To prepare monoclonal antibody to the N protein of porcine reproductive and respiratory syndrome virus(PRRSV),BALB/c mice were immunized with the purified N protein of the PRRSV-2 strain expressed by prokaryotic expression system.Mouse splenocytes were fused with myeloma cells using hybridoma technique,hybridoma cells were identified by indirect ELISA method and indirect immunofluorescence assay(IFA),and positive hybridoma cells were screened for subclones using the limited dilution method.The results showed that a monoclonal antibody cell line 4A7 was successfully obtained,and the results of Western blot and IFA indicated that the monoclonal antibody could accurately recognize the N proteins of type 1 and type 2 PRRSV.Mean-while,the N protein gene was truncated and expressed by prokaryotic expression system,and the amino acid sequence of the B-cell antigenic epitope recognized by 4A7 was screened as 51EKPHF55 using Western blot.Comparison of epitope amino acids in the N protein gene sequences of different strains revealed that the antigenic epitope 51EKPHF55 recognized by the monoclonal antibody 4A7 has no amino acid difference in the sequences of three subtypes among the PRRSV-1 strains and nine lineages among the PRRSV-2 strains,indicating a high degree of conservation.The results of the study provide a foundation the development of PRRSV diagnostic kits and novel vaccines.

Objective:To evaluate the efficacy and safety of a new plasma radiofrequency generator and a disposable mucosal incision knife for endoscopic submucosal dissection (ESD) of gastrointestinal lesions.Methods:This study is a prospective, randomized, non-inferiority multicenter study (Chinese Clinical Trial Registry No.: ChiCTR2000041394). The inclusion criteria for cases are as follows: (1) being able to understand and voluntarily participate in this clinical trial, and voluntarily signing the informed consent form; (2) being 18-70 years old, regardless of gender; (3) having a gastrointestinal lesion and meeting the endoscopic treatment conditions for the indications of endoscopic submucosal dissection (ESD). The exclusion criteria are: (1) meeting the contraindications for ESD; (2) having a surgical contraindication due to coagulation dysfunction or still taking anticoagulants 1 week before surgery; (3) having coagulation dysfunction, that is, the prothrombin time is prolonged by more than 3 seconds (more than 5 seconds in patients with liver disease), or the activated partial thromboplastin time is prolonged by more than 10 seconds, and the platelet count is < 70×10?/L; (4) having severe cardiopulmonary insufficiency and being unable to tolerate the surgery; (5) being a reproductive-aged woman with a positive blood or urine pregnancy test or a lactating woman; (6) the investigator deems that there are other factors that are not suitable for inclusion or affect the subject's participation. Patients who underwent ESD treatment for gastrointestinal lesions from March 2019 to April 2023 at the Endoscopy Center of Zhongshan Hospital Affiliated to Fudan University, the Department of Gastroenterology of Minhang District Central Hospital in Shanghai, and the Department of Gastroenterology of Quzhou People's Hospital were prospectively included. The experimental group used a new plasma radiofrequency therapy instrument and a disposable mucosal incision knife, while the control group used a high-frequency electrosurgical system and a disposable mucosal incision knife. The primary efficacy indicator was the en bloc resection success rate, the secondary efficacy indicators included the coagulation success rate, and the operation stability of the plasma radiofrequency therapy instrument and the disposable mucosal incision knife; the safety indicators included the incidence of intraoperative bleeding, intraoperative perforation, and postoperative complications. Results:The study cohort comprised 194 patients, 95 in the experimental group and 99 in the control group. Analysis of the full set showed rates of en bloc resection of 97.89% (93/95) and 96.97% (96/99) in the experimental and control groups, respectively; the difference being 0.53% (-5.58%, 6.64%). Analysis of the compliance set showed rates of en bloc resection of 97.83% (90/92) and 96.88% (93/96) in the experimental and control groups, respectively; the difference being 0.58% (-5.79%, 6.94%). These data indicate that the rate of en bloc resection in the experimental group using plasma radiofrequency therapy and disposable mucosal incision was not inferior to that of the control group. The rates of achieving intraoperative coagulation in the experimental and control groups were 7.14% (5/70) and 5.97% (4/67), respectively; this difference is not statistically significant ( P=1.000). The control group had a significantly better rate of using the same instruments throughout the procedure than did the experimental group ( P<0.001). In the safety analysis set, the incidences of intraoperative and postoperative adverse events did not differ significantly between the two groups (both P>0.05). Conclusion:Plasma radiofrequency therapy equipment and a disposable mucosal incision knife are safe and effective instruments for performing ESD of gastrointestinal lesions.

