ObjectiveTo investigate the impact of aberrant SKI expression and its mutations on the biological characteristics of cholangiocarcinoma cell lines QBC939 and RBE， and to explore the underlying molecular mechanisms. MethodsThe Gene Expression Profiling Interactive Analysis 2 （GEPIA2） database was utilized to analyze SKI expression and its clinical relevance in cholangiocarcinoma patients. Lentiviral transduction was employed to establish QBC939 and RBE cell lines with stable SKI overexpression， mutation， or knockdown. Cell proliferation was assessed using CCK-8， colony formation， and EdU assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Cell migration was evaluated using Transwell and wound healing assays. The effect of SKI over-expression， mutation， or knockdown on key proteins （SMAD2， SMAD3， SMAD4） in the transforming growth factor-β （TGF-β）/Small mothers against decapentaplegic （SMAD） signaling pathway was examined by Western blot. ResultsCompared to SKI overexpression alone， the introduction of SKI mutations significantly promoted S-phase progression， enhanced proliferation and migration， and inhibited apoptosis. Mechanistically， SKI mutations suppressed the phosphorylation of SMAD2 and SMAD3 proteins， thereby inhibiting the transcriptional activity of the TGF-β signaling pathway. Conversely， SKI knockedown produced the opposite effects. ConclusionSKI gene mutation acts as a gain-of-function genetic alteration， exerting an oncogenic role in cholangiocarcinoma cells. The primary mechanism involves the inhibition of the TGF-β/SMAD signaling pathway， which in turn promotes proliferation and cell cycle progression， and suppresses apoptosis in QBC939 and RBE cells， ultimately driving tumor progression.

The receptor-binding region(RBD)of the spike protein(S)on the surface of porcine epi-demic diarrhea virus(PEDV)is a critical structural domain mediating viral invasion of host cells and serves as a key target for inducing neutralizing antibodies.In order to prepare antibodies that can be used to study the biological function of PEDV RBD and develop novel diagnostic and thera-peutic methods,recombinant RBD protein expressed in Sf9 insect cells was utilized as an immuno-gen to immunize BALB/c mice.Monoclonal antibodies(mAbs)were generated via hybridoma tech-nology,and positive hybridoma clones were screened using indirect ELISA.The reactivity of the mAbs was subsequently characterized.The results of ELISA,Western blot,and indirect immuno-fluorescence assay(IFA)showed that three monoclonal antibodies screened(3E5,4F9 and 5A5)had good reactivity with the virus and RBD protein.Antibody subtype results showed that 3E5 and 4F9 were of IgG1 subtypes and 5A5 was of IgM subtype.Neutralization assay further revealed that 3E5 monoclonal antibody had viral neutralizing activity.In this study,three monoclonal antibodies against PEDV RBD proteins were successfully prepared,providing the basis for the study of the bi-ological function of RBD proteins,PEDV serologic detection and vaccine development.

Objective:To analyze the effect of mouse nerve growth factor(mNGF)combined with rehabilitation training on the expression of BDNF and Caspase-3 in brain tissue of newborn rats with spastic cerebral palsy(SCP).Method:A total of 150 clean-grade SD rats aged 7days were randomly divided into the control group(group NS,n=40)and the model group(group T,n=110)using a random number table.Group T received directional injection of absolute ethanol(1μl/g)into the left intracranial pyramidal tract at the age of 7 days,while the NS group received an equal volume of saline.24 hours after injection(at 8 days old),the spastic cerebral palsy rat model was determined by modified Li Xiaojie et al.'s criteria combined with Longa scoring method.Ninety successfully modeled SD rats in group T were randomly divided into group A1,A2 and A3,with 30 each.The intervention began on the 1st day of the experiment(8 days old).Group A1 was given intramuscu-lar injection of mNGF(30ng/g),group A2 was given intramuscular injection of an equal dose of mNGF plus rehabilitation training,and group A3 was given intramuscular injection of an equal volume of saline(2.27ml/kg).The group NS was given intramuscular injection of the equal volume of normal saline.Intervention lasted until 21 days of age.At 8,14 and 21 days of age,HE staining was used to observe the dynamic changes in the morphology of cells in the internal capsule area of brain,TUNEL staining to observe the dynamic changes in the apoptosis rate of cells in the internal capsule area of brain,and Western Blot to monitor dy-namic changes in the expression of BDNF and Caspase-3 proteins in the hippocampus.Result:Model preparation showed that a total of 97 spastic cerebral palsy litters were identified in group T at 8 days old,while no rats with cerebral palsy in groupNS.TUNELstaining revealed that the apoptosis rates of the rats in group A1,A2 and A3 were significantly higher than in group NS at each age(P<0.01),and were significantly higher in group A3 than in group A1 and A2 at 14 and 21 days of age(P<0.01),with group A1 significantly higher than group A2(P<0.01).Western Blot showed that the relative expression of BDNF and Caspase-3 protein in group A1,A2 and A3 at 14 days old were higher than that in group NS(P<0.05).The rel-ative expression of BDNF protein in group A1 and A2 was higher than that in group A3(P<0.05),with group A2 was higher than that in group A1(P<0.05).There was no significant difference between group A2 and A1 at 21 days old(P>0.05).The relative expression of Caspase-3 protein in group A1 and A3 was higher than that in group A2(P<0.05).and group A3 was significantly higher than that in A1 group(P<0.05).Conclusion:It is speculated that both mNGF and combined rehabilitation training can inhibit neuronal apoptosis by promoting BDNF protein expression and reducing the activity of Caspase-3.The effect of mNGF combined with rehabilitation training is better than that of mNGF alone.

