1.Establishment and preliminary application of prokaryotic expression of BTV16 re-combinant VP2 protein,preparation of polyclonal antibody and indirect ELISA detection method
Mingzhu ZHANG ; Peng WANG ; Jiaxin TIAN ; Shigang CHEN ; Junduo BAO ; Xiangshu QIU ; Huijun LU ; Chang LI
Chinese Journal of Veterinary Science 2024;44(12):2549-2555
Bluetongue virus(BTV)is classified as a category Ⅱ animal epidemic disease in China,infecting multiple species and posing significant threats to the ruminant breeding industry.There are 29 serotypes of BTV,with BTV16 being one of the major serotypes currently prevalent in Chi-na.Bluetongue virus infection mainly manifests as a latent infection,making the establishment of ELISA assays crucial for epidemiological detection.In this study;the expression of the BTV16 VP2 protein was achieved using a prokaryotic expression system,and polyclonal antibodies were pre-pared using BALB/c mice.An indirect ELISA assay using VP2 protein as the encapsulated antigen was established and optimized.Clinical samples from the Guangxi Zhuang Autonomous Region were tested and analyzed for compliance with commercial kits.The results showed that the BTV16 VP2 protein was successfully expressed and purified,and the prepared polyclonal antibody exhibi-ted good immunogenicity.The ELISA assay had good specificity,with no cross-reactivity against ruminant diseases such as AKAV,FMDV and GETV.The critical values for negativity and positivity were determined to be 0.314,and the coefficients of variation(Cv)between batches and within batches were both less than 5%,indicating good reproducibility.The ELISA assay revealed a positive rate of 92.4%for 79 samples from the Guangxi Zhuang Autonomous Region,with a compliance rate of 98.7%when compared to the commercialized kit.In conclusion,this study suc-cessfully established an indirect ELISA method for BTV16,facilitating the detection of bovine clin-ical samples.
2.Exploration of Predictors to Identify and Screen High Risk Patients With Early Gastric Cancer Submucosal Invasion
Keyan CHEN ; Ye WANG ; Jing ZHANG ; Shigang DING
Chinese Journal of Minimally Invasive Surgery 2024;24(6):401-408
Objective To analyze the clinical,pathological,and endoscopic features of differentiated early gastric cancer,and to study predictors to identify and screen high risk patients with early gastric cancer submucosal infiltration.Methods A total of 172 patients with differentiated early gastric cancer treated by surgical or endoscopic submucosal dissection in our hospital from January 2017 to December 2022 were included,which were divided into the mucosal layer group(144 patients)and submucosal layer group(28 patients)based on postoperative pathology.The clinical,pathological,and white-light endoscopy(WLE)and linked color imaging(LCI)features of the 2 groups were compared.The color difference between the lesion and the surrounding mucosa was evaluated by using the Commission International de L'Eclairage(CIE)L*a*b*system.Indicators with significant differences were included to multifactor logistic stepwise regression analysis(forward method)for the identification and screening of predictors.Results A history of alcohol consumption(P=0.037),a history of smoking(P=0.035),thickening of the gastric wall on enhanced CT(P=0.032),a lesion located in the upper 1/3(P<0.001)or middle 1/3(P=0.009)part of the stomach,depressed macroscopic type(P<0.001),marked margin elevation(P=0.003),presence of fold changes(P=0.006),color difference ≥12.3 under WLE(P=0.003)and≥18.2 under LCI(P=0.002)were associated with submucosal infiltration.Multivariate analysis showed that lesions located in the upper 2/3 portion of the stomach(OR=5.463,95%CI:2.562-11.648,P<0.001),depressed macroscopic type(OR=5.992,95%CI:1.624-22.100,P=0.007),marked margin elevation(OR=4.338,95%CI:1.124-16.747,P=0.033),and color difference ≥18.2 under LCI(OR=4.675,95%CI:1.342-16.288,P=0.015)were independent risk factors for infiltration of submucosal layer of lesions.Conclusion Lesions with depressed macroscopic type,marked elevated margins,located in the upper 2/3 part of the stomach,and having a large color difference from the surrounding mucosa under LCI are high-risk lesions for submucosal infiltration and require more aggressive intervention.
