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2017 Vol. 47, No. 1 Published: 2017-01-25   1 Prokaryotic expression of recombinant PE38KDEL protein and preparation of its polyclonal antibody CEN Danwei, SONG Yiling, ZHANG Chanqiong, MAO Shanshan, YE Xiaoxian, CHEN Jun, CHEN Shao, ZHU Shanli, ZHANG Lifang.? Objective: To prepare PE38KDEL recombinant protein from Pseudomonas exotoxin A in prokaryotic expression system and prepare the PE38KDEL specific polyclonal antibody. Methods: The C-terminal 609-613 amine acid REDLK from Pseudomonas aeruginosa exotoxin (PE) genes (PE38) was mutanted to KDEL. After prokaryotic codon optimization (http://www.jcat.de), the full gene sequences were synthesized and then cloned into expression vector pET32a (+) prokaryotic expression vector inserted into Hind III and Xho I and constructed the recombinant plasmid pET32a (+)/PE38KDEL. After sequenced, the plasmid was transformed into E.coli BL21 (DE3) and induced by isopropyl beta-D-1-thiogalactoside (IPTG) to get the PE38KDEL protein. The recombinant protein was purified by Ni-NTA affinity chromatography, SDS-PAGE and Western blot were used to identify it. Then, the rabbits were immunized by the PE38KDEL purified protein. The sera were collected before and after immunized. The antibody reaction and titer were detected by ELISA. Results: The pET32a (+)/PE38KDEL recombinant plasmid was successfully constructed and the protein was expressed and purified in the prokaryotic expression system. The protein molecular weight was about 57 kDa which was in accordance with the expected theoretical value. The PE38KDEL polyclonal antibody was obtained by immunization of rabbits and the titer of it was as high as 1:60 000. Conclusion: The PE38KDEL recombinant protein can induce the rabbit to produce serum polyclonal antibody which has high titer and strong specificity and can be used for further study which laid a solid foundation on the biology and immunology function. 2017 Vol. 47 (1): 1-6 [Abstract] ( 754 ) HTML (1 KB)  PDF (9854 KB)  ( 685 ) 7 Effects of platelet-rich plasma on proliferation and adipogenic differentiation of adipose-derived stem cells in vitro LI Feng, HUANG Shengbin, ZHAO Shupan, Deng Hui. Objective: To evaluate the effects of platelet-rich plasma (PRP) on the proliferation and adipogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Methods: ASCs were isolated and cultured from human free fat tissue. The surface molecular markers of ASCs were determined with flow cytometry. PRP was prepared from human whole blood. Cell count kit-8 (CCK-8) and reverse transcription polymerase chain reaction (RT-PCR) were used to evaluate the influence of PRP (0, 10%, 20%, and 30%) in medium on ASC proliferation and adipogenic differentiation, respectively. Results: The platelet count in PRP was over 5 times of that in whole blood. CCK-8 showed that medium supplemented with PRP promoted ASCs proliferation significantly and the stimulatory potency of 20% PRP was the greatest. RT-PCR detected upregulated adipogenic-related genes, such as peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL) and adipophilin. Conclusion: These results indicates PRP facilitated the proliferation and adipogenic differentiation of ASCs, which is of great value for adipose tissue engineering. 2017 Vol. 47 (1): 7-13 [Abstract] ( 659 ) HTML (1 KB)  PDF (3123 KB)  ( 1039 ) 14 The role of Wnt7b/β-catenin signal pathway in the course of airway remodeling of asthma rats  ZHU Tingting, WENG Cuiye, JIA Xiaoxiao, ZENG Zeyu, WANG Lei, LI Changchong, ZHANG Weixi. Objective: To explore the role of Wnt7b/β-catenin signaling pathway in airway remodeling in asthma. Methods: This study enrolled 16 8-week-old healthy male Sprague-Dawley (SD) rats (SPF grade), weighting from 180-200 g, without specific pathogens, and assigned them into asthma group and normal control group. Asthmatic airway remodeling models were established in rats with ovalbumin while the normal control group was given spray of physiological saline. HE staining was used to observe pulmonary pathological structure of two groups of rats, and color medical image analysis was used to determine the thickness of bronchial wall (Wat) and the thickness of smooth muscles (Wam). Immunohistochemical method was used to detect the distribution and protein