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2019 Vol. 49, No. 5 Published: 2019-05-25   313 The effect of high glucose on autophagy of cultured murine myotubes LUO Shengnan, SU Zhen, HUANG Peipei, CHEN Ruyi, ZUO Yidan, HU Feifei Objective: To observe the effect of high glucose on the expression of autophagy in myotubes, and to investigate the expression of autophagy in myotubes cultured with high glucose for different time periods. Methods: Murine C2C12 myoblasts were induced into myotubes using differentiation medium, and myotubes were cultured with normal (5.5 mmol/L) and high dose (17 mmol/L or 30 mmol/L) of glucose, and cells were collected at 6, 12, 24, 36, 48, 60, and 72 h to detect autophagy-related proteins and mRNA expression. Additionally, murine myoblasts were transfected with tandem fluorescently labeled LC3 (mRFP-GFP-LC3) lentiviral vector. The red and green fluorescent spots in the cells were observed by laser confocal microscopy to monitor autophagic flux. Results: The autophagic flux in myotubes exposed to high glucose was divided into two phases. When myotubes incubated for 24 h with high glucose, the protein expression of LC3 and Beclin-1 was lower than that of the normal group (P<0.05); When C2C12 myotubes were cultured with 30 mmol/L of glucose for 48, 60 and 72 h, respectively, the levels of LC3 and Beclin-1 mRNA were significantly increased (P<0.05). In addition, localization and quantitative analysis of GFP and RFP fluorescent puncta in myotubes also demonstrated a significant increase in the synthesis of autophagosomes and autolysosomes after 48, 60 and 72 h of 30 mmol/L glucose culture, respectively. Conclusion: High glucose plays an important effect on autophagy in myotubes. Long-term hyperglycemia may eventually induce excessive autophagy. 2019 Vol. 49 (5): 313-320 [Abstract] ( 627 ) HTML (1 KB)  PDF (2218 KB)  ( 648 ) 321 The inhibiting effect of norspermidine on Candida albicans biofilm formation CAO Yanfan, ZHANG Keke, ZHANG Yanni, TANG Fengyu, HU Fengting, YE Qingsong. Objective: To study the effect of norspermidine on Candida albicans (C.albicans) biofilm formation and the underlying mechanisms. Methods: Crystal violet staining, XTT assay, colony-forming unit (CFU) count, scanning electron microscope (SEM) imaging and qRT-PCR was conducted to detect the effect of norspermidine on biofilm biomass, metabolic activity, colony number, fungal morphology and virulence-related gene expression of C.albicans biofilm. Results: Crystal violet staining results demonstrated that different concentrations of norspermidine had different inhibitory effects on C.albicans biofilm formation. XTT assay showed that norspermidine significantly restrained the metabolic activity of C.albicans biofilm. CFU counting results indicated that norspermidine severely reduced the number of C.albicans colonies in biofilm. SEM imaging displayed that norspermidine dramatically disturbed the formation of C.albicans biofilm and norspermidine in high concentration promoted the occurrence of yeast phase; qRT-PCR showed that norspermidine substantially interfered with the gene expression of ywp, hwp and als3, which were related to the virulence of C.albicans. Conclusion: Norspermidine could be used as a potential polyamine drug for anti- C.albicans biofilm as it could not only inhibit the formation of C.albicans biofilm but also interfere with the expression of its virulence-associated gene expression. 2019 Vol. 49 (5): 321-326 [Abstract] ( 937 ) HTML (1 KB)  PDF (1470 KB)  ( 876 ) 327 Honokiol-induced degradation of AML1-ETO oncoprotein through upregulation of UbcH8 in acute myeloid leukemia LI Haiying1, ZHOU Bin, WU Jianbo, XING Chongyun. Objective: To investigate the possible mechanism of the degradation of AML1-ETO protein induced by honokiol. Methods: Kasumi-1 cells were treated with 10, 20 and 40 μmol/L honokiol for 24 h and 48 h. The mRNA expression of AML1-ETO and UbcH8 and the protein expression were detected by qRT-PCR and Western blot respectively. Microarry analysis was used to select the target gene. To explore the role of UbcH8 in the degradation of AML1-ETO protein induced by honokiol, the expression of UbcH8 was knocked down by small hairpin RNAs (shRNAs) and overexpressed by MSCV-based vector to detect the related indicators. A xenograft mice model was treated by honokiol to detect tumorigenicity and related protein expression. Results: Honokiol decreased the protein expression of