To prepare monoclonal antibody to the N protein of porcine reproductive and respiratory syndrome virus(PRRSV),BALB/c mice were immunized with the purified N protein of the PRRSV-2 strain expressed by prokaryotic expression system.Mouse splenocytes were fused with myeloma cells using hybridoma technique,hybridoma cells were identified by indirect ELISA method and indirect immunofluorescence assay(IFA),and positive hybridoma cells were screened for subclones using the limited dilution method.The results showed that a monoclonal antibody cell line 4A7 was successfully obtained,and the results of Western blot and IFA indicated that the monoclonal antibody could accurately recognize the N proteins of type 1 and type 2 PRRSV.Mean-while,the N protein gene was truncated and expressed by prokaryotic expression system,and the amino acid sequence of the B-cell antigenic epitope recognized by 4A7 was screened as 51EKPHF55 using Western blot.Comparison of epitope amino acids in the N protein gene sequences of different strains revealed that the antigenic epitope 51EKPHF55 recognized by the monoclonal antibody 4A7 has no amino acid difference in the sequences of three subtypes among the PRRSV-1 strains and nine lineages among the PRRSV-2 strains,indicating a high degree of conservation.The results of the study provide a foundation the development of PRRSV diagnostic kits and novel vaccines.

Objective:To evaluate the efficacy and safety of a new plasma radiofrequency generator and a disposable mucosal incision knife for endoscopic submucosal dissection (ESD) of gastrointestinal lesions.Methods:This study is a prospective, randomized, non-inferiority multicenter study (Chinese Clinical Trial Registry No.: ChiCTR2000041394). The inclusion criteria for cases are as follows: (1) being able to understand and voluntarily participate in this clinical trial, and voluntarily signing the informed consent form; (2) being 18-70 years old, regardless of gender; (3) having a gastrointestinal lesion and meeting the endoscopic treatment conditions for the indications of endoscopic submucosal dissection (ESD). The exclusion criteria are: (1) meeting the contraindications for ESD; (2) having a surgical contraindication due to coagulation dysfunction or still taking anticoagulants 1 week before surgery; (3) having coagulation dysfunction, that is, the prothrombin time is prolonged by more than 3 seconds (more than 5 seconds in patients with liver disease), or the activated partial thromboplastin time is prolonged by more than 10 seconds, and the platelet count is < 70×10?/L; (4) having severe cardiopulmonary insufficiency and being unable to tolerate the surgery; (5) being a reproductive-aged woman with a positive blood or urine pregnancy test or a lactating woman; (6) the investigator deems that there are other factors that are not suitable for inclusion or affect the subject's participation. Patients who underwent ESD treatment for gastrointestinal lesions from March 2019 to April 2023 at the Endoscopy Center of Zhongshan Hospital Affiliated to Fudan University, the Department of Gastroenterology of Minhang District Central Hospital in Shanghai, and the Department of Gastroenterology of Quzhou People's Hospital were prospectively included. The experimental group used a new plasma radiofrequency therapy instrument and a disposable mucosal incision knife, while the control group used a high-frequency electrosurgical system and a disposable mucosal incision knife. The primary efficacy indicator was the en bloc resection success rate, the secondary efficacy indicators included the coagulation success rate, and the operation stability of the plasma radiofrequency therapy instrument and the disposable mucosal incision knife; the safety indicators included the incidence of intraoperative bleeding, intraoperative perforation, and postoperative complications. Results:The study cohort comprised 194 patients, 95 in the experimental group and 99 in the control group. Analysis of the full set showed rates of en bloc resection of 97.89% (93/95) and 96.97% (96/99) in the experimental and control groups, respectively; the difference being 0.53% (-5.58%, 6.64%). Analysis of the compliance set showed rates of en bloc resection of 97.83% (90/92) and 96.88% (93/96) in the experimental and control groups, respectively; the difference being 0.58% (-5.79%, 6.94%). These data indicate that the rate of en bloc resection in the experimental group using plasma radiofrequency therapy and disposable mucosal incision was not inferior to that of the control group. The rates of achieving intraoperative coagulation in the experimental and control groups were 7.14% (5/70) and 5.97% (4/67), respectively; this difference is not statistically significant ( P=1.000). The control group had a significantly better rate of using the same instruments throughout the procedure than did the experimental group ( P<0.001). In the safety analysis set, the incidences of intraoperative and postoperative adverse events did not differ significantly between the two groups (both P>0.05). Conclusion:Plasma radiofrequency therapy equipment and a disposable mucosal incision knife are safe and effective instruments for performing ESD of gastrointestinal lesions.