expression of Wnt7b, β-catenin and c-Myc in the lung tissues, qRT-PCR was used to determine the mRNA expressions of Wnt7b, β-catenin and c-Myc. Results: Wat and Wam of asthma group were significant higher than those in normal group. The protein expression of Wnt7b, β-catenin, c-Myc detected by IHC in asthma group were significant higher than those in normal group (P<0.01, respectively). The mRNA expression of Wnt7b, β-catenin, c-Myc detected by qRT-PCR in asthma group were significant higher than those in normal group (P<0.01, respectively). There were significantly positive correlation between Wat and the protein expression of Wnt7b, β-catenin and c-Myc, also between Wam and the protein expression of Wnt7b, β-catenin and c-Myc. Conclusion: Wnt7b, β-catenin and c-Myc play important roles in airway remodeling in asthma. 2017 Vol. 47 (1): 14-18 [Abstract] ( 777 ) HTML (1 KB)  PDF (2149 KB)  ( 924 ) 19 Correlation of oxidized low-density lipoproteins with coronary artery lesion in children with Kawasaki disease HE Yuee, CHU Maoping, ZHANG Yuanhai, WU Rongzhou, XIANG Rulian. Objective: To observe the expression of plasma oxidized low-density lipoprotein (oxLDL) in children with Kawasaki disease and to analyze the correlation of oxLDL with coronary artery lesions. Methods: Sixty cases diagnosed as Kawasaki disease (KD group) were divided into coronary artery lesion group (CAL group) and non-coronary artery lesion group (NCAL group) based on two-dimensional echocardiography. The venous blood was collected in acute stage and sub-acute stage (one week after the use of intravenous immunoglobulin). Twenty healthy controls and twenty fever control were enrolled into the study, and their venous blood was collected. Oxidized low- density liprproteins (oxLDL) was measured with enzyme linked immunosorbent assay (ELISA). Results: Compared with normal control group and fever control group, plasma oxLDL levels were higher in children with Kawasaki disease, whether acute or sub-acute, the difference was statistically significant (P<0.05). After the use of intravenous immunoglobulin plasma oxLDL levels have decreased, but the difference was not statistically significant compared with the acute stage (P>0.05). Compared with non-coronary artery lesion group, plasma oxLDL levels were significantly higher in coronary artery lesion group (P<0.05). Conclusion: Oxidized LDL plays an important role in the coronary artery sustained damage, the high-dose intravenous immunoglobulin can not completely relieve endothelial dysfunction in Kawasaki disease. 2017 Vol. 47 (1): 19-22 [Abstract] ( 759 ) HTML (1 KB)  PDF (1765 KB)  ( 868 ) 23 Application of the knee joint control training based on Bobath concept framework in rehabilitation of spastic hemiplegia patients FAN Liubo, JIANG Yiqing, LIU Baohua, HAN Wensheng, LIU Suzhi, WANG Lingling, LU Zhan. Objective: To observe the effects of the knee joint control training based on Bobath concept framework intervening on motor control of spastic hemiplegia patients by analyzing the sEMG signals. Methods: Forty-six patients with spastic hemiplegia from July 2013 to December 2014 in Taizhou Hospital Affiliated to Wenzhou Medical University were randomly divided into two groups. There were 23 patients in observation group which were treated by the traditional rehabilitation training and control training of knee joint, and 23 patients in control group which were treated only by the traditional rehabilitation therapy training. Modified Ashworth scale (MAS), clinic spasticity index (CSI) and Fugl-Meyer assessment of lower extremity (FMA-LE) were used as outcome measure pre-training and post-training after 6 weeks by two appointed raters. The electromyography signals were collected by surface electrode and then processed by linear time-and frequency-domain method. Real-time shear wave ultrasound elasticity imaging of vastus lateralis, vastus medialis, rectus femoris and biceps femoris in longitudinal section was performed in therapy group and control group, then the results were compared and analyzed. Results: After treatment for 6 week. Lower FAM-LE scores in both groups were significantly increased, and the lower extremity MAS and CSI in both groups were decreased significantly as compared with those before treatment (P<0.05). More observation in observation group (P<0.05). Longitudinal Young’s modulus values of the vastus lateralis, vastus medialis, rectus femoris and biceps femoris of therapy group were lower than those of control group (P<0.01). Cocontraction ratio were decreased before treatment during knee maximum isometric voluntary contraction (P<0.05), and observation group were more apparently than control group (P<0.05). Conclusion: The knee joint control training based on Bobath concept framework can relieve the muscle tonus, effectively improve stroke patients lower limb function ,and improve the quality of life of spastic hemiplegia patients. 