AML1-ETO in a time- and concentration- dependent manner, and reduced the stability of AML1-ETO protein, but did not affect the mRNA expression of AML1-ETO in kasumi-1 cell. We identified UbcH8 as the target gene by honokiol through microarray analysis. Honokiol obviously increased the expression of UbcH8 by 4-fold. Furthermore, overexpression of UbcH8 decreased the expression of AML1-ETO and knockdown of UbcH8 by shRNAs prevented honokiol-induced degradation of AML-ETO, suggesting that UbcH8 plays a critical role in the degradation of AML1-ETO by honokiol. Finally, honokiol decreased xenograft tumor size in a xenograft leukemia mouse model. Meanwhile, the protein of AML1-ETO was significantly reduced, while UBCH8 expression was elevated. Conclusion: Honokiol can degrade AML1-ETO oncoprotein through upregulation of UbcH8 in acute myeloid leukemia. 2019 Vol. 49 (5): 327-332 [Abstract] ( 616 ) HTML (1 KB)  PDF (1726 KB)  ( 630 ) 333 The design, synthesis and biological activity of small molecule inhibitors targeting STAT3 signaling pathway HE Qin, ZHANG Huan, DU Xuze, GUO Taoning, YE Faqing. Objective: Based on the lead compound, STAT3 small molecule inhibitor S3I-V4-01, a series of novel target compounds were designed and synthesized and their biological activity were tested by preliminary in vitro cell experiments so as to screen the more potent compounds. Methods: A series of target compounds were synthesized by nucleophilic substitution reaction; Luciferase reporter gene assay was used to measure the effect of target compounds on STAT3 signal transduction pathway; MTT assay was used to detect the inhibitory effect of target compounds on proliferation of human epidermal squamous cell carcinoma A431 and non-small cell lung cancer cell lines A549 cells. Results: In this study, eight target compounds (Z2-Z5 and H2-H5) were synthesized. Among them, the dominant compound H3 could effectively inhibit the phosphorylation of STAT3 and block the STAT3 signal transduction pathway and inhibit the proliferation of A431 and A549 cells. Conclusion: Compound H3 had stronger p-STAT3 inhibition and anti-proliferation ability than S3I-V4-01, and it could also be optimized in H3 structure to further improve biological activity. 2019 Vol. 49 (5): 333-338,343 [Abstract] ( 721 ) HTML (1 KB)  PDF (1356 KB)  ( 794 ) 339 Phenotypic and genetic analysis of congenital hypofibrinogenemia associated with a novel heterozygous mutation in fibrinogen β Chain ZHU Liqing, ZHANG Haiyue, LUO Shasha, FANG Weiwei, LIU Siqi, SU Kankan, YANG Lihong, WANG Mingshan. Objective: To identify the genetic defect underlying congenital hypofibrinogenamia in a Chinese pedigree. Methods: Totally 6 family members were enrolled in this study. Routine coagulation tests, including activated partial thromboplastin time (APTT), thrombin time (TT), the prothrombin time (PT), D-Dimer (DD) and fibrinogen degradation products (FDPs) were determined. The activity of fibrinogen (Fg:C) was measured using Clauss method and fibrinogen antigen (Fg:Ag) was measured using immunoturbidimetry. All exons and exon-intron boundaries of the fibrinogen Aα, Bβ and γ chain gene were amplified using PCR, followed by direct sequencing. Suspected mutation was further confirmed by reverse sequencing. The mutant fibrinogen was analyzed by the Swiss-PdbViewer, ClustalX-2.1-win and other online software. Results: PT and TT were prolonged in the proband. Her functional fibrinogen (Fg:C) and antigen fibrinogen (Fg:Ag) levels were reduced, which was 0.82 g/L and 1.19 g/L, respectively. Genetic analysis revealed a heterozygous T>G change at nucleotide 425 in exon 3 of FGB gene in the proband, predicting a novel Leu121Arg mutation. This mutation was also found in her father and son. Model analysis showed that the Leu121Arg mutation added a hydrogen bonding among Try117, Met118, Trp125 and Leu121. Moreover, the mutation also changed the mutual electrostatic forces, affecting the folding and instability of the mutant fibrinogen. Conclusion: The heterozygous Leu121Arg mutation leading to the hypofibrinogenemia in this pedigree. 