3.Establishment and preliminary application of prokaryotic expression of BTV16 re-combinant VP2 protein,preparation of polyclonal antibody and indirect ELISA detection method
Mingzhu ZHANG ; Peng WANG ; Jiaxin TIAN ; Shigang CHEN ; Junduo BAO ; Xiangshu QIU ; Huijun LU ; Chang LI
Chinese Journal of Veterinary Science 2024;44(12):2549-2555
Bluetongue virus(BTV)is classified as a category Ⅱ animal epidemic disease in China,infecting multiple species and posing significant threats to the ruminant breeding industry.There are 29 serotypes of BTV,with BTV16 being one of the major serotypes currently prevalent in Chi-na.Bluetongue virus infection mainly manifests as a latent infection,making the establishment of ELISA assays crucial for epidemiological detection.In this study;the expression of the BTV16 VP2 protein was achieved using a prokaryotic expression system,and polyclonal antibodies were pre-pared using BALB/c mice.An indirect ELISA assay using VP2 protein as the encapsulated antigen was established and optimized.Clinical samples from the Guangxi Zhuang Autonomous Region were tested and analyzed for compliance with commercial kits.The results showed that the BTV16 VP2 protein was successfully expressed and purified,and the prepared polyclonal antibody exhibi-ted good immunogenicity.The ELISA assay had good specificity,with no cross-reactivity against ruminant diseases such as AKAV,FMDV and GETV.The critical values for negativity and positivity were determined to be 0.314,and the coefficients of variation(Cv)between batches and within batches were both less than 5%,indicating good reproducibility.The ELISA assay revealed a positive rate of 92.4%for 79 samples from the Guangxi Zhuang Autonomous Region,with a compliance rate of 98.7%when compared to the commercialized kit.In conclusion,this study suc-cessfully established an indirect ELISA method for BTV16,facilitating the detection of bovine clin-ical samples.
4.Research on medical radioisotope production technology by medium and high-energy cyclotron and solid target
Tianjue ZHANG ; Kai WEN ; Jingyuan LIU ; Li HUO ; Chengwei MA ; Junyi CHEN ; Jiantao BA ; Xianlu JIA ; Guofang SONG ; Haiqiong ZHANG ; Sumin WEI ; Lei WANG ; Suping ZHANG ; Shigang HOU ; He ZHANG ; Jingfeng WANG ; Lei CAO ; Guang YANG ; Zhibo LIU
Chinese Journal of Nuclear Medicine and Molecular Imaging 2022;42(6):340-346
In this paper, the domestic and international demand and development trend of clinical diagnostic radionuclides are analyzed, and the medium and high-energy cyclotrons, adequate and systematic facilities, and preparation techniques required for the production of medical radionuclides based on solid targets are introduced. This paper focuses on the research and development carried out by some important medical institutions and scientific research institutes in China over the years in the aspects of medium and high-energy cyclotrons, beam transmission lines, high-power irradiation target stations and new medical isotope production processes etc. It also looks forward to some new directions for the development of medical radionuclides in China during the 14th Five-Year Plan period.
