Objective To establish high performance liquid chromatography(HPLC)characteristic chromatograms of Xinqingduyin Granules(composed of Taraxaci Herba,Lonicerae Japonicae Flos,Chrysanthemi Indici Flos,etc.)and content determination of chicory acid and glycyrrhizic acid,and to optimize the preparation process of Xinqingduyin Granules.Methods Using the characteristic chromatograms of Xinqingduyin and the retention rate of chicory acid and glycyrrhizic acid as indexes,we carried out orthogonal experiment to optimize the extraction process of Xinqingduyin,and studied the concentration process.The molding process of Xinqingduyin Granules was conducted by screening the types and dosage of auxiliary materials,then three batches of pilot experiments were carried out.Results HPLC characteristic chromatograms of Xinqingshuyin Granules and the determination methods of chicory acid and glycyrrhizic acid were established.The optimal preparation technology was as follows:8 times amount of water was added,the drug was decocted for 3 times,with 1 hour per time.After the extract was concentrated under reduced pressure at 80℃,the appropriate amount of steviol glycoside and lactose was added into the extract and mixed.One-step granulation and packaging were adopted.The retention rates of chicoric acid and glycyrrhizic acid in the 3 batches of Xinqingduyin Granules,which were prepared on the pilot scale,were(54.56±1.63)%and(54.96±1.08)%,and the rate of finished product was(87.47±0.49)%,respectively.The quality is uniform,and the characteristic map of Xinqingduyin Granules showed high similarity with that of decoction prepared from the same batch of slices.Conclusion The optimized preparation technology is reasonable,feasible and reproducible.This preparation can be used to obtain the granule with similar materials of Xinqingduyin decoction.

OBJECTIVES: Hyperhomocysteinaemia (Hcy) is an independent risk factor for cardiovascular and cerebrovascular diseases. MicroRNA (miR)-18a-5p is closely related to cardiovascular diseases. This study aims to investigate the effects of miR-18a-5p on homocysteine (Hcy)-induced myocardial cells injury. METHODS: H9c2 cells were transfected with miR-18a-5p mimic/miR-18a-5p mimic negative control (NC) or combined with Hcy for intervention, and untreated cells were set as a control group. The transfection efficiency was verified by real-time RT-PCR, and cell counting kit-8 (CCK-8) assay was used to determine cell viability. Flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) levels. Western blotting was performed to measure the protein levels of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin1, p62, Bax, Bcl-2, and Notch2. Dual luciferase reporter assay was used to detect the interaction of miR-18a-5p with Notch2. RESULTS: Compared with the control, treatment with Hcy or transfection with miR-18a-5p mimic alone, or combined treatment with Hcy and miR-18a-5p mimic/miR-18a-5p mimic NC significantly reduced the H9c2 cell viability, promoted apoptosis and ROS production, up-regulated the expressions of Bax and Beclin, down-regulated the expressions of Bcl-2, p62, and Notch2, and increased the ratio of LC3-II/LC3-I (all P<0.05). Compared with the combined intervention of miR-18a-5p mimic NC and Hcy group, the above indexes were more significantly changed in the combined intervention of miR-18a-5p mimic and Hcy group, and the difference between the 2 groups was statistically significant (all P<0.05). There is a targeted binding between Notch2 and miR-18a-5p. CONCLUSIONS MiR-18a-5p could induce autophagy and apoptosis via increasing ROS production in cardiomyocytes, and aggravate Hcy-induced myocardial injury. Notch2 is a target of miR-18a-5p.
Apoptosis/genetics* ; Autophagy/genetics* ; bcl-2-Associated X Protein ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Reactive Oxygen Species ; Rats ; Animals ; Myocytes, Cardiac/drug effects* ; Homocysteine/adverse effects* ; Hyperhomocysteinemia