The receptor-binding region(RBD)of the spike protein(S)on the surface of porcine epi-demic diarrhea virus(PEDV)is a critical structural domain mediating viral invasion of host cells and serves as a key target for inducing neutralizing antibodies.In order to prepare antibodies that can be used to study the biological function of PEDV RBD and develop novel diagnostic and thera-peutic methods,recombinant RBD protein expressed in Sf9 insect cells was utilized as an immuno-gen to immunize BALB/c mice.Monoclonal antibodies(mAbs)were generated via hybridoma tech-nology,and positive hybridoma clones were screened using indirect ELISA.The reactivity of the mAbs was subsequently characterized.The results of ELISA,Western blot,and indirect immuno-fluorescence assay(IFA)showed that three monoclonal antibodies screened(3E5,4F9 and 5A5)had good reactivity with the virus and RBD protein.Antibody subtype results showed that 3E5 and 4F9 were of IgG1 subtypes and 5A5 was of IgM subtype.Neutralization assay further revealed that 3E5 monoclonal antibody had viral neutralizing activity.In this study,three monoclonal antibodies against PEDV RBD proteins were successfully prepared,providing the basis for the study of the bi-ological function of RBD proteins,PEDV serologic detection and vaccine development.

Objective:To analyze the effect of mouse nerve growth factor(mNGF)combined with rehabilitation training on the expression of BDNF and Caspase-3 in brain tissue of newborn rats with spastic cerebral palsy(SCP).Method:A total of 150 clean-grade SD rats aged 7days were randomly divided into the control group(group NS,n=40)and the model group(group T,n=110)using a random number table.Group T received directional injection of absolute ethanol(1μl/g)into the left intracranial pyramidal tract at the age of 7 days,while the NS group received an equal volume of saline.24 hours after injection(at 8 days old),the spastic cerebral palsy rat model was determined by modified Li Xiaojie et al.'s criteria combined with Longa scoring method.Ninety successfully modeled SD rats in group T were randomly divided into group A1,A2 and A3,with 30 each.The intervention began on the 1st day of the experiment(8 days old).Group A1 was given intramuscu-lar injection of mNGF(30ng/g),group A2 was given intramuscular injection of an equal dose of mNGF plus rehabilitation training,and group A3 was given intramuscular injection of an equal volume of saline(2.27ml/kg).The group NS was given intramuscular injection of the equal volume of normal saline.Intervention lasted until 21 days of age.At 8,14 and 21 days of age,HE staining was used to observe the dynamic changes in the morphology of cells in the internal capsule area of brain,TUNEL staining to observe the dynamic changes in the apoptosis rate of cells in the internal capsule area of brain,and Western Blot to monitor dy-namic changes in the expression of BDNF and Caspase-3 proteins in the hippocampus.Result:Model preparation showed that a total of 97 spastic cerebral palsy litters were identified in group T at 8 days old,while no rats with cerebral palsy in groupNS.TUNELstaining revealed that the apoptosis rates of the rats in group A1,A2 and A3 were significantly higher than in group NS at each age(P<0.01),and were significantly higher in group A3 than in group A1 and A2 at 14 and 21 days of age(P<0.01),with group A1 significantly higher than group A2(P<0.01).Western Blot showed that the relative expression of BDNF and Caspase-3 protein in group A1,A2 and A3 at 14 days old were higher than that in group NS(P<0.05).The rel-ative expression of BDNF protein in group A1 and A2 was higher than that in group A3(P<0.05),with group A2 was higher than that in group A1(P<0.05).There was no significant difference between group A2 and A1 at 21 days old(P>0.05).The relative expression of Caspase-3 protein in group A1 and A3 was higher than that in group A2(P<0.05).and group A3 was significantly higher than that in A1 group(P<0.05).Conclusion:It is speculated that both mNGF and combined rehabilitation training can inhibit neuronal apoptosis by promoting BDNF protein expression and reducing the activity of Caspase-3.The effect of mNGF combined with rehabilitation training is better than that of mNGF alone.

