Objective To construct the double hairpin siRNA and green fluorescent protein(GFP) expressing vector pU6/double-siRNA/Neo/GFP/1A/2A to interfere 1A and 2A gene of Coxsackie virus B4.Methods 21 bp fragments of the Coxsackie virus B4 2A and 1A gene were chosen as the targets and 65 bp complimentary fragments were synthesized,then the target gene fragments were cloned into pSilencer2.1U6 Neo and pGCsi-U6/Neo/GFP/siNeGative,respectively,then the double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A /2A was constructed by restrict endonuclease digestion,elctrophoresis isolation and reclaimer,ligatied by T4 DNA ligase;then the double siRNA expressing plasmid was transfected into Hela cells,and the GFP was observed under fluorescent microscope.Results The correct results showed that the recombinant plasmid had the correct special fragments and DNA sequence detected by restrict endonuclease digestion, electrophoresis and DNA sequencing;and GFP was also observed in Hela cells tansfected with pU6/double-siRNA/Neo/GFP/1A /2A under fluorescent microscope more than 15 d after transfection.Conclusion The double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A/2A is constructed successfully;it has the correct target viral gene sequences and can express GFP gene in Hela cells more than 15 d after transfection.

Objective To explore the effects of comprehensive rehabilitation on patients with stable chronic obstructive pulmonary disease (COPD) in rural communities.Methods Between June 2010 and June 2012,212 stable COPD patients were randomly divided into management group (n =107) and control group (n =105).All patients received conveutional treatment.In addition,107 stable COPD patients relying on new rural cooperative medical service station in management group underwent community comprehensive rehabilitation including training in respiratory function,exercise training,nutrition intervention and psychological care.Effect of lung function,symptoms (times of acute attack & hospitalization) and quality-of-life (QOL) rated by SF-36 questionnaire between two groups were recorded at Month 6.Results The score changes of QOL had significant inter-group differences between 2 groups for PCS (scores for physical component summary) and MCS (scores for mental component summary) (11.4 ±8.2 vs.1.6 ± 1.2,5.5 ±3.5 vs.2.2 ±0.9,all P ＜0.01).In the scores for physical component summary,the score change of physical function,physiological function,body pain and general health were significantly different between two groups (6.7 ±4.3 vs.1.2 ±0.8,10.9 ±6.3 vs.1.9 ± 1.5,6.4 ±4.7 vs.3.6 ±2.7,3.2 ±2.7 vs.1.6 ± 1.1,P ＜ 0.01).In the scores for mental component summary,the score change of vitality,emotional function,social function and mental health were significantly different between two groups (4.9±3.2 vs.1.9±1.4,2.7±2.1 vs.1.6±1.1,11.6 ±9.2 vs.3.6 ±2.3,6.7 ±4.3 vs.1.4±0.9,P＜0.01).The times of acute attack and hospitalization were obviously lower than those of the control group.No significant inter-group difference existed in lung function.Conclusion Comprehensive rehabilitation may improve the QOL in stable COPD patients in rural communities and reduce their times of acute attack.

Objective To evaluate the effectiveness of management for patients with chronic obstructive pulmonary disease (COPD) in stage Ⅰ at new rural cooperative medical facilities.Methods A quasi-experiment design was used for the evaluation of patients registering at Dongcun and Fangyuan subdistrict offices in Haiyang.And 79 COPD cases from Dongcun sub-district offices were selected into the management group while 76 cases from Fangyuan the control group.The measures at the new rural cooperative medical facilities included smoking-free publicity,avoiding a hazardous environment,application of influenza vaccines and training of patients and local general practitioners in essences of COPD prevention and treatment.Except for routine treatment,no special measures were taken for the control group.ResultsSixty-one patients in the management group and 60 in the control group completed a 3-year follow-up.Fifteen patients were smokers during their initial visits in the management group and 13 quitted smoking after a 3-year management.Meanwhile 19 patients were smokers during their initial visits in the control group and 3 of them quitted smoking within the same period.The decrement of BODE index between post-management at year 2&3 and between pre-management was higher in the management group versus the control group within the same period (P ＜ 0.01).The annual average frequencies of common cold,acute attack and physician consultation were obviously lower post-management at year 3 than those pre-management and the control group within the same period ( P ＜ 0.05 ).Lung functions of two groups deteriorated as compared with those 3 years before ( P ＜ 0.05).But no statistical difference existed between two groups (P ＞ 0.05).ConclusionThe management of COPD patients in stage Ⅰ at new rural cooperative medical facilities can improve their quality of life and reduce their frequencies of common cold,acute attack and physician consultation.