Objective By prokaryotic expression and purifying the human bocavirus recombinant protein VP2, to establish the indirect enzyme-linked immunoassay for detection of virus. Methods We amplified the human bocavirus recombinant protein VP2 gene fragments from WHL-1 template by PCR , and cloned into the expression vector pET28a, then conversed into the BL21 (DE3) and expressed the fusion protein detected by Western Blot detection , the obtained the antibody and detected the human bocavirus in serum in Guanghzhou area in healthy people. Results The Recombinant prokaryotic expression identified correct by double enzyme, and it could occur specific reaction with the virus positive serum. The best optimal antigen coating concentration were serum multiples and blocking BSA was 2 mg/mL , 1 ∶ 200 and 1%. The best working dilution of enzyme-labeled secondary antibody was 1 ∶ 4 000. The best working hours was 1h. This detection method had good specificity and reproducibility. The cut-off of the indirect ELISA method was 0.1 and the sensitivity and specificity of the developed ELISA method were 92% and 98% respectively. The coincidence rate of determination results by the developed kit and control kit was 97%. Conclusion The competitive ELISA established by prokaryotic expressing and purifying the human bocavirus protein VP2 protein , provides a basis in detecting the human bocavirus serum antibody.

Objective To discuss the correlation between the level of serum cystatin C and lipids in patients with system lupus erythematosus.Methods Used automatic biochemical analyzer to detect serum cystatin C (CysC),triglyceride (TG),total cholesterol (TC),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol (LDL-C)and hsCRP levels in 136 cases of SLE patients and 113 cases of healthy people.Data obtained using SPSS13.0 software to carry on sta-tistical analysis.Results Outcome of SLE patients group compared with healthy controls,hsCRP (13.5 ± 4.85 mg/L vs 2.03±0.88 mg/L),CysC (2.63±1.95 mg/L vs 0.85±0.37 mg/L),LDL-C (3.06±1.21 mmol/L vs 2.33±0.41 mmol/L),TC (5.32±2.63 mmol/L vs 4.02±1.67 mmol/L)and TG (1.92±0.83 mmol/L vs 1.44±0.8 mmol/L)were signifi-cantly higher the difference between groups was statistically significant(t=2.45~12.4,P <0.05).Compared with healthy controls,HDL-C (1.12±0.31 mmol/L vs 1.52±0.85 mmol/L)was decreased (P <0.01).In SLE patients group,the ser-um CysC level and hsCRP,TC,TG and LDL-C were positively correlated,and the level of HDL-C was negative to the level of CysC.The health control group was no significant correlation.Conclusion Serum lipid levels of SLE patients were posi-tive to the level of CysC.Suggest that joint detection of SLE patients serum CysC and blood lipids index is helpful to the di-agnosis of SLE treatment and condition monitoring.

Objective To discuss the clinical value of the expression level of acute lower respiratory tract Boka virus (HBoV)in‐fectecd children whose detection serum specific antibody .Methods 904 cases of children with acute lower respiratory tract Boka vi‐rus infection hospitalized from March 2011 to July 2014 who were selected as study objects ,serum ,sputum ,bronchoalveolar lavage fluid HBoV DNA positive were as the gold standard for diagnosis of acute lower respiratory tract infection in HBoV ,the positive serum HBoV antibody of HBoV in children with acute lower respiratory tract infection was defined as the observation group ,serum HBoV antibody negative acute lower respiratory tract infection in children with HBoV was defined as the control group ,the correla‐tion between serum HBoV antibody and acute lower respiratory tract HBoV infection children whose clinical characteristics were analyzed .Results Serum HBoV antibody in the diagnosis of acute lower respiratory tract infection of HBoV whose sensitivity ,spe‐cificity ,positive predictive value ,and negative predictive value ,accuracy of diagnosis were separately 60 .32% 、90 .25% 、31 .67% 、96 .81% 、88 .16% .In the general data ,between the observation group and the control group in gender ,age ,hospitalization time , there were no significant differences(P>0 .05) .In the clinical manifestations ,nasal congestion and runny nose ,cough ,fever ,vomi‐ting and diarrhea ,shortness of breath ,breathing difficulties whose occur rates had no significant differences between the observation group and the control group(P>0 .05) ,the incidence of wheezing of the observation group was significantly higher than that of the control group ,the difference had statistical significance (P<0 .05) .The comparison of clinical diagnosis between the observation group and the control group had no significant difference(P>0 .05) .Conclusion Serum HBoV antibody is in favor of acute lower respiratory tract infection of HBoV in the diagnosis of exclusion ,and the serum HBoV antibody positive and acute lower respiratory tract infection of HBoV have a certain relationship in children with wheezing symptoms .