2017 Vol. 47 (1): 23-28 [Abstract] ( 819 ) HTML (1 KB)  PDF (2858 KB)  ( 1300 ) 29 Effect of imiquimod on cervical condyloma acuminate accompanied with high-risk HPV infections and the content of HPV DNA YU Jianqin, HU Yan, ZHOU Qiangyong, XU Yunsheng Objective: To explore the effect of imiquimod on cervical condyloma acuminate accompanied with high-risk HPV infections, and the content of HPV-DNA. Methods: One hundred and twenty female patients with cervical condyloma acuminate and high-risk HPV infections were randomly divided into treatment goup and control group, and each group was administered either 5% imiquimod local treatment or high frequency cauterization. The dropping of verrucous, wound healing, recurrence and adverse reactions between the two groups were analyzed. HPV gene sequencing was conducted, and the viral load of HPV-DNA was compared before and after treatment between the two groups. Results: At 4, 8, 12 weeks after treatment, the clearance rate of verrucous were 25.0% (13/52), 65.4% (34/52), and 84.6% (44/52) in the treatment group, respectively. Meanwhile, the clearance rate of verrucous were 30.9% (17/55), 70.9% (39/55), and 74.5% (41/55), respectively. There were no significant differences between two groups (P>0.05). The recurrence rate was 10.2% (5/52) in the treatment group, which was significantly lower compared to the control group (P<0.05). HPV gene sequencing showed that HPV type 16 was most common in cervical condyloma acuminate in our hospital, followed by type 52 and 58. After 3 and 6 months follow-up, HPV negative conversion rate in treatment group was 28.2% (15/52) and 59.6% (31/52), and statistically significant over that of 12.7% (7/55) and 27.3% (15/55) in the control group. Besides, the viral load of HPV-DNA in the treatment group was significantly lower compared to the control group (P<0.05). Conclusion: 5% imiquimod can remove verrucous of cervical condyloma acuminate and additionally prevent recurrence and cure high-risk HPV. This result shows that 5% imiquimod is effective in the treatment and the prevention of relapse cervical condyloma acuminate. 2017 Vol. 47 (1): 29-32 [Abstract] ( 910 ) HTML (1 KB)  PDF (2705 KB)  ( 1434 ) 33 Effects of epirubicin combined with cyclophosphamide on tumor formation and caveolin-1 expression in MDA-MB-231 breast cancer cells LI Jinhai, CHEN Huichun, ZHANG Haifeng, ZHAI Huawei, LIN Suifang, DAI Huawei. Objective: To investigate the effects of epirubicin combined with cyclophosphamide on tumor formation and the expression of caveolin-1 in MDA-MB-231 breast cancer cells. Methods: Human breast cancer xenografts in nude mice were established by using MDA-MB-231 cells. Epirubicin group, cyclophosphamide group, and epirubicin+cyclophosphamide group, and saline control group were set up. By using immunohistochemistry the expression of caveolin-1 was detected in xenografts. Image signal acquisition and analysis system was applied for image scanning analysis. Results: The rate of tumor suppressor in epirubicin group, cyclophosphamide group and epirubicin+cyclophosphamide group was 31.28%, 33.42% and 66.14% respectively. Immunohistochemistry demonstrated that the caveolin-1 expression in epirubicin+cyclophosphamide group (253.22±38.52) was significantly increased compared with saline control group (31.73±2.28), epirubicin group (157.52±32.69) and cyclophosphamide group (148.23±41.66) (P<0.05). Conclusion: Epirubicin and cyclophosphamide can inhibit the growth of MDA-MB-231 cells with a synergic effect. The caveolin-1 expression is increased significantly in epirubicin+cyclophosphamide group, indicating caveolin-1 protein may play an important role in tumor cells growth. 