ObjectiveTo evaluate the effect and safety of Jiuxin Pill （救心丸） on exercise tolerance and quality of life in patients with stable angina pectoris （SAP）. MethodsA randomised， double-blind， placebo-controlled， multicentre study design was used to enroll 170 patients of SAP from nine centres， which were divided into 85 patients each in the trial group and control group with 1∶1 ratio. Both groups maintained the original western medicine treatment plan， and added Jiuxin Pill or placebo respectively， 2 pills （0.05 g） each time twicely for 28 days. The main outcomes were total exercise time （TED） in the exercise treadmill test and Seattle Angina Questionnaire （SAQ） scores including physical limitation （PL）， angina stability （AS）， angina frequency （AF）， treatment satisfaction （TS）， and disease perception （DP）. The secondary outcomes were exercise treadmill test indicators including heart rate recovery in 1 min （HRR1）， metabolic equivalents （METs）， maximum magnitude of ST-segment depression， and the Borg rating of perceived exertion scale， the average number of angina attacks per week， withdrawal and reduction rate of nitroglycerin， traditional Chinese medicine syndrome scores， incidence of major adverse cardiovascular events. Safety indicators were evaluated and the occurrence of adverse events during the trial was recorded. Data was collected before treatment， day 28±2 in treatment period， and follow-up at day 56 which is 28±2 days after treatment period finished. ResultsEighty-four and eighty-five patients respectively from trial group and control group were included to the full analysis set （FAS） and safety analysis set （SS）. Compared with the group before treatment and with the control group after treatment， the trial group had higher TED， HRR1， and METs， and lower maximum magnitude of ST-segment depression and Borg rating of perceived exertion scores after treatment （P＜0.01）. Compared with the group before treatment and with the control group after treatment and at follow-up， the total SAQ score and scores of AS， AF， TS and DP of the trial group after treatment and at follow-up elevated， while the average number of angina attacks per week and traditional Chinese medicine syndrome scores reduced （P＜0.01）. There was no statistically significant difference in the withdrawal and reduction rate of nitroglycerin between groups （P＞0.05）. Major adverse cardiovascular events occurred in 1 case （1/84， 1.19%） in the trial group and 1 case （1/85， 1.18%） in the control group， and the difference between groups was not statistically significant （P＞0.05）. A total of 3 cases of adverse events occurred in the trial group （3/84， 3.57%）， and a total of 6 cases of adverse events occurred in the control group （6/85， 7.06%）， and there was no statistically significant difference in the incidence of adverse events between groups （P＞0.05）. ConclusionIn the treatment of SAP， Jiuxin Pill combined with conventional western medicine can further enhance exercise tolerance， improve quality of life， and demonstrate great safety.

