Objective To optimize the culture process of insect cell-baculovirus expression vector system(BEVS) in order to further improve the protein expression of recombinant hepatitis E virus-like particles(HEV-LPs), and to scale up and verify the process.Methods Serum-free suspension culture of Sf9 insect cells was carried out and a 7 L bioreactor was established through shaking flask cascade amplification. The parameters such as viable cell density, culture temperature and recombinant virus inoculation ratio were optimized when the cells were inoculated with recombinant baculovirus. The cell growth status was observed and the expression level of the target protein was analyzed. The target protein was detected by SDSPAGE, Western blot, high-performance liquid chromatography(HPLC) and transmission electron microscope(TEM). The morphology of HEV-LPs and protein yield were integrated, and the suitable expression parameters of recombinant HEV-LPs were determined to prepare three batches of 7 L recombinant hepatitis E vaccine.Results The optimal parameters for the cultivation of insect cells in a 7 L bioreactor were as follows: stirring 90 r/min, pH(6. 2 ± 0. 1), optimal incubation temperature 27 ℃, cell density of recombinant baculovirus inoculation 6. 0 × 10~6 cells/mL, and dissolved oxygen 90%. Under this condition, the relative molecular mass of the expressed HEV-LPs target protein was about 58 000, which had a specific binding with HEV mouse monoclonal antibody. The final yield was 60-70 mg/L, with the purity of more than 95%. Spherical particles with a diameter of about 20 nm were observed under TEM. The HEV-LPs recombinant protein prepared by the optimized parameters had stable yield and uniform particle morphology.Conclusion The culture and expression process of recombinant hepatitis E vaccine was successfully optimized, and the stability of the amplification process was verified, which lays a foundation for the development of recombinant hepatitis E vaccine production process.

Background Recent studies have demonstrated that glaucoma progression had great relationship with corneal biomechanical properties.However,there are not enough studies about corneal biomechanical properties in different types of glaucoma.Objective This study was to evaluate the corneal biomechanical properties in chronic primary angle-closure glaucoma (CPACG) and primary open angle glaucoma (POAG).Methods Prospective observational study was adopted.Sixty-eight cases (68 eyes) with CPACG and 69 POAG patients (69 eyes) from Zhongshan Ophthalmic Center were investigated by a novel technique named Corvis-ST which can measure corneal deformation.The Cronbach's αt coefficient and intraclass correlation coefficient (ICC) were used to evaluate the repeatability of the parameters from Corvis-ST.The differences of corneal biomechanical properties between POAG and CPACG were evaluated by independent-samples t test.Multiple regression analysis was used to assess the association between parameters of deformation response and clinical factors.This study protocol was approved by Ethic Committee of Zhongshan Ophthalmic Center and complied with Helsinki Declaration.Written informed consent was obtained from each patient prior to any medical examination.Results There were no statistic differences about age,sex and intraocular pressure (IOP) between CPACG and POAG patients (all at P＞0.05).However,differences existed in the number of anti-glaucoma medications (t =-2.388,P =0.020).Measurements of central corneal thickness (CCT),deformation amplitude (DA),and peak distance (PD) demonstrated excellent repeatability (ICC≥ 0.8).There were no statistic differences about CCT,first applanation time/length/velocity (A1 T,A1 L,A1V),second applanation time/length/velocity (A2T,A2L,A2V),DA between CPACG and POAG patients (all at P＞ 0.05).The highest concavity time was shorter (t =2.920,P =0.005),PD was bigger (t =-2.453,P =0.017),and central curvature radius (CCR) was smaller (t =1.997,P =0.050) in POAG than those in the CPACG patients.DA,A1V,A2T and PD were negatively associated with IOP (r =-0.709,-0.531,-0.645,-0.554;all at P＜0.001).However,A1T and A2V were positively associated with IOP (r=0.744,0.546;both at P＜0.001).And CCR was positively associated with CCT (r =0.181,P =0.039).Conclusions It is useable to evaluate corneal biomechanical properties of glaucoma by Corvis ST.Under the same level of IOP,the corneal concavity of POAG is easier than that of CPACG,which indicates that cornea in POAG patients is more deformable.