2017 Vol. 47 (1): 33-36 [Abstract] ( 873 ) HTML (1 KB)  PDF (2040 KB)  ( 1489 ) 37 The effect of different positions of injector catheter tip on carbon dioxide elimination during high-conventional frequency jet ventilation  ZHOU Yamei, LIU Le, CHEN Limei, XU Xuzhong. Objective: To compare the effect of carbon dioxide elimination during high-conventional frequency jet ventilation with different injector positions in trachea. Methods: Twenty patients undergoing scheduled surgery were selected in this study, with exclusion of thoracic or laparoscopic surgery. High-conventional frequency jet ventilation was applied after general anesthesia. The study objects were randomly divided into two groups named group A and group B by crossover design. In group A, the injection catheter tip was first placed in the endotracheal tube about 3 cm above carina for 40 minutes applying high-conventional frequency jet ventilation, then the injection catheter tip was placed proximal to vocal cord level for another 40 minutes for the same. After that, IPPV was carried out to the end of the surgery. In group B, the only difference is that the injection catheter tip placement sequence was opposite to group A. PETCO2, PIP, MAP, HR, SpO2 and the results of blood gas analysis were recorded at three defined time points including the time before jet ventilation (T0), 20 minutes after jet ventilation (T1), 40 minutes after jet ventilation (T2). Results: The value of PETCO2 at T2 and T1 was significantly higher than that at T0 (P<0.05), and the value at T2 was significantly higher than that at T1 (P<0.05) when the injector head was placed about 3 cm above carina. While the value of PETCO2 at T2 and T1 was significantly lower than that at T0 (P<0.05) when the injector tip was located proximal to the vocal cord level. Placement sequences of injection catheter tip in these two groups made no statistical differences in values of d-PaCO2 (P>0.05), d-PaO2 (P>0.05), d-PH (P>0.05), d-HCO3- (P>0.05). However, there were significant differences in d-PaCO2 (P<0.01), d-PH (P<0.01), d-HCO3- (P<0.01). Conclusion: It is easier to eliminate carbon dioxide in blood when the injector tip was located proximal to the vocal cord level than that was placed about 3 cm above carina where the level of carbon dioxide in blood increased during the high-conventional frequency jet ventilation. 2017 Vol. 47 (1): 37-41 [Abstract] ( 598 ) HTML (1 KB)  PDF (1584 KB)  ( 870 ) 42 Comparision of the structures and properties of four kinds of lignans in Schisandra Chinensis (Turcz.) Baill JIN Lisi, WENG Jinyue, ZHENG Jingsheng, WANG Chaojie Objective: To analyze the structures and spectral difference of four kinds of lignans in Schisandra Chinensis (Turcz.) Baill. through theoretical calculation. Methods: The geometrical structure, ultraviolet-visible spectrum, infrared vibrational spectrum and electronic structure of four kinds of lignans in Schisandra Chinensis were studied at the level of DFT/B3LYP/6-31+G** in gas phase and methanol, the solvent effects were considered by polarizable continuum model (PCM). Results: The optimized geometrical structure parameters of the lignans were close to each other in gas phase and in methanol. The calculated UV-Vis spectral characteristic peaks were similar to each other but Schisantherin A was higher than the other three. To the IR vibrational spectra, the peak strength increased in methanol. All lignans were polar molecules, and only the LUMO of Schisantherin A located at benzoyl group. All computed data were agreed well with available reported data. Conclusion: The results of deep understanding of the structure and properties of four kinds of lignans in Schisandra Chinensis (Turcz.) Baill. have certain scientific significance.   