Porcine deltacoronavirus(PDCoV),a newly discovered virus in recent years,can cause severe diarrhea,vomiting,dehydration and even death in piglets.S protein is an important structur-al protein of PDCoV,which determines the host or tissue tropism of the virus,and is an important target for the development of PDCoV vaccines and detection methods.In order to prepare mono-clonal antibody(MAb)against the S protein of PDCoV,the recombinant plasmid of S protein was constructed based on the extracellular domain sequence of S protein of PDCoV epidemic strain in China and transformed into DH10Bac competent cells.The recombinant bacmid was identified by blue-white spot screening and PCR.BALB/c mice were immunized with S protein,and the spleen cells of immunized mice were fused with myeloma cells.The positive hybridoma cells that secreted stable antibodies were screened by indirect ELISA and subcloning.Five hybridoma cell superna-tants(MAb)specifically recognizing S protein(2E5,4D5,5D10,2F7 and 5A9)were identified by Western blot and immunofluorescence assay(IFA).Subsequently,the neutralization test showed that three of the monoclonal antibodies(2E5,4D5 and 5D10)could neutralize the virus to varying degrees.The S protein was successfully expressed and 5 monoclonal antibodies that can stably se-crete and specifically bind to PDCoV and S protein were prepared,which laid an important founda-tion for further research on the structure and function of S protein,the development of detection methods for PDCoV infection,and the prevention or treatment of PDCoV infection.

Objective To explore the activation of tumor necrosis factor-α(TNF-α)signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells.Methods A human proximal renal tubular cell(HK2)model of lentivirus-mediated NPHP1 knockdown(NPHP1KD)was constructed,and the expressions of TNF-α,p38,and C/EBPβ and the inflammatory factors CXCL5,CCL20,IL-1β,IL-6 and MCP-1 were detected using RT-qPCR,Western blotting or enzyme-linked immunosorbent assay.A small interfering RNA(siRNA)was transfected in wild-type and NPHP1KDHK2 cells,and the changes in the expressions of TNF-α,p38,and C/EBPβ and the inflammatory factors were examined.Results NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α,C/EBPβ,CXCL5,IL-1β,and IL-6(P<0.05),protein expressions of phospho-p38 and C/EBPβ(P<0.05),and IL-6 level in the culture supernatant(P<0.05),and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ(P<0.05).Conclusion TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells,and C/EBPβ may serve as a key transcription factor to mediate these changes.

Objective To explore the activation of tumor necrosis factor-α(TNF-α)signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells.Methods A human proximal renal tubular cell(HK2)model of lentivirus-mediated NPHP1 knockdown(NPHP1KD)was constructed,and the expressions of TNF-α,p38,and C/EBPβ and the inflammatory factors CXCL5,CCL20,IL-1β,IL-6 and MCP-1 were detected using RT-qPCR,Western blotting or enzyme-linked immunosorbent assay.A small interfering RNA(siRNA)was transfected in wild-type and NPHP1KDHK2 cells,and the changes in the expressions of TNF-α,p38,and C/EBPβ and the inflammatory factors were examined.Results NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α,C/EBPβ,CXCL5,IL-1β,and IL-6(P<0.05),protein expressions of phospho-p38 and C/EBPβ(P<0.05),and IL-6 level in the culture supernatant(P<0.05),and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ(P<0.05).Conclusion TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells,and C/EBPβ may serve as a key transcription factor to mediate these changes.