2019 Vol. 49 (5): 339-343 [Abstract] ( 792 ) HTML (1 KB)  PDF (1366 KB)  ( 796 ) 344 Analysis on the care ability status and its influencing factors of family caregivers for the hospitalized newly disabled elderly before discharge LI Hui, LI Xiuyue, YIN Zhiqin, GUAN Yaqi, ZHENG Tingting. Objective: To explore the status and influencing factors of care ability of family caregivers for the newly disabled elderly in hospital before discharge, and to provide basis for nursing staff to take pertinent intervention measures. Methods: Using the convenient sampling method, this study recruited 206 hospitalized newly disabled elderly patients and their caregivers from the First Affiliated Hospital of Wenzhou Medical University and Taizhou Hospital Affiliated to Wenzhou Medical University from July 2017 to May 2018. General information questionnaire and family caregiver task inventory were used to investigate these participants. The influencing factors of caregivers' care ability including demographic sociological data, disease data and others were analyzed. Multiple linear stepwise regression analysis was used to explore the main influencing factors. Results: The total score of caregivers’ care ability was 15.0±6.8, with significant differences (P<0.05) compared with the Hong Kong norm. The results of multivariate linear stepwise regression analysis showed that the factors affecting the care ability of family caregivers mainly include the patients’ economic level, self-care ability on admission and the education level and average daily care time of the caregiver, accounting for 32% of the total variation of caregivers’ care ability. Conclusion: Family caregivers’ care ability for the hospitalized newly disabled elderly is at low level before discharge. Nursing staff should give particular guide to the caregivers for the disabled elderly with low self-care ability on admission and poor economic conditions, and also to the caregivers with low educational level and short care time. 2019 Vol. 49 (5): 344-349,355 [Abstract] ( 830 ) HTML (1 KB)  PDF (1318 KB)  ( 922 ) 350 Vascularization mechanism of periosteal decellularized bioscaffold implanted in bone defect mice ZHANG Xueming, CHEN Junhao, CHEN Lei, LI Xiaohang, WANG Kaicheng, LIN Qiongqiong, JIN Keke Objective: To observe the reparative effect of periosteal decellularized bioscaffold on bone defect in mice and the process of angiogenesis in the scaffold and to elucidate the possible mechanisms underlying the angiogenesis. Methods: The periosteal decellularized biological scaffolds were obtained using sequential treatment including physical freeze-thaw, chemical and biological enzyme reagents processing. To investigate in vitro effects of periosteal decellularized scaffold extract on human umbilical vein endothelial cells (HUVECs), wound healing assay was used to evaluate the presence of pro-vascularized biological factors in scaffolds; Meanwhile, Enzyme-linked immunosorbent assay (ELISA) was used to detect vascular endothelial growth factor (VEGF) in scaffolds. The bone reparative ability of the periosteal decellularized bioscaffold due to its pro-vascularization effect was further evaluated in a mouse femur bone defect model. A 0.5 mm diameter single cortical bone defect was prepared at the distal femur of the mouse, in which the periosteal decellularized scaffold was implanted, followed by layer-by-lay suture. The bone defects without material implanted were set as the control group. On the day 7, 14, 21 and 28 after surgery, the materials were harvested, fixed, decalcified, embedded, and sectioned. The bone repair was evaluated by HE staining. Immunofluorescence staining of von Willebrand Factor (vWF) was performed to observe the vascularization within the defect area. Results: Cell experiments showed that the decellularized periosteum scaffold extract had no obvious inhibitory effect on the proliferation of HUVEC. The results of cell wound healing test showed that the migration area of the scaffold extract group was larger than that of the control group, suggesting that the decellularized periosteum scaffold extract can effectively promote the migration of HUVEC. Moreover, VEGF was present in the scaffold extract at a concentration of 210 pg/mL. In animal experiments, HE staining demonstrated that decellularized periosteal scaffolds can promote the growth of blood vessels and new bone formation in the bone defect area. Immunofluorescence staining further proved that the repair activity of the decellularized periosteum scaffold was associated with the process of vascularization, and the density of blood vessels in the decellularized periosteal scaffold increased first and then decreased. Conclusion: Decellularized periosteal scaffolds can vascularize and promote healing of bone defects. VEGF may be a key factor in its angiogenesis process. 