Objective Sialic acid and its derivatives in mammalian cells mainly include N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN), among which Neu5Ac can be catalyzed by cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) for the synthesis of Neu5Gc. In this study, the transcriptional level of CMAH in different tissues of BALB/c mice were determined by relative fluorescence quantitative PCR, to provide a reference for further analysis of Neu5Gc levels in different tissues. Methods The mRNA sequence of CMAH was searched in the NCBI database and specific primers were designed. The mouse β-actin was selected as an internal control, and the transcriptional levels of CMAH in 9 different organ tissues of BALB/c mice were detected by relative fluorescence quantitative PCR using SYBR Green dye. Results Among the 9 mouse organs, the transcriptional level of CMAH in the liver tissue was the highest, which was 2. 46 times higher of that in the spleen, 3. 17 times of the kidney, 5. 14 times of the trachea, 11. 70 times of the lung, 21. 12 times of the myocardium, 31. 37 times of the skeletal muscles, 66. 90 times of the small intestine and 1056. 99 times of the brain tissue, respectively. Conclusions CMAH is transcribed in many organ tissues of mice, and its transcriptional levels vary in a quite wide range.

To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED₅₀ of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.

ObjectiveTo investigate the effects of Guizhi Jia Longgu Mulitang on the expression difference of interleukin-10 （IL-10） and tumor necrosis factor -α （TNF-α） in related organs of insomnia rats with sensory dysfunction dominated by lung and study the mechanism of Guizhi Jia Longgu Mulitang in improving insomnia. MethodSD rats were randomly divided into the blank group， model group， western medicine group， and traditional Chinese medicine （TCM） group， with 10 rats in each group. The rats were deprived of sleep by shallow water environment method in a long platform， and the modeling lasted for 42 d. The blank group and model group were given 0.05 mL·kg^-1 normal saline by gavage， and the western medicine group and TCM group were given drugs during modeling. To be specific， the western medicine group was given 0.105 mg·kg^-1 dexzopiclone tablet by gavage， while the TCM group was given 7 600 mg·kg^-1 Guizhi Jia Longgu Mulitang by gavage， both lasting for 28 days. After successful modeling， the Morris water maze experiment was performed on the 42nd day to detect the motion and spatial memory ability of rats. The levels of IL-10 and TNF-α in serum were detected by enzyme-linked immunosorbent assay （ELISA）. The protein expression of IL-10 and TNF-α in the lung and brain tissue of rats was detected by Western blot. The levels of IL-10 and TNF-α in the lung and brain tissue of rats were detected by immunohistochemistry. ResultCompared with the blank group， the sleep stages non-rapid eye movement ( NREM ) and rapid eye movement ( REM ) of the model group were significantly shortened （P<0.5， P<0.01）， and the wake stage was significantly increased （P<0.01）. The total time and distance of platform exploration were significantly increased （P<0.01）. In the target quadrant （the third quadrant）， the percentage of exploration time and the times of crossing the platform were significantly decreased （P<0.01）. ELISA results showed that the serum IL-10 level was significantly decreased （P<0.01）， and TNF-α level was significantly increased （P<0.01）. The results of Western blot showed that the protein expression of IL-10 in brain and lung tissue of rats was significantly decreased （P<0.01）， and the protein expression of TNF-α was significantly increased （P<0.01）. The results of immunohistochemistry showed that the expression of IL-10 in the brain and lung tissue of rats was significantly decreased （P<0.01）， and that of TNF-α was significantly increased （P<0.01）. Compared with the model group， the NREM stage and REM stage of the western medicine group and the TCM group were significantly increased （P<0.5， P<0.01）， and the wake stage was shortened （P<0.5）. The total time and distance of platform exploration were significantly decreased （P<0.01）. In the target quadrant （the third quadrant）， the percentage of exploration time and the times of crossing the platform were significantly increased （P<0.05， P<0.01）. The serum IL-10 level was significantly increased （P<0.01）， and the serum TNF-α level was significantly decreased according to the ELISA results （P<0.01）. The results of Western blot showed that the protein expression of IL-10 in brain tissue and lung tissue was significantly increased （P<0.01）， and the protein expression of TNF-α was significantly decreased （P<0.01）. The results of immunohistochemistry showed that the expression of IL-10 in brain tissue and lung tissue was significantly increased （P<0.05， P<0.01）， and the expression of TNF-α was significantly decreased （P<0.05， P<0.01）. ConclusionGuizhi Jia Longgu Mulitang can improve the expression of IL-10 and TNF-α in brain and lung tissue of insomnia rats with sensory dysfunction dominated by lung， prolong sleep time， and then improve insomnia. The mechanism may be related to improving the expression level of inflammatory factors.