Objective Targeting the cell cycle and autophagy,Kangai injection(KAI)was investigated through PI3K/Akt/mTOR Signaling pathways improve the molecular mechanism of cisplatin resistance in human lung adenocarcinoma A549/DDP cells.Methods A549/DDP cells were randomly divided into control group,cisplatin group(20 μmol·L-1 cisplatin),low concentration KAI combined with cisplatin(15 mg·mL-1 KAI+20 μmol·L-1 cisplatin),medium concentration KAI combined with cisplatin(25 mg·mL-1 KAI+20 μmol·L-1 cisplatin)and high concentration KAI combined with cisplatin(50 mg·mL-1 KAI+20 μmol·L-1 cisplatin).The survival rate of cells was detected by CCK-8 method,the cell cycle distribution was detected by flow cytometry,the expression of p53 protein was detected by immunocytochemistry,and the expression levels of protein p53、autophagy-related protein LC3 and key molecules of autophagy pathway(mTOR,p-mTOR,Akt and p-Akt)were detected by Western blotting.Besides,the inhibitor of PI3K/Akt signaling pathway was used to further verify the molecular mechanism of KAI in reversing cisplatin in A549/DDP cells.Results Compared with the control group,cisplatin group and different concentrations of KAI combined with cisplatin groups effectively inhibited cell viability(P<0.05);compared with the cisplatin group,increased the cell proportion in S+G2/M phase(P<0.05),the mean integral absorbance of p53 protein(P<0.05),and the expression level of autophagy-related protein LC3Ⅱ(P<0.05),but decreased the expression levels of p-mTOR and p-Akt(both P<0.01).Moreover,LY294002,the inhibitor of PI3K/Akt signaling pathway,could effectively inhibit the up-regulation of LC3Ⅱ expression and cell death induced by KAI combined with cisplatin(both P<0.05).Conclusion KAI combined with cisplatin can induce cell cycle arrest and autophagic death by inhibiting PI3K/Akt/mTOR signaling pathway,and improve cisplatin resistance in NSCLC.

Objective Targeting the cell cycle and autophagy,Kangai injection(KAI)was investigated through PI3K/Akt/mTOR Signaling pathways improve the molecular mechanism of cisplatin resistance in human lung adenocarcinoma A549/DDP cells.Methods A549/DDP cells were randomly divided into control group,cisplatin group(20 μmol·L-1 cisplatin),low concentration KAI combined with cisplatin(15 mg·mL-1 KAI+20 μmol·L-1 cisplatin),medium concentration KAI combined with cisplatin(25 mg·mL-1 KAI+20 μmol·L-1 cisplatin)and high concentration KAI combined with cisplatin(50 mg·mL-1 KAI+20 μmol·L-1 cisplatin).The survival rate of cells was detected by CCK-8 method,the cell cycle distribution was detected by flow cytometry,the expression of p53 protein was detected by immunocytochemistry,and the expression levels of protein p53、autophagy-related protein LC3 and key molecules of autophagy pathway(mTOR,p-mTOR,Akt and p-Akt)were detected by Western blotting.Besides,the inhibitor of PI3K/Akt signaling pathway was used to further verify the molecular mechanism of KAI in reversing cisplatin in A549/DDP cells.Results Compared with the control group,cisplatin group and different concentrations of KAI combined with cisplatin groups effectively inhibited cell viability(P<0.05);compared with the cisplatin group,increased the cell proportion in S+G2/M phase(P<0.05),the mean integral absorbance of p53 protein(P<0.05),and the expression level of autophagy-related protein LC3Ⅱ(P<0.05),but decreased the expression levels of p-mTOR and p-Akt(both P<0.01).Moreover,LY294002,the inhibitor of PI3K/Akt signaling pathway,could effectively inhibit the up-regulation of LC3Ⅱ expression and cell death induced by KAI combined with cisplatin(both P<0.05).Conclusion KAI combined with cisplatin can induce cell cycle arrest and autophagic death by inhibiting PI3K/Akt/mTOR signaling pathway,and improve cisplatin resistance in NSCLC.

