Objective To study the antigenicity of RNA polymerase and the relationship between the protein and the SARS-CoV replication. Methods The fragment was amplified by PCR, ligated with the prokaryotic expression vector pET32a+ and transformed into E. coli BL-21 .The expressed fusion protein identified by Western-blot and ELISA was used to detect the anti-SARS CoV IgG in different sera. Results The fusion protein was expressed successfully in E. coli BL-21. Detected by ELISA, the positive percentage of the anti-SARS IgG in the healthy donors, patients, infected animals and the rhesus administrated with the inactivated-virus were 0%, 95%, 100% and 0%, respectively. Conclusion Good antigenicity was shown in the expressed RNA polymerase. It can be used to diagnose the infection and to demonstrate the replication of SARS-CoV virus.

BACKGROUND:Autologous conditioned serum (ACS) is a new and safe biological therapy method mainly used for osteoarthritis, rheumatoid arthritis and other similar diseases. OBJECTIVE:To explore the purification method and cytokine profile of ACS. METHODS:Blood obtained equipment (containing 3 mm of glass beads) was used to collect peripheral blood samples that were mixed uniformly and then placed in a 37℃ thermostatic incubator for 24 hours, finally filtered to obtain ACS. The quantitative determination of interleukin (IL)-1Ra, IL-6, IL-8, and IL-1β was conducted, and the 36 kinds of cytokines were semi-quantitatively detected by cytokine antibody array. RESULTS AND CONCLUSION: The levels of various cytokines in ACS were obviously increased, among which, IL-1Ra level (important for alleviating osteoarthritis) was increased from 140.25 ng/L to 1125 ng/L(increased by above 8 times); IL-6 level was increased from 389.5 ng/L to 2802 ng/L; IL-8 level was not detected firstly and finally increased to 2822 ng/L. Cytokine antibody array results showed that at least 10 kinds of cytokines in ACS were changed, and most of cytokines were on a rise, and few in decline. These results indicate that the ACS contains IL-1Ra and a variety of cytokines, and some chemokines in the ACS recruit more immune cells to the inflammation sites and accelerate the inflammatory process, further exerting therapeutic effects.

Objective To investigate the regulatory effects of icariin (ICA) combined with the Panax notoginseng saponins (PNS) on immunological function in mice and provide some experimental evidences for the combination mechanism improving the spatial learning and memory abilities of Alzheimer's disease (AD) animal model. Methods Based on serum pharmacological method, the ICR mice were individually ig administrated with ICA+PNS [(40+320), (80+640), and (160+ 1 280 ) mg/kg] or ICA (80 mg/kg), and PNS (640 mg/kg) only for 7 d. Drug-containing serum was prepared and effects on spleen lymphocyte proliferation of Bable/c mice induced by concanavalin-A (ConA) or lipopolysaccharide (LPS) and on interleukin-2 (IL-2) secretion were observed in vitro. Meanwhile, the immunological organ indexes of treated mice were evaluated. Results Drug-containing serum of ICA+PNS [(80+640) and (160+ 1 280 ) mg/kg] could improve the spleen lymphocyte proliferation induced by ConA (P0.05). Drug-containing serum of ICA+PNS [(80+640) and (160+ 1 280) mg/kg] could improve IL-2 production (P0.05), respectively. Conclusion ICA combined with PNS could improve immunological function selectively and promote T cell function in mice.

Objective To observe the therapeutic efficacy and safety of the glucagon-like peptide-1 ( GLP-1 )analogue combined with isulin for treating obese or overweight type 2 diabetes.Methods 40 cases with obese or overweight type 2 diabetic patients( body mass index(BMI) ≥24) who have previously used human insulin,and experienced poor glycemic control( ≥7.5％ ) were selected.They were randomly divided into two groups.A group treated by GLP-1 analogue liraglutide plus insulin,and B group by continuous adjustment of insulin.12 weeks later,fasting blood glucose(FBG),2-hour plasma glucose (2hPG),hemoglobin A 1 C (HbA1c),body weight were observed and the incidence of hypoglycemia,insulin dosage were recorded.Results Weight and insulin dosage of group A was significantly lower than those of group B after treatment 12 week( t =2.738,2.865,all P ＜ 0.01 ).Numbers of hypoglycemia events were 6 in group A(30％ ),and 9 in group B(45％ ),but there was no statistical significance between the two groups ( P ＞ 0.05).Conclusion The addition of liraglutide to insulin in patients with obese or overweight type 2 diabetes is associated with reductions in weight and insulin dosage,without increase risk of hypoglycemia.This treatment proved effective and safe.