Irinotecan is an anticancer topoisomerase I inhibitor that acts as a prodrug of the active metabolite, SN-38. Unfortunately, the limited utility of irinotecan is attributed to its pH-dependent stability, short half-life and dose-limiting toxicity. To address this problem, a novel trivalent PEGylated prodrug (PEG-[Irinotecan]₃) has been synthesized and its full-profile pharmacokinetics, antitumor activity and toxicity compared with those of irinotecan. The results show that after intravenous administration to rats, PEG-[Irinotecan]₃ undergoes stepwise loss of irinotecan to form PEG-[Irinotecan]_3‒x (x = 1,2) and PEG-[linker] during which time the released irinotecan undergoes conversion to SN-38. As compared with conventional irinotecan, PEG-[Irinotecan]₃ displays extended release of irinotecan and efficient formation of SN-38 with significantly improved AUC and half-life. In a colorectal cancer-bearing model in nude mice, the tumor concentrations of irinotecan and SN-38 produced by PEG-[Irinotecan]₃ were respectively 86.2 and 2293 times higher at 48 h than produced by irinotecan. In summary, PEG-[Irinotecan]₃ displays superior pharmacokinetic characteristics and antitumor activity with lower toxicity than irinotecan. This supports the view that PEG-[Irinotecan]₃ is a superior anticancer drug to irinotecan and it has entered the phase II trial stage.

BACKGROUNDThis multicenter prospective study aimed to assess the utility of dynamic enhanced magnetic resonance imaging (MRI) prior to breast-conserving surgery for breast cancer.

METHODSThe research subjects were drawn from patients with primary early resectable breast cancer treated in the breast disease centers of six three-level hospitals in Beijing from 1 January 2010 to 31 December 2012. The participants were allocated to a breast-conserving surgery group (breast-conserving group) or a total mastectomy group (total mastectomy group). Enhanced MRI was used to measure breast volume, longest diameter of tumor and tumor volume. The correlations between these measurements and those derived from histopathologic findings were assessed. The relationships between the success rate of breast-conserving surgery and MRI- and pathology-based measurement results were statistically analyzed in the breast-conserving group.

RESULTSThe study included 461 cases in the total mastectomy group and 195 in the breast-conserving group. Allocation to these groups was based on clinical indications and patient preferences. The cut-off for concurrence between MRI- and pathology-based measurements of the longest diameter of tumor was set at 0.3 cm. In the total mastectomy group, the confidence interval for 95% concurrence of these measurements was 35.41%-44.63%. Correlation coefficients for MRI and histopathology-based measurements of breast volume, tumor volume and tumor volume/breast volume ratio were r = 0.861, 0.569, and 0.600, respectively (all P < 0.001). In the breast-conserving group, with 0.30 cm taken as the cut-off for concurrence, the 95% confidence interval for MRI and pathology-based measurements of the longest diameter of tumor was 29.98%-44.01%. The subjective and objective success rates for breast-conserving surgery were 100% and 88.54%, respectively.

CONCLUSIONSThere were significant correlations between dynamic enhanced MRI- and histopathology-based measurements of the longest diameter of breast lesions, breast and tumor volumes, and breast volume/tumor volume ratios. Preoperative MRI examination improves the success rate of breast-conserving surgery.

Adult ; Breast Neoplasms ; surgery ; Female ; Humans ; Magnetic Resonance Imaging ; methods ; Middle Aged ; Prospective Studies