2019 Vol. 49 (5): 350-355 [Abstract] ( 727 ) HTML (1 KB)  PDF (1802 KB)  ( 692 ) 356 The value of imaging examination techniques in diagnosing of type II painful accessory navicular bone CHEN Shiyu, ZANG Guoli, XU Weiying, HU Min, XU Qing, ZHAO Jia, WANG Jiefeng Objective: To evaluate the diagnostic value of X-ray, CT, MRI and ultrasonography in type II painful accessory navicular bone (PANB). Methods: The X-ray, CT, MRI, and ultrasonic diagnostic data of 45 cases (from 42 patients) of type II PANB were retrospectively analyzed. The sensitivity and accuracy of PANB were compared between the four groups. Results: The sensitivity of X-ray, CT, MRI, ultrasonography diagnosis of type II PANB was 68.9% (31/45), 88.9% (40/45), 95.6% (43/45), 91.1% (41/45), respectively. The sensitivity of X-ray was lower than that of CT, MRI and ultrasonography, with statistical difference (P<0.05). The sensitivity of CT, MRI and ultrasonography in diagnosing type II PANB was high and there was no significant difference between them (P>0.05). The maximum diameter of positive cases measured respectively by X-ray, CT, MRI and ultrasonography was (11.5±6.6)mm, (9.5±7.3)mm, (9.3±7.1)mm, (11.8±6.8)mm, with no statistically significant difference between all groups (P>0.05). Conclusion: X-ray is less sensitive to the diagnosis of type II PANB. CT has higher sensitivity, but its imaging capability of soft tissue is poor. MRI has the highest sensitivity and can display the bone marrow edema and soft tissue lesions, but it costs more; Ultrasonography diagnosis, being highly sensitive, able to find the accessory bone, the fibrous junction and soft tissue lesions, can be used for dynamic multi angle scanning. In addition, ultrasonography is more convenient and less expensive. Therefore, it is a recommended screening method. 2019 Vol. 49 (5): 356-359,366 [Abstract] ( 659 ) HTML (1 KB)  PDF (1348 KB)  ( 896 ) 360 Preparation and bioactivity evaluation of MIP-3α-SA fusion protein SONG Zhichun, DAI Shu, WANG Peng, GAO Jimin. Objective: To prepare the streptavidin-tag MIP-3α fusion protein MIP-3α-SA, and to study its biological function and activity. Methods: The recombinant expression vector of MIP-3α-SA-pET21 was constructed and induced to express MIP-3α-SA fusion protein in Escherichia coli. MIP-3α-SA fusion protein was purified by the Ni-NTA affinity chromatography and refolded by gradient dialysis of urea. The fusion protein was identified by amide gel electrophoresis (SDS-PAGE), Western blot and silver ammonia staining. In vitro lymphocyte chemotaxis assays was used to detect the chemotactic activity of the fusion protein, and flow cytometry (FCM) analysis was performed to detect the modification efficiency of the fusion protein on biotin RM-1 cell surface anchoring. Results: The MIP-3α-SA fusion protein expression was induced with high efficiency in E. coli Rosetta (DE3), which accounted for 30% of the total bacterial protein. The purity of the fusion protein was 95% after purification by nickel column affinity chromatography. After renaturation by urea gradient, the fusion protein was verified to have dual activity, namely: MIP-3α-SA stimulated human lymphocyte chemotactic activity has a dose dependent manner and SA-mediated efficient binding to the surface has biotinylated RM-1 cell function (modification of the surface anchoring efficiency of more than 95%). Conclusion: The MIP-3α-SA bifunctional fusion protein has dual activity, which lays the foundation for the research and development of MIP-3α surface-modified tumor cell vaccine. 2019 Vol. 49 (5): 360-366 [Abstract] ( 807 ) HTML (1 KB)  PDF (1453 KB)  ( 702 ) 367 Repair of multiple skin defects with joined donor grafts in equal size ZHOU Qiaochu, LI Zhiming, CHEN Zhiwei, XU Jie Objective: To explore coalescent methods of a malpositioned joining and the application of skin graft in equal size for preparing donor graft of multiple skin defects. Methods: Three patients in the Wenzhou Hospital of Traditional Chinese Integrated Western Medicine from January 2014 to July 2018 with multiple skin malignant tumors were selected. It’s used in combination with coalescent methods of a malpositioned joining for the first level and application of skin graft in equal size to the second level. Results: By coalescent methods of a malpositioned joining and the application of skin graft in equal size, the width of donor graft was reduced. The donor site was sutured directly. The donor graft in multiple places was avoided. All achieved complete survival of skin grafts. Conclusion: For preparing donor graft of multiple skin defect, the applications with coalescent methods of a malpositioned joining andthe application of skin graft in equal size may be a fine choice. 