Objective To investigate the effects of methylthioadenosine phosphorylase (MTAP) on invasion and migration in breast cancer cells.Methods Human breast cancer cell line MCF-7 cells were treated with MTAPtargeted siRNA to diminish MTAP mRNA.MCF-7 cells proliferation was evaluated by cell counting kit-8,the analysis of cells invasion and migration was performed using Transwell chamber.The expressions of MTAP and matrix metalloproteinase 1 (MMP1) in cell extracts were detected by Western blotting.The experimental divided into blank contrd group,negative control group,MTAP-siRNA experimental group.Results The MCF-7 cells growth was promoted after knockdown the MTAP mRNA.MATP-siRNA experimental group 450 nm absorbance values at 24h,48 hand72 h of the control group were (112.3±11.9)％,(144.4±8.4)％,(169.3±9.4)％ respectively.Cell invasion analysis by Transwell chamber showed 570 nm absorbance values were 0.49 ± 0.06 (control),0.45 ± 0.07 (negative control) and 0.87 ± 0.07 (MTAP-siRNA) respectively.Cell migration analysis by Transwell chamber showed 570 nm absorbance values were 0.46 ± 0.06 (control),0.49 ± 0.08 (negative control)and 0.75 ± 0.07 (MTAP-siRNA) respectively.The expression of MMP1 in MCF-7 cells was upregulated after knockdown the MTAP mRNA.Conclusion The knockdown of MTAP in MCF-7 cell can increase the cells invasion and migration,and this may involve the the MMP1.

Objective To improve the canines myocardial infarction curative effect by using ultrasound targeted microbubble destruction (UTMD) combining with nuclear localization signal(NLS) peptide to increase hAng-1 gene transfection efficiency.Methods Forty-six canines were randomly divided into 4 groups (n=10 in each group) after the models of myocardial infarction were prepared.Group A were untreated control;Group B were transfected with hAng-1;Group C were transfected with UTMD+hAng-1;Group D were transfected with UTMD+NLS nuclear localization signal+hAng-1.The therapeutic agents were intravenously injected at one week after myocardial infarction in each group,and the ultrasound were irradiated at the precardium in group C and D.①Echocardiography was used before and at one week after myocardial infarction and 28 days after gene transfection.Two-dimensional echocardiography was used to detect cardiac function,the left ventricular ejection fraction,the left ventricular wall motion and the myocardial contrast echocardiography were used to detect myocardial perfusion of all canines in the four groups.②On twenty-eight days after gene transfection,mRNA and Western Blot were used to detect the expression of hAng-1.Immunohistochemistry was used to detect capillary density of peri-infarct area and microvessel density (MVD).Masson′s trichromatic staining and gross specimen were used to evaluate the degree and the area of myocardial fibrosis.The gene transfection efficiency and the curative effect of all the four groups were evaluated and compared.Results ①Before myocardial infarction,in four groups canine ventricular wall motion and cardiac function were normal,and myocardial filling defect was not showed by myocardial contrast echocardiography.At one week after myocardial infarction,the left ventricular anterior and interval walls motion and the left ventricular ejection fraction in the four groups were significantly decreased.Myocardial contrast echocardiography showed anterior and interval walls myocardial filling defect.There was no significant difference among the four groups(P>0.05);On 28 days after gene transfection the left ventricular ejection fraction in the four group were increased in an order of group A,B,C,D,there was significant difference when comparing group C and D with other groups separately(P<0.05).Myocardial contrast echocardiography showed much contrast filling in the infarction and surrounding area in group D,a little contrast filling in group C and filling defect in group A and group B,there was significant difference when comparing group C and D with other groups separately(P<0.05).②RT-PCR and Western Blot showed the hAng-1 mRNA and protein expression in group D were higher than those in the other group.There was significant difference when comparing group C and group D with other groups separately(P<0.05).③Immunofluorescence showed the capillary densities were(4.7±1.6)/mm2,(11.2±2.8)/mm2,(70.0±6.4)/mm2 and (85.3±7.0)/mm2 in group A,group B,group C and group D.The differences were statistically significant compared group C and group D with other groups (P<0.05).④Masson′s trichromatic staining and cardiac gross specimen showed that the degree and area of myocardial fibrosis were gradually reduced in an order of group A,B,C,D.Conclusions UTMD and NLS peptide could effectively transfect hAng-1 gene and it provided a novel strategy of gene treatment for ischemic heart disease.