Objective To evaluate the value of HbA2 level determined by capillary electrophoresis (Hb-CE)in screening and diagnosis of thalassemia.Methods HbA2 level of 249 thalassaemia carriers and 142 healthy controls confirmed by molecular biological detection were determined by Hb-CE method.The thalassaemia carrier subjects were divided into different groups and subgroups according to their results of gene detection.The sensitivity,specificity,accuracy,positive predictive value and negative predictive value for the diagnosis ofα-thalassemia,β-thalassaemia,α,β-thalassaemia were calculated under different HbA2 cut-off value.Results Mean value of HbA2 in healthy controls was (3.03±0.27)%.Mean values of HbA2 inα-thalassemia group and its subgroups of silentα-thalassemia,standardα-thalassemia and hemoglobin H disease were (2.38± 0.55)%,(2.61±0.46)%,(2.47 ± 0.32)% and (1.07 ± 0.17)%,respectively.Mean values of HbA2 inβ-thalassaemia group and itsβ0 subgroup,β+ subgroup were (5.65±0.47)%,(5.71±0.48)% and (5.56±0.43)%.Mean value of HbA2 in compoundαandβ-thalassaemia group was (5.7±0.82)%.Compared with healthy controls,HbA2 level inα-thalassemia group,silentα-thalassemia subgroup,standardα-thalassemia subgroup and hemoglobin H disease group decreased signifi-cantly (t values of 11.73,5.02,12.91 and 33.46,respectively,P<0.01).HbA2 level in hemaglobin H disease was signifi-cantly lower than silent and standardα-thalassemia subgroups (t values of 15.62 and 21.31,respectively,P<0.01),but there were no differences in HbA2 level between silent and standardα-thalassemia subgroups (t=1.50,P>0.05).HbA2 level inβ-thalassaemia group,β0 subgroup,β+ subgroup and compoundαandβ-thalassaemia group increased significantly (t values of 55.12,44.33,38.94 and 9.10,respectively,P<0.01),but there were no differences in HbA2 level betweenβ0 andβ+ subgroups (t=1.79,P>0.05).Of 249 thalassemia carriers,all 124β-thalassaemia carriers were distinguished with ele-vated HbA2 level (>3.5%)determined by Hb-CE and only 57 were distinguished from 117α-thalassemia carriers by Hb-CE.Under the cut-off value of 2.5%,the sensitivity,specificity,positive predictive value,negative predictive value and accu-racy for the diagnosis ofα-thalassemia were 48.72%,97.18%,93.44%,69.70%,75.29%,respectively.Under the cut-off value of 3.5%,they were 100.00%,98.59%,98.41%,100%,and 99.25% for the diagnosis ofβ-thalassaemia,respectively. The analysis of ROC curve showed that the optimal HbA2 cut-off values for diagnosis ofα,β-thalassaemia by capillary elec-trophoresis were 2.8% and 3.7%,respectively.Conclusion When no abnormal bands,the elevated HbA2 (>3.7% in this study)determined by Hb-CE could be used as a marker forβ-thalassaemia diagnosis,but theβ-thalassaemia co-existingα-thalassemia could not be differentiated fromβ-thalassaemia diagnosis.Decreased HbA2 level (<1.5% in this study)and HbH band could be used for the diagnosis of hemoglobin H disease.Only HbA2 determination by Hb-CE has no clinical sig-nificance for the screen and diagnosis ofα-thalassemia.

Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.

