its mechanism.Methods:Female Kunming mice were divided into: control group, sham operation group and osteoporosis group. Ovariectomy mouse models were established and identified by bilateral ovariectomy; Mouse primary osteoblasts were extracted and identified; Cells was transfected and detected the miR-204 expression levels; MTT was used to detect the viability of each group of cells; Alkaline phosphatase (ALP) activity was detected in each cell group; Cell flowmetry was used to detect apoptosis in each group; Cell flowmetry was used to detect the activity of Caspase-3 in each group of cells; Interaction between miR-204, β-catenin and LRP-5 was detected by dual luciferase reporter gene. Western blot was used to detect the expression of Wnt signaling pathway-related proteins.Results:The bone mineral density of the osteoporosis group was significantly lower than that of the control group and the sham operation group ( P=0.007, P=0.057) , indicating that the osteoporosis mice were successfully modeled; The expression level of miR-204 was significantly increased in the miR-204 mimics group ( P=0.007) , and decreased in the miR-204 inhibitor group ( P=0.031) ; The activity of bone cell and ALP activity of miR-204 mimics increased ( P=0.007, P=0.043) , and the activity of bone cell and ALP decreased by miR-204 inhibitor ( P=0.007, P=0.035) ; The invasive ability of miR-204 mimics was significantly increased ( P=0.006) , and the invasive ability of miR-204 inhibitor was decreased ( P=0.036) ; The apoptosis ability and Caspase-3 activity of miR-204 mimics were decreased ( P=0.041, P=0.045) , and the apoptosis ability and Caspase-3 activity of bone cells were enhanced by miR-204 inhibitor ( P=0.005, P=0.039) ; There were targeting relationship between miR-204 and β-catenin, LRP-5. The expressions of β-catenin and LRP-5 protein in osteoblasts of miR-204 mimics were up-regulated ( P=0.043, P=0.009) , and the expression of β-catenin and LRP-5 protein in bone cells of miR-204 inhibitor was down-regulated ( P=0.041, P=0.032) . Conclusion:miR-204 maybe promote the proliferation and differentiation of osteoblasts, activate Wnt signaling pathway, and has certain protective effect on osteoporosis.

Objective:To investigate the effect and potential mechanism of serum exosome-derived cirC_0009362 on the osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) .Methods:Serum samples from patients with osteoporosis (OP) were collected and exosomes were isolated. The expression level of circ_0009362 in exosomes was detected by qRT-PCR. hBMSCs osteogenesis was induced and the expression of circ_0009362 and miR-29b-3p was detected. The interaction between circ_0009362 and miR-29b-3p was detected by dual luciferase reporter assay. Alkaline phosphatase (ALP) kit was used to detect ALP activity and alizarin red (ARS) staining was used to detect calcium deposition.Results:Compared with healthy control group, the expression of circ_0009362 in serum exosomes of OP patients was increased, and the expression of circ_0009362 was decreased after inducing hBMSCs osteogenesis (all P<0.05) . The ALP activity and the percentage of calcium deposition in hBMSCs were decreased by exosomes, and this effect was achieved by secreting circ_0009362. The effect of exosomes was partially offset by circ_0009362 expression in knockdown exosomes (all P<0.05) . The expression of miR-29b-3p was increased after inducing hBMSCs osteogenesis ( P<0.05) . Circ_0009362 had a targeting relationship with miR-29b-3p, and exosomes inhibited the expression of miR-29b-3p by secreting circ_0009362. The ALP activity and the percentage of calcium deposition in hBMSCs were promoted by overexpression of miR-29b-3p, which was partially offset by exosomes (all P<0.05) . Conclusion:Serum exosomes of OP patients inhibit the osteogenic differentiation of hBMSCs by secreting circ_0009362 to down-regulate the expression of miR-29b-3p.