2017 Vol. 47 (1): 42-46 [Abstract] ( 824 ) HTML (1 KB)  PDF (4032 KB)  ( 1069 ) 47 Stablishment of vascular endothelial cell injury model induced by high methionine feed diet in rats REN Xiaoli, Yang Bo, CHEN Xiwen, LOU Yongliang. Objective: To investigate the effects of the diet rich in different proportions of methionine on endothelial injury in sprague-dawley (SD) rats. Methods: Male SD rats were randomly divided into the normal diet group (CR), 1% methionine loading diet group (M1 R), 3% methionine loading diet group (M3 R) and 5% methionine loading diet group (M5 R), and blood in tail vein were drawn at 0, 2nd, 4th, 6th and 8th week, respectively. After 8 weeks, serum concentrations of homocysteine (Hcy) were measured, and the endothelial ultrastructure changes in aorta wall were observed using scanning electron microscope. Results: A significant interaction between high methionine diet and feeding duration on endothelial injury was found (P<0.05), and serum levels of Hcy were significantly increased in rats fed with high methionine diet (P<0.05), with feeding duration increasing. Compared with CR, serum level of Hcy in M3 and M5 R was significantly higher at 2nd week (P<0.01), whereas serum Hcy in M1 R was significantly higher at 4th week (P<0.05). Endothelial cells in M1 R were imperfectly unanimous in size, and the cell surface shrinked. Endothelium in M3 R and M5 R were found to be damaged by worms kinds of harming typical, with enclose wall thrombus and the fat deposition. Conclusion: High dietary intake of methionine is significantly associated with endothelial injury, which suggests that a method setting up endothelial injury animal models would be established.   2017 Vol. 47 (1): 47-51 [Abstract] ( 655 ) HTML (1 KB)  PDF (3122 KB)  ( 1085 ) 52 Study on fatigue condition and its influencing factors in multiple sclerosis patients CHEN Kelong, FAN Yongping. Objective: To study fatigue condition and discuss its influencing factors in multiple sclerosis (MS) patients. Methods: The modified fatigue impact scale (MFIS), data of disease-related factors and general profile registration form were used to conduct a face-to-face questionnaire investigation in 99 MS patients to collect demographic characteristics, MS-related information, fatigue degree, depression degree, impacts on quality of life (QOL) and other clinical data. SPSS15.0 software was then used for statistical analysis of the influencing factors. Results: Gender, age and relapse amount showed no statistical correlation with occurrence of fatigue (P>0.05); three variables, including disease course, disability level and depression degree were independently correlated with occurrence of fatigue. The fatigue occurrence in patients with a disease course of 5-10 years is 4.76 times of that in patients with a disease course≤5 years; the fatigue occurrence in moderately and severely disabled patients was 10.81 times of that in mildly disabled patients; and the occurrence of fatigue increases for 2.32 times if the depression degree increases for one scale. With respect to QOL, scores of both physical and psychological symptoms in patients with fatigue were higher than those in patients without fatigue, showing statistically significant differences (P<0.01). Conclusion: MS patients with long disease course, disability and severe depression degree have severer fatigue and fatigue poses obvious impacts on QOL. Disease course, disability level and depression degree are all influencing factors for MS-related fatigue. 2017 Vol. 47 (1): 52-55 [Abstract] ( 704 ) HTML (1 KB)  PDF (1580 KB)  ( 925 ) 56 The case-control study of influencing factors for birth defects in Lishui  ZHANG Yali, LAN Juhong, LYU Zhuochao, XIAO Lihua Objective: To investigate the risk factors of birth defects in Lishui city, and to provide scientific evidence for intervention. Methods: A 1:3 case-control study was conducted, 601 mothers of infants with birth defects and 1803 controls were investigated retrospectively. Risk factors were analyzed by multivariate Logistic regression. Results: The incidence of birth defects was 29.79‰, the former five types of birth defects were congenital heart disease, polydactyly, cleft lip, syndactyly and, microtia. Logistic regression showed that maternal age (OR=3.752, 95%CI: 1.247-11.293, P=0.019), the health condition in the first trimester of pregnancy (OR= 2.997, 95%CI: 1.084-8.287, P=0.034), smoking or passive smoking during pregnancy (OR=2.205, 95%CI: 1.088-4.470, P=0.028), exposure to toxic factors during pregnancy (OR=3.582, 95%CI: 2.071-4.983, P=0.047), and face-painting, dying fingernails and dying hair in pregnancy (OR=3.659, 95%CI: 2.064-6.488, P<0.001) were the risk factors. Conclusion: Maternal age, the health condition in the first trimester of pregnancy, smoking or passive smoking during pregnancy, exposure to toxic factors in pregnancy, and face-painting, dying fingernails and dying the hair in pregnancy are the risk factors of birth defects. 