Objective To study the genetic diversity in Fujian Sanming Sarcandra Glabra from different geographical provenances;To construct genetic relationship diagram.Methods DNA in leaves was extracted by CTAB. Analysis and evaluation of DNA molecular level of 45 samples of different geographical provenances were conducted by ISSR method. POPgen32 software was used to calculate the genetic diversity and establish gene trees. NTSYS software was used to carry out cluster analysis.Results Six ISSR primers amplified 630 bands. Genetic diversity analysis showed that the average effective number of alleles of 45 varieties was 1.620 2;the average Nei's genetic diversity index was 0.364 5;the average Shannon information index was 0.543 2. Each point had different levels of genetic diversity. Nei's genetic diversity index of the maximum value was 0.497 2, and the minimum value was 0.107 8;Maximum Shannon information index was 0.690 3, and the minimum value was 0.219 2. Cluster analysis results showed that 45 varieties and 14 loci band data were the primitive matrix. 630 genetic similarity coefficients between two different species were obtained. The maximum similarity coefficient among different groups was 1.0, and the minimum was 0.516.Conclusion Different varieties of Fujian Sanming Sarcandra Glabra exist abundant genetic variation and has the molecular basis of abundant species. Using 0.610 as the threshold value can divide Sarcandra Glabra from 45 different geographical provenances into 6 groups. The genetic distance and geographical distance was not related.

Hymenolepis nana and Hymenolepis diminuta are globally widespread zoonotic cestodes. Rodents are the main reservoir host of these cestodes. Brown rats (Rattus norvegicus) are the best known and most common rats, and usually live wherever humans live, especially in less than desirable hygiene conditions. Due to the little information of the 2 hymenolepidid species in brown rats in China, the aim of this study was to understand the prevalence and genetic characterization of H. nana and H. diminuta in brown rats in Heilongjiang Province, China. Total 114 fecal samples were collected from brown rats in Heilongjiang Province. All the samples were subjected to morphological examinations by microscopy and genetic analysis by PCR amplification of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene. In total, 6.1% (7/114) and 14.9% (17/114) of samples were positive for H. nana and H. diminuta, respectively. Among them, 7 and 3 H. nana isolates were successfully amplified and sequenced at the COX1 and ITS2 loci, respectively. No nucleotide variations were found among H. nana isolates at either of the 2 loci. Seventeen H. diminuta isolates produced 2 different COX1 sequences while 7 ITS2 sequences obtained were identical to each other. The present results of H. nana and H. diminuta infections in brown rats implied the risk of zoonotic transmission of hymenolepiasis in China. These molecular data will be helpful to deeply study intra-specific variations within Hymenolepis cestodes in the future.
Animals ; Cestoda ; China* ; Electron Transport Complex IV ; Genes, Mitochondrial ; Genes, rRNA ; Humans ; Hygiene ; Hymenolepiasis ; Hymenolepis diminuta ; Hymenolepis nana* ; Hymenolepis* ; Microscopy ; Polymerase Chain Reaction ; Prevalence* ; Rats* ; Rodentia

Objective:To prepare the multilayer alginate chitosan microspheres loading vascular endothelial growth factor (VEGF) and vancomycin (VAN), and to study in vitro release characteristics.Methods:The microspheres were prepared by emulsion cross-linking and self-assembly techniques.The effects of sodium alginate concentration, calcium chloride concentration, oil/water ratio and span80 concentration on the entrapment efficiency(EE) and drug loading(DL) of VEGF and VAN were investigated by orthogonal experimental design to optimize the preparation process.The surface morphology and particle size of microspheres were observed by scanning electron microscope (SEM).Self-assembly was detected by Fourier transform infrared spectroscope (FTIR).The EE, DL and in vitro release of VEGF and VAN were detected by ELISA double antibody sandwich method and ultraviolet spectrophotometry,and the cumulative release curve was drawn.Results:The prepared microspheres were yellowish brown powder.The SEM results showed that the microspheres were spherical, the surface was smoothy, and the dispersity was better.The average particle size was about 50 μm.Sodium alginate concentration of 1.0 g·mL-1, CaCl2 concentration of 8 g·mL-1, oil to water ratio of 3∶1, and span80 concentration of 2% were the best formula.The EE of VEGF and VAN were 49.63% and 16.67%, respectively.In vitro, the cumulative release last 16.5 d and 12.5 d respectively and the amount reached up to 95%.Conclusion:The multilayer alginate chitosan microspheres loading VEGF and VAN present several advantages, such as smaller particle size, higher EE and better controlled release.