Objective To evaluate the Flavivirus specific monoclonal antibody(McAb) 2A10 as detective antibody for simultaneously identify tick borne encephalitis virus( TBEV), Japanese encephalitis virus( JEV), dengue ( DEN )-2, DEN-4 and yellow fever virus ( YFV ) by antibody microarray technique.Methods The antibody microarray was developed by spotting TBEV, JEV, DEN-2, DEN-4 and YFV specific McAb on chip as capture antibodies. After incubating with cultured viral supernatants of the above viruses, CY3 labeled detective antibody 2A10 was added to the chips. After reaction, the antibody microarray was scanned and the results were analyzed. By comparing the signal intensities of different spots on chips,the detecting titre and sensitivity of 2A10 for Flavivirus were determined, and the value of 2A10 in detection of Flavivirus was evaluated. Results The hybridization results demonstrated that the titre of 2A10 for Flavi2A10 was specific for Flavivirus and could be used as universal detective antibody for Flavivirus on antibody microarray.

Objective To evaluate the effects of recombinant human bone morphogenetic-2-polylactide sustained release nanospheres (rhBMP-2-PLA-Ns) on cultured rabbit osteoblasts in vitro. Methods Rabbit osteoblasts were cultured in vitro, and rhBMP-2-PLA-Ns was added into the medium of the third generation of rabbit osteoblasts. The expression of the proliferating cell nuclear antigen (PCNA) was examined by immunofluoreacence staining, and the formation of tuberculums observed with alizarin red staining. Western blot was used to detect the effects of rhBMP-2-PLA-Ns on the expression of vascular endothelial growth factor (VEGF), which was compared with that in rhBMP-2 group and blank group. Results There was no significant difference in the number of osteoblasts with positive PCNA expression among three groups five days later. Ten days later, the number of osteoblasts with positive PCNA expression in rhBMP-2-PLA-Ns group was significantly higher than that in rhBMP-2 group and blank group, which indicated that rhBMP-2-PLA-Ns could enhance the expression of PCNA. Compared with rhBMP-2 group and blank group, rhBMP-2-PLA-Ns could significantly enhance the formation of tuberculums, with statistical difference (P<0.05). The expression of VEGF was detected in all three groups, and the level of the VEGF expression in rhBMP-2-PLA-Ns group was significantly higher than that in the other 2 groups (P<0.05). Conclusion The biological activity of rhBMP-2-PLA-Ns is superior than that of rhBMP2, and rhBMP-2-PLA-Ns can promote the proliferation, mineralization of osteoblasts and the secretion of VEGF, which has a better prospect in facilitating traumatic bone healing.

Objective To study the effect of rhein on the process of tubular epithelial-mesenchymat transformation in kidney of diabetic rats. Methods Wistar male rats were randomly assigned to 3 groups: Control group (N group, n=12),diabetic group(D group, n=12), rhein treatment group(R group, n=12).The rats of rhein treatment group were treated with daily intragastric administration of periment. The excretion of urinary protein and serum creatine were measured. Histological changes of renal tissue were observed by HE and MASSON stain. Immunohistochemistry was performed to investigate the expression of E-cadherin, α-SMA,FN and TGF-β1 in kidney. Results Compared with the control group, the tubulointerstitial injury and the accumulation of extraeellular matrix protein in diabetic models were obvious(P＜0.01).Compared with the control group, the expression of E-cadherin was decreased significantly and the expression of α-SMA,FN and TGF-β1 was increased significantly in diabetic group. E-cadherin was negatively correlated with TGF-β1(rs=-0.60,P＜0.05),α-SMA and FN was positively correlated with TGF-β1(rs=0.88,P＜0.05;rs:0.91,P＜0.01).In comparison with diabetic group,rhein could up-regulate the expression of E-cad and down-regulate the expression of α-SMA and FN in renal tubular epithelial cells(P＜0.01).Conclusion Rhein could protect kidney by ameliorating interstitial fibrosis in diabetic rats. The mechanism may be depend on down-regulating the expression of TGF-β1 and suppressing tubular epithelial-mesenchymal transformation.