5.Effect of biglycan on neural apoptosis in mice with early brain injury after subarachnoid hemorrhage
Yuke XIE ; Kecheng GUO ; Jianhua PENG ; Jinwei PANG ; Jian ZHOU ; Long GU ; Lifang ZHANG ; Shigang YIN ; Ligang CHEN ; Yong JIANG
Chinese Journal of Neuromedicine 2020;19(2):138-145
Objective To investigate the effect ofbiglycan (BGN) on neural apoptosis in mice with early brain injury (EBI) after subarachnoid hemorrhage (SAH).Methods SAH models were induced by endovascular perforation in young male C57BL/6J mice.(1) Totally,48 mice were randomly divided into sham-operated group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group,and SAH 72 h group (n=8);the BGN protein and mRNA expressions were detected by Western blotting and real-time quantitative PCR (qRT-PCR).(2) Totally,16 mice were randomly divided into sham-operated group and SAH 48 h group (n=8);immunofluorescent double staining was conducted to explore the BGN expression in the neurons of brain tissues.(3) Totally,24 mice were randomly divided into sham-operated group,sham+control lentivirus group,and sham+BGN lentivirus group (n=8);BGN lentiviral vector and control lentivirus were administered intracerebroventricularly 7 d before sham-operation;qRT-PCR was performed to explore the BGN mRNA expression.(4) Totally,48 mice were randomly divided into sham-operated group,SAH+control lentivirus group,and SAH+BGN lentivirus group (n=16);BGN lentiviral vector and control lentivirus were administered intracerebroventricularly 7 d before SAH;neurological scores were detected by modified Garcia scale and beam balance tests;TUNEL was used to detect the neuronal apoptosis,and Western blotting was performed to explore the expressions of nuclear transcription factor kappa B (NF-κB) and phosphorylated-(p-) NF-κB.Results (1) Mice in the SAH 48 h group had the highest BGN protein and mRNA expressions,which showed statistical differences as compared with the sham-operated group (P<0.05).(2) A majority of BGN expressions were detected in the neurons 48 h after SAH.(3) The sham+BGN lentivirus group had statistically lower BGN mRNA expression than the sham+control lentivirus group (P<0.05).(4) As compared with those in the SAH+control lentivirus group,both scores of modified Garcia scale and beam balance tests were significantly higher in SAH+BGN lentivirus group (6.125±1.246 vs.13.000±1.309;1.125±1.126 vs.2.875±0.835),and neural apoptosis ratio and ratio of p-NF-κB/NF-κB were significantly lower in the SAH+BGN lentivirus group (51.950%±11.166% vs.31.938%±7.705%;1.161±0.156 vs.0.886±0.142,P<0.05).Conclusion Inhibition of BGN can effectively reduce neuronal apoptosis in mice with EBI after SAH,and attenuate neurological deficits.
6. Accuracy of ultrasound-determined end-diastolic velocity of central retinal artery in diagnosing postoperative low cerebral perfusion pressure in patients with craniocerebral trauma
Mengnan YU ; Yanjun DENG ; Hua LI ; Shigang QIAO ; Chen WANG
Chinese Journal of Anesthesiology 2019;39(8):982-984
Objective:
To evaluate the accuracy of ultrasound-determined end-diastolic velocity (EDV) of central retinal artery (CRA) in diagnosing postoperative low cerebral perfusion pressure (CPP) in the patients with craniocerebral trauma.
Methods:
Forty-nine patients of both sexes with brain injury, aged 18-64 yr, with body mass index of 18.5-23.9 kg/m2, were enrolled.The peak systolic velocity and EDV of CRA were determined using ultrasound at 1 day after operation.Mean arterial pressure and intracranial pressure were recorded, and CPP was calculated (CPP=mean arterial pressure-intracranial pressure).