Objective:To analyze the influence of serological indexes on the liver cirrhosis (LC) in patients with chronic hepatitis B, and to construct a nomogram model.Methods:The clinical data of 220 patients with chronic hepatitis B in Xianning Central Hospital from January 2019 to December 2021 were retrospectively analyzed. Among them, 42 patients developed LC (LC group), and 178 cases did not develop LC (non-LC group). The patient′s fasting peripheral venous blood was taken in the morning. The platelet, red blood cell, white blood cell, fasting blood glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), triacylglycerol (TG), total cholesterol (TC), total bilirubin (TBiL), albumin, globulin, alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), prothrombin time (PT), thrombin time (TT), D-dimer (D-D), alpha-fetoprotein (AFP) and C-reactive protein (CRP) were detected. Receiver operating characteristic (ROC) curve was used to analyze the efficacy of each index in predicting LC in patients with chronic hepatitis B. Multivariate Logistic regression was used to analyze the independent risk factors for LC in patients with chronic hepatitis B. The R software "rms" package was used to construct a nomogram model to predict the LC in patients with chronic hepatitis B, the correction curve was used to internally verify the prediction model, and the decision curve evaluated the efficacy of the prediction model.Results:The TBiL, ALP, GGT, PT, TT, D-D, AFP and CRP in LC group were significantly higher than those in non-LC group: (50.57 ± 5.61) μmol/L vs. (46.69 ± 3.92) μmol/L, (105.23 ± 30.60) U/L vs. (75.14 ± 26.45) U/L, (68.73 ± 19.47) U/L vs. (50.39 ± 14.21) U/L, (13.88 ± 1.98) s vs. (13.01 ± 2.10) s, (18.88 ± 2.56) s vs. (15.98 ± 2.43) s, (2.62 ± 1.04) mg/L vs. (1.34 ± 0.63) mg/L, (4.19 ± 1.95) μg/L vs. (2.66 ± 1.21) μg/L and (8.54 ± 1.22) mg/L vs. (7.47 ± 0.79) mg/L, the platelet, ALT, AST and albumin were significantly lower than those in the non-LC group: (129.63 ± 32.66) × 10 9/L vs. (183.53 ± 56.31) ×10 9/L, (131.27 ± 22.19) U/L vs. (157.57 ± 38.67) U/L, (112.76 ± 19.57) U/L vs. (125.16 ± 21.84) U/L and (29.79 ± 6.17) g/L vs. (33.52 ± 5.89) g/L, and there were statistical differences ( P<0.01 or <0.05); there were no statistical differences in red blood cell, white blood cell, fasting blood glucose, TG, TC and globulin between the two groups ( P>0.05). ROC curve analysis result showed that the area under the curve (AUC) of AFP, platelet, ALT, AST, ALP, GGT, TBiL, albumin, D-D, CRP, PT and TT for predicting LC in patients with chronic hepatitis B were 0.731, 0.798, 0.723, 0.676, 0.766, 0.762, 0.710, 0.673, 0.856, 0.759, 0.603 and 0.786, and the optimal cut-off values were 4.64 μg/L, 162.56 × 10 9/L, 155.67 U/L, 122.37 U/L, 95.17 U/L, 68.96 U/L, 49.95 μmol/L, 28.8 g/L, 1.64 mg/L, 8.55 mg/L, 12 s and 18 s. Multivariate Logistic regression analysis result showed that AFP (>4.64 μg/L), platelet (≤162.56 × 10 9/L), ALP (>95.17 U/L), GGT (>68.96 U/L), D-D (>1.64 mg/L) and TT (>18 s) were independent risk factors for LC in patients with chronic hepatitis B ( OR = 1.278, 1.428, 1.488, 1.356, 1.513 and 1.369; 95% CI 1.109 to 1.369, 1.269 to 1.623, 1.217 to 1.894, 1.127 to 1.669, 1.342 to 1.878 and 1.169 to 1.583; P<0.05 or <0.01). The AFP, platelet, ALP, GGT, D-D and TT were used as predictors to construct a nomogram model for predicting the LC in patients with chronic hepatitis B. The correction curve of the nomogram model to predict the LC in patients with chronic hepatitis B was close to the ideal curve (C-index was 0.739, 95% CI 0.615 to 0.876); the decision curve analysis result showed that the prediction model had higher clinical net benefit when the risk threshold > 0.26 than a single index, and that it had significantly additional clinical net benefit. Conclusions:The AFP, platelets, ALP, GGT, D-D and TT are independent risk factors for LC in patients with chronic hepatitis B, and the nomogram model constructed based on these factors could provide important guidance for the prevention and treatment of LC in patients with chronic hepatitis B.