2019 Vol. 49 (5): 367-370 [Abstract] ( 588 ) HTML (1 KB)  PDF (1371 KB)  ( 674 ) 371 The effectiveness of integrated intervention with psychotherapy and drug therapy for senile depression GONG Benhong, SHI Sumi, CHEN Guangqiang, LIU Lingjing, WANG Wenxia, TIAN Haijia, ZHANG Yu, CHEN Mulin Objective: To explore the effectiveness of integrated psychotherapy for senile depressive disorder so as to promote rapid recovery of social function and cure rate of the elderly patients with depression. Methods: There were 365 senile depressive disorder patients who were screened by using the old age Depression Scale, of whom 120 cases who met the diagnostic criteria of ICD-10 depression were pre-identified and randomly grouped in the form of voluntary group. Integrated psychotherapy plus drug therapy (experimental group) and simple antidepressant treatment (control group) were carried out. HAMD and GWB were used to assess the status, with statistical analysis by spss19.0. Results: After the 2-week treatment in the experimental group and the control group, there was significant difference in HAMD score reduction rate between the two groups. In the 2nd week after integrated psychotherapy was added to the experimental group, there was obvious difference in subtraction, compared with the control group. After 12 weeks of treatment with comprehensive intervention in both groups, the overall happiness index was significantly different from that before treatment (P<0.01) in the experimental group, but not in the control group. Conclusion: Integrated psychotherapy and comprehensive intervention of drugs are more effective in the treatment of senile depressive disorder. 2019 Vol. 49 (5): 371-373 [Abstract] ( 656 ) HTML (1 KB)  PDF (1193 KB)  ( 780 ) 376 Investigation and analysis of drug compliance of type 2 diabetes mellitus patients in the Glucose Optimization Management Project SHEN Wen, ZHOU Lingli, JIANG Zipei Objective: To investigate and analyze the medication compliance of type 2 diabetes mellitus patients. Method: A total of 243 patients with type 2 diabetes in the First Affiliated Hospital of Wenzhou Medical University who participated in the “glucose optimization management project” (launched by the Chinese Medical Doctor Association and Sanofi) were divided into two groups depending on whether they had stopped taking drugs during the 12-week follow-up period. The general situation and clinical characteristics of the two groups before the use of basic insulin were compared and further analyzed by multiple logistic regression model. The differences between the two groups before the end of the 12-week follow-up period were observed. The causes of drug withdrawal and the distribution of time of drug withdrawal, the causes of drug withdrawal in different previous treatment schemes and the causes of drug withdrawal in different current treatment schemes were analyzed. Results: Compared with the group without discontinuation, the discontinuation group was found to have higher proportion of males, younger age, higher body mass index (BMI), shorter course of disease, lower initial fasting blood glucose and insulin dosage and fewer systemic complications (all P<0.05). In the discontinuation group, the number of discontinuation was the largest in the first week. The main reason for discontinuation was to change the treatment plan into oral hypoglycemic drugs only. Multivariate logistic regression analysis showed that shorter course of disease, higher initial fasting blood glucose, higher BMI and lower initial insulin dosage were the influencing factors of drug withdrawal (P<0.05), and shorter course of disease was the most important influencing factor (P<0.001). Conclusion: In the glucose optimization management project, poor compliance with medication occurred in patients with shorter course of disease, higher initial fasting blood glucose, higher BMI and lower initial insulin dosage. The main reason for discontinuation is to change the treatment plan into taking oral hypoglycemic drugs only. 2019 Vol. 49 (5): 376-380 [Abstract] ( 727 ) HTML (1 KB)  PDF (1264 KB)  ( 691 )

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