Results:
EDV was positively correlated with CPP (
7.Role of microRNA-1-mediated AMP-activated protein kinase pathway in cardiac fibroblasts induced by high glucose in rats
Jia QIU ; An WANG ; Yingna XU ; Shigang QIAO ; Jianzhong AN ; Hua LI ; Chen WANG
Chinese Critical Care Medicine 2018;30(2):145-150
Objective To investigate the role of microRNA-1 (miR-1) in cardiac fibroblasts induced by high glucose in rats. Methods The primary fibroblasts were cultured from the apical tissue of 1-3 day-old Sprague-Dawley (SD) rats. The cells which were passaged to generation 3 or 4, were randomly divided into normal glucose+lentivector-vehicle group (CON+Lv-Vehicle group), normal glucose+lentivector-miR-1 group (CON+Lv-miR1 group), high glucose+lentivector-vehicle group (HG+Lv-Vehicle group), high glucose+lentivector-miR-1 group (HG+Lv-miR1 group), high glucose+Lv-Vehicle+inhibitor group (HG+Lv-Vehicle+CC group), and high glucose+lentivector-miR-1+inhibitor group (HG+Lv-miR1+CC group). The myocardial fibroblasts were cultured in the concentration of 5.5 mmol/L glucose (normal glucose) or 25.0 mmol/L (high glucose) DMEM medium. Then lentiviral vector containing miR-1 silent sequence or the same volume of lentiviral vector was inoculated into the cells. The AMP activated protein kinase (AMPK) inhibitor Compound C (20 μmol/L) was added to the medium at 12 hours before sampling in inhibitor groups. The expression of phosphorylation of AMPK (p-AMPK), collagenⅠandⅢ, matrix metalloproteinase (MMP-2, MMP-9), and autophagy flux related protein LC3B-Ⅱ and p62/SQSTM1 were measured by Western Blot. Results The purity of rat myocardial fibroblasts in vitro was 97%. Compared with CON+Lv-Vehicle group, there was no significant difference in the expression of p-AMPK in CON+Lv-miR1 group, the expression of p-AMPK in HG+Lv-Vehicle group was significantly decreased (p-AMPK/t-AMPK: 44.72±3.29 vs. 100.00±7.77, 1 < 0.01). The expression of p-AMPK in HG+Lv-miR1 group was higher than that in HG+Lv-Vehicle group (p-AMPK/t-AMPK:60.52±5.16 vs. 44.72±3.29, 1 < 0.05). Compared with HG+Lv-Vehicle group, the expressions of collagen, MMP, LC3B-Ⅱand p62/SQSTM1 in HG+Lv-miR1 group were significantly decreased; after the treatment with AMPK inhibitor, the expressions of collagen, MMP, LC3B-Ⅱ, p62/SQSTM1 were significantly increased (HG+Lv-Vehicle+CC group vs. HG+Lv-Vehicle group: collagen Ⅰ/β-actin: 158.74±13.21 vs. 100.00±7.64, collagenⅢ/β-actin: 177.38± 17.31 vs. 100.00±5.18, MMP-2/β-actin: 130.09±14.31 vs. 100.00±10.47, MMP-9/β-actin: 215.54±20.92 vs. 100.00±11.28, LC3B-Ⅱ/β-actin: 159.34±13.83 vs. 100.00±6.44, p62/SQSTM1/β-actin: 201.01±24.02 vs. 100.00±8.62; HG+Lv-miR1+CC group vs. HG+Lv-miR1 group: collagenⅠ/β-actin: 108.69±9.93 vs. 80.83±7.24, collagenⅢ/β-actin: 127.68±10.46 vs. 81.56±9.97, MMP-2/β-actin: 106.66±10.21 vs. 74.80±7.43, MMP-9/β-actin: 145.65±11.56 vs. 74.63±10.55, LC3B-Ⅱ/β-actin: 150.15±13.28 vs. 22.98±2.87, p62/SQSTM1/β-actin: 130.48±10.74 vs. 49.90±2.27, all 1 < 0.05). Conclusion miR-1 gene silencing inhibits myocardial fibrosis induced by high glucose, its mechanism may be related to the up-regulation of p-AMPK, which can recover autophagy flux.
8.Value of ratio of optic nerve sheath diameter to eyeball transverse diameter in assessing intracranial pressure in patients with brain injury: measurement using ultrasound
Jie DU ; Yanjun DENG ; Xiaoxin HE ; Shigang QIAO ; Chen WANG
Chinese Journal of Anesthesiology 2018;38(11):1369-1371
Objective To evaluate the value of the ratio of optic nerve sheath diameter to eyeball transverse diameter (ONSD/ETD) in assessing intracranial pressure using ultrasound in the patients with brain injury.Methods Forty-six patients of both sexes with brain injury in the surgical intensive care unit,were selected.Craniotomy was performed within 24 h after admission to hospital,and the intracranial pressure probe was placed in the lateral cerebral ventricle of the enrolled patients who were aged 18-80 yr,with Glasgow Coma Scale score 3-15.ONSD and ETD were measured within 3 days after operation,three times a day,and the intracranial pressure was simultaneously recorded.The correlation between ONSD/ETD ratio and intracranial pressure were analyzed using the Pearson correlation analysis.The receiver operating characteristic curve was used to evaluate the accuracy of ONSD/ETD ratio in assessing the intracranial hypertension (intracranial pressure>20 mmHg).Results The ONSD/ETD ratio was positively correlated with intracranial pressure (r =0.720,P<0.01).The area under the receiver operating characteristic curve was 0.900 (95% confidence interval 0.854-0.946),and the threshold was 0.248 (sensitivity 89.4%,specificity 77.5%).Conclusion The ONSD/ETD ratio produces higher accuracy in assessing intracranial pressure in the patients with brain injury.