2017 Vol. 47 (1): 56-60 [Abstract] ( 708 ) HTML (1 KB)  PDF (1678 KB)  ( 869 ) 61 Clinical application of LAMP and Xpert Mtb/RIF in early detection of pulmonary tuberculosis infection ZHANG Weiyang, ZHONG Jianping, YANG Guobiao, JIN Faxiang, XU Wenfang, WANG Huajun. Objective: To compare the clinical application of DNA loop mediated isothermal amplification (LAMP) technology and Xpert MTB/RIF in early detection of the infectious source of pulmonary tuberculosis. Methods: Three hundred TB sputum samples were detected using Xpert Mtb/RIF detection, MGIT liquid culture and LAMP technology, of which 100 cases of smear-positive specimens, 200 cases of smear-negative specimens. Results: In 100 cases of TB smear-positive specimens, the positive rate was 96.0% with LAMP technology, and the positive rate was 97.0% with Xpert Mtb/RIF detection (P>0.05). In 200 cases of smear-negative specimens, the positive rate was 53.5% with LAMP technology, and the positive rate was 25.0% with Xpert Mtb/RIF detection (P<0.01). Conclusion: In the diagnosis of smear positive specimens, the sensitivity of LAMP technology and Mtb RIF/Xpert was similar. In the diagnosis of smear negative specimens, LAMP technology is better than Mtb Xpert/RIF. 2017 Vol. 47 (1): 61-63 [Abstract] ( 1031 ) HTML (1 KB)  PDF (1927 KB)  ( 1291 ) 64 Analysis on thoracoscopic cardiac surgical procedures in 28 cases WANG Chunguo, ZHANG Jian, JIN Jiang, Objective: To evaluate the efficacy and safety of thoracoscopic cardiac surgical procedures under extracorporeal circulation. Methods: From November 2011 to June 2015, 28 patients underwent thoracoscopic cardiac surgery under extracorporeal circulation. These procedures included 1 case of left atrial myxoma, 1 case of tricuspid valvuloplasty, 2 cases of interatrial septum defect, mitral valve repair for 2 patients and mitral valve replacement for 22 patients. Thirty degree thoracoscopes and femoral extracorpereal circulation were used. The aorta was cross-clamped and the myocardium was protected by coronary perfusion with cold crystal or blood cardioplegie. Results: The operation successed in all 28 patients. Cardiopulmonary bypass time was from 68 min to 120 min with the mean of (94.6±20.58) min. Aortic cross-clamp time was from 40 min to 87 min with the mean of (63.8±15) min. The volume of bleeding was 200-500 (350±119.52) mL. The volume of chest drainage in 24 hours was 20-350 mL. The postoperative hospital stay was 7-29 (13.1±7.45) d. Postoperative complications occurred in 3 cases, including systolic anterior motion (SAM) in 1 patients, 1 case of pulmonary infection and arrhythmia in 1 case. Conclusion: Thoracoscopic cardiac surgical procedures for cardiac surical is feasible and safe, especially in the secondary cardiac valvular surgery. 2017 Vol. 47 (1): 64-66 [Abstract] ( 763 ) HTML (1 KB)  PDF (1798 KB)  ( 877 ) 67 Evaluation and analysis of patients with continuous ambulatory peritoneal dialysis WANG Tingting, CAO Yang, XU Qin, LIN Jun, MAO Jiejin, ZHU Qiuhong, ZHAO Weiyu, LI Lei, JI Feng Guide::null Objective: To investigate the nutritional status of continuous ambulatory peritoneal dialysis (CAPD) patients in the First Affiliated Hospital of Medical School of Zhejiang University, then providing nutrition interventions and dietary guidance for the patients. Methods: A total of 114 CAPD patients were collected. Their nutritional status was assessed and grouped by subjective global assessment (SGA). Then anthropometric measures and laboratory parameters were also collected and performed statistical analysis. Results: The incidence of malnutrition in CAPD patients was 51.75%, among which, severe malnutrition was 19.30%, mild-moderate malnutrition was 32.45%. Compared to good nutrition group, BMI, upperarm circumference, red blood cell count, hemoglobin, albumin level of patients with malnutrition were decreased, the difference was statistically significant (P<0.05). Conclusion: The incidence of malnutrition in CAPD patients is high, which is mainly manifested by weight loss, anemia, low serum albumin. 2017 Vol. 47 (1): 67-70 [Abstract] ( 686 ) HTML (1 KB)  PDF (1631 KB)  ( 901 )

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