Mucin-type O-glycosylation is one of the most common post-translational modifications in proteins,capable of altering protein conformation and biological functions.It plays a crucial role in biological processes such as cell signaling,cell adhesion,and immune responses.Polypeptide N-acetylgalactosaminyltransferase 3(GALNT3),as the initiating enzyme of mucin-type O-glycosylation,is of paramount importance in maintaining the homeostasis of human cells and tissues.Dysfunction of GALNT3 has been found to play a role in various diseases,such as calcium-phosphorus metabolism disorders and atherosclerosis.Additionally,GALNT3 is abnormally expressed in several types of tumors,including colorectal cancer,lung cancer,and ovarian cancer.Its expression is associated with the clinical pathological features of patients and poor prognosis,making it a potential biomarker for early tumor diagnosis and prognosis evaluation.Further research shows that GALNT3 can both regulate glycosylation levels to reduce adhesion between tumor cells and activate multiple metabolism-related pathways,promoting tumor cell invasion and metastasis.This review summarizes the role of GALNT3 in the development of malignant tumors and discusses the prospects and challenges of developing anti-tumor drugs targeting GALNT3.

Objective To observe the effect of extracorporeal shockwave therapy (ESWT) in regulating endothelial cell phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression on lower ex-tremity vascular lesion and its possible mechanism.Methods Twenty-four 2-month-old healthy male SD rats were randomly divided into the three groups:control group (group A),diabetes angiopathy group (group B),diabetes angiopathy+ESWT group (group C).The group B and C were fed with high fat and high sugar and intraperitoneally injected with streptozotocin 60 mg/kg to establish the rat model of diabetes vascular lesion. The group C received ESWT at 1 week (T1),2 weeks (T2),3 weeks (T3) and 4 weeks (T4) after modeling,and the blood stream velocity of rat femoral artery vascular lesion area and vascular internal diameter were measured at T4 by ultrasound.At the end of ESWT,the rats were immediately killed for taking their femoral arteries and gastrocnemius.The structures of the femoral arteries in each group were observed under electron microscopy.Western blot was used to detect the expression levels of PTEN,PI3K and Akt proteins,while qRT-PCR was used to detect the expression levels of PTEN mRNA.Immunofluorescence was used to detect the expression level of CD31 in gastrocnemius muscle .Results The peak systolic flow velocity and end-dias-tolic flow velocity of femoral artery at T4 in group B and C were significantly lower than those in group A (P<0.05),but group C was higher than group B (P<0.05).The internal diameter of femoral artery had no statistical difference among 3 groups (P>0.05).The PTEN expression level in group B and group C was sig-nificantly lower than that in group A (P<0.05),while group C was higher than group B (P<0.05).The ex-pression levels of PI3K and Akt in group B were higher than those in group A (P<0.05),and group C was lower than group B (P<0.05).The PTEN mRNA expression level in group B and group C was significantly lower than that in group A (P<0.05),but group C was higher than group B (P<0.05).Under electron mi-croscopy,it was observed that after ESWT,the endothelial cell damage in group C was obvious when com-pared with group B.The CD31 expression level in group B and group C was significantly lower than that in group A (P<0.05),but group C was higher than group B (P<0.05).Conclusion ESWT could improve the vascular function,increase the peak velocity during systolic period of femoral artery in diabetes rats and im-prove the microvessel density of gastrocnemius muscle by up-regulating PTEN in lower extremity artery and down-regulating PI3K and Akt in diabetes rats.