Objective:To understand the relationship between the RNA shedding time of SARS-CoV-2 infected persons and the single nucleotide mutation of the virus, the population of infected persons, underlying diseases and other factors, so as to provide more clues for the study of SARS-CoV-2 infection dynamics.Methods:The data of epidemiology, clinical manifestations, and underlying diseases of infected persons in a cluster epidemic of COVID-19 in Jiangsu province from July to September 2021 were collected. Nasopharyngeal swab samples of cases were collected, and the whole genome of the virus was sequenced by second-generation sequencing technology. The online analysis platform was used to judge the virus type and analyze the mutation site, and Cox proportional risk model was used to analyze the relationship between the RNA shedding time of SARS-CoV-2 and various research factors.Results:There were 350 persons who finally obtained the whole genome sequence of the virus in this COVID-19 outbreak, of which 60.3% were female, the median age was 49 years old (interquartile range, IQR: 37-65 years old)), and the median time of virus shedding was 33 days ( IQR, 26-44 days). The whole-genome sequencing analysis showed that compared with the Wuhan reference strain sequence, the infected persons’ sequence had 34~41 nucleotide mutation sites, belonging to VOC/Delta variant (B.1.617.2 evolutionary branch), and C346T, C1060T, T2803C, T7513C, A29681C were the main single nucleotide polymorphisms (SNPs) of this epidemic. Cox regression analysis showed that age, underlying disease, clinical classification, vaccination, SNP T2803C and T7513C had an impact on the RNA shedding time of SARS-CoV-2. The adjusted multivariate Cox regression result showed that age [ HR=0.73, 95% CI (0.55, 0.95)] and T7513C [ HR=0.37, 95% CI (0.18, 0.77)] were still the risk factors for the extension of SARS-CoV-2 RNA shedding time. Conclusions:This study analyzed the effects of the individual factors and viral single nucleotide variations on the time of viral RNA shedding. Those who were older, suffered from hypertension, had more severe clinical symptoms, were not vaccinated or incompletely vaccinated, and had T7513C mutation in the infected virus, had a risk of a long RNA shedding time of SARS-CoV-2, which should be given special attention and follow-up after rehabilitation.

Objective : To investigate the effects of Lin28 overexpression on the proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs) through mTOR signaling pathway. Methods : After transfecting lentiviral vectors of Lin28 gene in hDPSCs , the relative expression of Lin28 was detected by Real⁃time PCR. CCK⁃8 assay was applied to detect the effect on cell proliferation. qRT⁃PCR was used to research the expression levels of alkaline phosphatase (ALP) , osteopontin (OPN) and osteocalcin (OCN) . Western blot assay was processed to investigate the effects on the relative expression levels of ALP and OPN proteins. Alizarin red staining was utilized to detect the mineralized nodules. Results : Compared with the control group , the cell proliferation of transfection group was promoted (P < 0. 05) ; The mRNA and protein expression levels of ALP , OPN and OCN in transfection group were significantly lower than those in control group (P < 0. 05) , the expression level of ALP apparently decreased after the addition of mTOR inhibitor rapamycin (P < 0. 05) ; Alizarin red staining showed that the size and number of mineralized nodules formed in transfection group were markedly declined compared with empty carrier group (P < 0. 05) . Conclusion Overexpression of Lin28 can inhibit the osteogenic differentiation of hDP⁃ SCs through suppress mTOR signaling pathway.