9.Role of autophagosomes clearance in delayed cardioprotection in rats with ischemia-reperfusion injury by sevoflurane preconditioning in vivo
Shigang QIAO ; Bo SUN ; Haihua SHAN ; An WANG ; Jia QIU ; Chen WANG
Chinese Journal of Emergency Medicine 2017;26(1):65-70
Objective To evaluate role of autophagosomes clearance in delayed cardioprotection by sevoflurane preconditioning in rats with ischemia-reperfusion injury in vivo.Methods Forty-five adult male Sprague-Dawley rats,weighing 270-350 g,were randomly (random number) divided into 3 groups:sham operation group (sham group),ischemia-reperfusion group (CON group),sevoflurane preconditioning group (SWOP group).Myocardial ischemia was induced by 30 min occlusion of left anterior descending branch (LAD) of coronary artery followed by reperfusion for 2 h,and myocardial infarct size was stained by triphenyltetrazolium chloride.Cardiomyocyte apoptosis was evaluated by terminal deoxyribonucleotidyl transferase-mediated biotin-16dUTP nick-end labeling.Autophagosomes were detected under transmission electron microscope.Expression of LC3-Ⅱ,cathepsin B,p62 and cleaved caspase-3 were assessed by western blotting.Statistical analysis were performed using one or two way analysis of variance (SPSS 15.0,Chicago,USA) test followed by Dunnet-t or LSD-t test.Results Sevoflurane preconditioning reduced myocardial infarct size and the number of autophagosomes (P =0.027),attenuated cardiomyocyte apoptosis (P =0.042).Sevoflurane preconditioning decreased the level of LC3-Ⅱ (P =0.033),p62 (P =0.041)and cleaved caspase-3 (P =0.037),but increased the level of cathepsin B (P =0.046).Conclusions Delayed cardioprotection by sevoflurane preconditioning increased myocardial clearance of autophagosomes against the delayed ischemia reperfusion injury.
10.Effects of ovarian induction with Raloxifene versus Clomiphene Citrate on endometrial receptivity in mouse endometrium
Yingjie XIAN ; Cairong CHEN ; Xiaoqing LIANG ; Xiuqin ZHOU ; Shigang WU ; ia Qiux YAN ; Xiaoying ZHAO
The Journal of Practical Medicine 2017;33(24):4053-4056
Objective To investigate the effects of ovarian induction with raloxifene(RAL)versus clomi-phene citrate(CC)on the endometrial receptivity in mouse endometrium during perimplantation period. Methods 48 female Kun-ming mouse were randomly divided into four groups in equal number:RAL 240 mg group,RAL 180 mg group,CC group,natural conception group(NC),all treated with ovulation induction after drug administration.Successfully mated female mouse were killed,and uterus samples were collected for HE stain-ing and immunohistochemistry. Results HE staining showed that the endometrial morphology in the RAL 180 mg group and RAL 240 mg group and NC group were better than that of CC group.The expressions of COX-2 and LP-AR3 in the RAL 180 mg group and RAL 240 mg group were similar to NC group,without significant difference among the three groups(P > 0.05). But in the CC group,it was statistically significantly lower than other three groups(P<0.05),indicating ovarian induction with RAL did not decrease the expressions of COX-2 and LPAR3 in endometrium. Conclusion The mechanism of ovulation induction with RAL is similar to CC,but RAL has fewer adverse effects on the endometrial receptivity compared with CC.

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