BACKGROUND:Procolagen type 1 N-terminal propeptide (P1NP) and β-colagen special sequence(β-CrossLaps) are two bone metabolic markers that are closely related to osteoporosis. Combined detection of bone metabolic markers and bone mineral density is of clinical significance for the diagnosis of osteoporosis. Bone metabolic markers are ideal indicators to predict fractures, which can compensate for the lack of bone density test. OBJECTIVE:To introduce the application of bone metabolic markers in the monitoring of drug efficacy on the treatment of osteoporosis as wel as in the prediction of fracture risks in recent 20 years and to explore the clinical values of P1NP and β-CrossLaps to assess the therapeutic efficacy on osteoporosis and risks for osteoporotic fractures. METHODS:A computer-based search of CNKI and SCI databases were performed for relevant articles published from 2000 to 2014 using the keywords of “serum bone metabolic markers; osteoporosis; bone mineral density” in Chinese and English, respectively. Finaly, 44 articles meeting the inclusive criteria were reviewed. RESULTS AND CONCLUSION:This paper analyzes the source and detection mechanisms of P1NPand β-CrossLaps and then compares their advantages in the therapeutic effect assessment of osteoporosis. Serum bone metabolic markers cannot only reflect the dynamic changes of bone metabolism, but also have earlier changes than the bone mineral density. Both P1NPand β-CrossLaps are very important for assessing the early diagnosis of osteoporosis as wel as anti-osteoporosis drug efficacy.

Objective To evaluate the application of real-time three-dimensional transesophageal echocardiography (RT-3D TEE)in left atrial appendage (LAA)closure.Methods After excluding valvular disease,21 of 36 atrial fibrillation (AF)patients suffered the percutaneous LAA closure were chosen to measure left ventricular ejection fraction (LVEF)and to observe if the thrombus or the cloudiness echo will occur.The multiple planner reconstruction function was applied to rebuild sections and to observe the LAA anatomical morphology and its internal structure.During operation,the Flexi Slice function was used to measure the dimensions of LAA ostium,RT-3D mode was used to monitor sheath transport and closure device release,and to immediately evaluate the effect of blocking and complications after the operation. Results The exam before the closure procedure showed 5 of 36 patients' left ventricular ejection fraction (LVEF)<40% and one or more thrombus or the cloudiness echo were found in other 10 patients' which were thus excluded from the study.RT-3D TEE examination in the 21 patients revealed 8 single-lobe cases, 8 double-lobes cases and 5 patients with multi-lobe.Two patients of them need a special device.The maximum dimensions of LAA ostium was (22.24±4.35)mm,the fixed plate size of conventional LAmbreTM device was (28.26±5.23)mm.All patients choiced an average (1 .1 ±0.30)closure device and get the best sealing effect evaluated by RT-3D TEE immediately after operation,10 cases of them has about 1 -3 mm residual leakage exist around the closure.None of the patients with a new pericardial effusion and cardiac tapenade,no closure shift and embolism events.Conclusions RT-3D TEE can real-time and dynamically observe LAA anatomical morphology and its internal structure,compared with other imaging techniques, RT-3D TEE has irreplaceable advantages in the percutaneous LAA closure.