Objective : To investigate the effects of C-type lectin domain family 5，member A( CLEC5A) on the pro- liferation，apoptosis，and cell cycle of leukemia cell lines THP-1 and K562，and the underlying mechanism． Methods : The expression of CLEC5A in leukemia patients was investigated in the GEPIA database. Recombined plasmid containing CLEC5A was transfected into THP-1 and K562 cells for overexpression of CLEC5A．Small interfering RNA(siRNA) was used to knock down the endogenous CLEC5A in leukemia cells．CCK-8 and EdU assays were used to assess the leukemia cells proliferation．Flow cytometry was used to assess cell cycle．Flow cytometry was used to assess cell apoptosis under hydrogen peroxide( H2 O2 ) stress．The RNA sequencing( RNA-seq) and pathway enrichment analysis were used to analyze the signal pathways of significant enrichment of up-regulated or down-reg- ulated genes after knocking down CLEC5A gene．Protein expression levels of several members in AKT1 / mTOR and p53 signaling pathways were detected by Western blot assays. Results : CLEC5A was significantly up-regulated in bone marrow tissues of leukemia patients compared to the matched non-tumor tissues of healthy individuals．Knock- down of CLEC5A significantly reduced the proliferation(all P＜0. 01) and S phase progression(all P＜0. 05) ，and increased the apoptosis(all P＜0. 001) under H2 O2 stress，in THP-1 and K562 cells．Conversely，overexpression of CLEC5A significantly increased the proliferation(all P ＜0. 001) and S phase progression ( all P ＜0. 01) ，and re- duced the apoptosis(all P＜0. 01) under H2 O2 stress，in THP-1 and K562 cells．The uregulated genes were sig- nificantly enriched in AKT1-mTOR and other signal pathways after knocking down CLEC5A，while the down-regula- ted genes were significantly enriched in cell cycle signal pathways．CLEC5A in leukemia cells significantly reduced the genes expression levels of BAX and p53，and significantly induced the gene expression levels of BCL-2 and phosphorylation levels of AKT1 and mTOR proteins． Conclusion CLEC5A increases the cell cycle and proliferation and inhibits cells apoptosis in THP-1 and K562 cells，and the mechanism may be related to activating the AKT / mTOR and p53 signaling pathways.

Objective To establish the enzyme-linked immunosorbent assay (ELISA) for the detection of serum alcohol dehydrogenase (ADH) antibody and evaluate its role in its diagnosis of autoimmune hepatitis( AIH ). Methods The reactivity between yeast ADH and human anti-ADH serum antibody was tested by Western blot analysis. ELISA was established using yeast ADH. The method was applied in serums of 67 AIH patients,94 primary biliary cirrhosis(PBC) patients, 199 chronic hepatitis B (CHB) patients, 132 chronic hepatitis(CHC) patients, 24 alcohol hepatitis disease(ALD) patients, 99 connective tissue disease(CTD) patients and 31 healthy individuals. The positive rate of ADH antibody in the patients and healthy individuals was measured. The χ2 test was used to compare the positive rates. Results The ELISA method for detecting human anti-ADH serum antibody was established successfully and the optimum reaction conditions were defined. Western blot showed that yeast ADH has cross reactivity with human anti-ADH antibody. The positive rate of anti-ADH antibody in the AIH group [59. 7% ,40/67 ] was higher than that in the normal control group(0,χ2 = 31. 271 ,P <0. 05), PBC group (6. 4% ,χ2 =54. 492,P <0. 05), CHB group( 14. 1% ,χ2 =54. 848,P <0. 05) ,CHC group(21.2% ,χ2 = 29.269,P<0.05), ALl) group ( 25. 0% ,χ2 =8.512,P <0.05)and CTD group ( 43. 4% ,χ2 =4.229, P <0. 05). Conclusions Compared with the PBC, CHB, CHC, ALD and CTD group, the anti-ADH antibody positive rate in the serums of AIH was significantly increased. The antibody may be helpful to the diagnosis of autoimmune hepatitis.