Gallbladder cancer is a highly malignant tumor of the digestive system,and its incidence in China has been increasing year by year.Due to the lack of specific symptoms in the early stage,early diagnosis is difficult for gallbladder cancer and it is often at advanced stage once discovered.Surgery is still the only method to cure gallbladder cancer.Therefore,early diagnosis is particularly important for radical resection and improving overall prognosis of gallbladder cancer patients.However,there is still no specific molecular diagnostic target for gallbladder cancer.The continuous improvement of research and surgical skills have made the operational procedure of gallbladder cancer more standardized.With the increasing understanding on the epidemiology and pathogenesis of gallbladder cancer and the improvement of imaging,radiotherapy and chemotherapy technology,the comprehensive diagnosis and treatment of gallbladder cancer has also developed rapidly in recent years.However,high-quality clinical research is still needed.Comprehensive treatment model including radical surgery,adjuvant radiotherapy and chemotherapy,targeted therapy and immunotherapy will be the trend of gallbladder cancer treatment in the future.This article summarizes the advances in comprehensive diagnosis and treatment of gallbladder cancer by reviewing literatures.

Objective To develop, optimize and preliminarily verify an indirect immunofluores-cence assay ( IFA) for detecting the titer of recombinant baculovirus. Methods Conditions for performing IFA, including cell concentration, co-incubation time, reaction temperature, dilution ratio, reaction time and types of fixative solution, blocking liquid and antibodies, were optimized to establish an IFA method for the detection of baculovirus titer. Specificity, accuracy, reproducibility and intermediate precision of the es-tablished assay were verified. And the results were compared with those of baculovirus rapid titer kit. Re-sults The optimal cell concentration for coating was 0. 6×106 cell/ml, and the optimal reaction time be-tween viruses with cells was 3 d. The optimal conditions for conducting IFA were as follows: formaldehyde buffered acetone (-20℃) was used as fixative, normal goat serum was used as blocking liquid and the first and second generation antibodies at a dilution of 1 : 200 were incubated at 37℃ for 1 h, respectively. Spe-cific fluorescence was observed only in baculovirus but not in others by using the method. No significant difference in virus titers was observed between the established IFA and baculovirus rapid titer kit. The two methods showed a good linear correlation (R2=0. 996). Coefficients of variation for evaluating the reproduc-ibility and intermediate precision were less than 10％. Conclusion IFA for the detection of baculovirus ti-ter was established with good specificity, accuracy, reproducibility and intermediate precision.

Objective:To investigate the influencing factors of prolonged postoperative ileus (PPOI) in patients undergoing pancreaticoduodenectomy (PD) during hospitalization.Methods:The data of 339 patients underwent PD admitted to the Department of Hepatobiliary Surgery of the Second Hospital of Hebei Medical University from January 2018 to September 2023 were retrospectively analyzed, including 204 males and 135 females, aged (60.6±11.2) years. Among the 339 patients, 112 (33.0%) had pancreatic tumors, 94 (27.7%) had Vater ampullary tumors, 82 (24.2%) had common bile duct tumors, and 51 (15.0%) had duodenal tumors. A total of 339 patients with PPOI were included in the PPOI group ( n=43) and those without PPOI were included in the control group ( n=296). The two groups were compared in terms of age, PD operation (open or laparoscopic), gastrojejunostomy (retrocolic or antecolic gastrojejunostomy), grade B or C pancreatic fistula, hypokalemia, and postoperative use of patient-controlled intravenous analgesia (PCIA). The index comparing P<0.05 between the two groups was further included in the multivariate logistic regression analysis to analyze the influencing factors of PPOI in PD patients. Results:There were statistically significant differences in age >70 years, PD operation, gastrojejunostomy, grade B or C pancreatic fistula, hypokalemia, and postoperative use of PCIA between the two groups (all P<0.05). Multivariate logistic regression analysis showed grade B or C pancreatic fistula ( OR=3.17, 95% CI: 1.48-6.82), open surgery ( OR=2.90, 95% CI: 1.35-6.24), retrocolic gastrojejunostomy ( OR=2.47, 95% CI: 1.23-4.95), postoperative usage of PCIA ( OR=2.61, 95% CI: 1.21-5.62), age >70 years ( OR=2.47, 95% CI: 1.71-5.19) had a high risk of PPOI during postoperative hospitalization (all P<0.05). Conclusion:Postoperative grade B or C pancreatic fistula, open surgery, retrocolic gastrojejunostomy (compares with antecolic gastrojejunostomy), postoperative using PCIA, and age >70 years are independent risk factors for PPOI in patients undergoing PD during postoperative hospitalization.