Objective To explore the clinical value of two‐dimensional transesophageal echocardiography (2D‐TEE) and real‐time three‐dimensional transesophageal echocardiography (RT3D‐TEE) for the left atrial appendage (LAA) closure procedures by the visualization of LAA shape by 2D‐TEE and RT3D‐TEE and the comparison between the measurement of LAA ostium and the sized LAmbreTM device during the procedure .Methods Forty‐one atrial fibrillation patients ,who had undergone 2D‐TEE examination at our hospital ,were enrolled in the study .At the mid‐esophageal ,dimensions of inner and outer ostium and depth of LAA were measured at the 2D‐TEE views of 0 ,45 ,90 and 135 degree respectively . RT3D‐TEE views were acquired and the maximal and the minimal dimensions of LAA inner ostium were measured .The measurement by RT3D‐TEE and 2D‐TEE were compared to find the difference and correlation .Eleven of 41 patients who have complied with the requirements for the LAA closure ,were undergone the procedures ,measured the dimension of LAA inner ostium at selective angiography intraoperative .Sizes of closure disks of the closure device and the measurement at selective angiography were recorded to compare the measurement at RT3D‐TEE and 2D‐TEE .Results Forty‐one atrial fibrillation patients were completed TEE examination successfully .Inner ostial dimension of LAA was (20.0±04.3)cm,(19.7±03.8)cm,(21.2±04.6)cm,(23.0±05.0)cmat2D‐TEEviewsof0,45,90and135 degree ,respectively .The maximum dimensions of LAA inner ostium by RT3D‐TEE was (2 4.9 ± 0 5.2)cm . At 2D‐TEE views ,the maximum dimensions of LAA inner ostium was at 135 degree ,there was a difference between it and the measurement by RT3D‐TEE ( P =0 0.12) .Monitoring by TEE ,LAA closure procedures with LAmbreTM device were successful for all 11 patients ,the landing zone by selective angiography was (2 4.9 ± 0 4.4)cm ,and the appropriate sized closure disk of the LAmbreTM device was 2 4. - 3 6. cm . Correlation between the measurements by RT3D‐TEE and selective angiography and the sized closure disk were r =0 8.16 ,P =0 0.02 and r =0 9.14 ,P =0 0.00 ,respectively .Correlation between the measurements by 2D‐TEE and selective angiography and the sized closure disk were r =0 6.93 ,P =0 0.18 and r =0 6.88 , P=0 0.19 ,respectively .Conclusions There was better correlation among the measurements by RT3D‐TEE and selective angiography and the size of closure device .Therefore ,compared to 2D‐TEE ,the guidance of RT3D‐TEE was more accurate during LAA closure procedures for LAmbreTM device selection .

Objective To explore the diagnostic value of MRI special use of breast and uhrasonography in axillary lymph node metastasis of early breast cancer.Methods Clinical data of 136 Ⅰ-Ⅲ A breast cancer patients accepted MRI examination before surgery had been retrospectively studied,analysing diagnostic value of MRI and ultrasonography in axillary lymph node metastasis of early breast cancer.Results The sensitivity,specificity,and accuracy obtained by MRI were 83.3％,88.6％ and 86.3％.And these data of ultrasonography were 73.1％,76.7％ and 75.0％.The sensitivity,specificity,and accuracy obtained MRI were better than that of ultrasonography.The sensitivity,specificity,and accuracy of ≥ 50 years old patients were 70.0％,77.8％ and 75.0％.And ＜ 50 years old patients were 85.7％,92.3％ and 88.9％.The sensitivity,specificity,and accuracy of ＜ 50 years old patients were better than ≥ 50 years old patients.Conclusions The MRI special use of breast have an important value in axillary lymph node metastasis of early breast cancer,especially to gounger than 50 years old patients.It can provide a scientific basis of the clinical accurate treatment for early breast cancer patients.