This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.
Animals ; Smad3 Protein/genetics* ; CRISPR-Cas Systems/genetics* ; Mice ; Podocytes/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cell Line ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics*

This paper elaborated the structural principles,structures and working principles of the A5 type passive training system for hand function through discussed the clinical application of passive training system for hand function in the rehabilitation of patients with stroke.It analyzed a series of occurring common faults during system operation,such as system errors caused by missing components,improper sensitivity settings for spasticity,abnormal connections for equipment,motor failures,jam and pause of robotic hand,and faults of lifting table,and conducted fault analysis for them.After that,this paper proposed corresponding handling strategies and solutions for faults,which included software configuration,calibration and detection of robotic hand sensors,repair of connection fault,and regular maintenance and replacement of system's components.It provided theoretical basis for the maintenance and optimization of the passive training system for hand function.Thus,it can rapid remove troubles,and reduce the equipment's failure rate of passive training system for hand function,and improve the equipment's normal utilization rate,and ensure favorable performance of equipment in long-term use.

Objective To evaluate the risk of right heart failure after heart transplantation by establishing nomogram based on preoperative pulmonary artery pressure.Methods A total of 184 patients undergoing heart transplantation were retrospectively collected and divided into the training group(126 cases)and the verification group(58 cases).Patients in the training set were divided into the right heart failure group(60 cases)and the non-right heart failure group(66 cases)according to whether right heart failure occurred after operation.The differences of clinical data between the two groups were compared,and the influencing factors of right heart failure occurred after operation in the training set were screened by Lasso-Logistic regression.According to the screened influencing factors,nomograms were drawn,and the predictive efficiency of the model was evaluated by using the receiver's operating characteristic(ROC)curve,calibration curve,receiver's operating characteristic(ROC)curve and clinical decision curve.Further vertification of the clinical application effect of centralized evaluation model was conducted.Results The Lasso-Logistic regression analysis identified the following independent risk factors for right heart failure after heart transplantation:elevated total bilirubin(OR=2.649,95％CI:1.339-5.239),increased mean pulmonary artery pressure(OR=3.082,95％CI:1.608-5.910),elevated pulmonary artery resistance(OR=3.171,95％CI:1.710-5.879),and widened right ventricular outflow tract diameter(OR=2.681,95％CI:1.361-5.281),all of which demonstrated statistical significance(P＜0.05).The nomogram model was constructed accordingly.The AUC of the nomogram model was 0.846(95％CI:0.813-0.947).The calibration curve demonstrated good fit via the goodness-of-fit test(Hosmer-Lemeshow x2=0.862,P=0.361).Clinical decision curve analysis revealed that the net benefit rate remained＞0 when the high-risk threshold probability ranged from 1％to 95％,indicating favorable clinical utility of this nomogram model.Based on the model predictions,among 58 heart transplant patients in the validation cohort,34 were classified as high-risk for right heart failure and 24 as low-risk.Actual diagnosis results showed 29 cases with right heart failure and 29 without.The Kappa coefficient reached 0.483(95％CI:0.261-0.705),demonstrating high consistency between model predictions and actual clinical outcomes.Conclusion Preoperative pulmonary systolic pressure increase is an independent risk factor for right heart failure after heart transplantation.A nomogram prediction model for right heart failure after heart transplantation is established by combining other clinical risk factors,and it has good prediction efficiency.

Objective The aim of this study is to detect the expression of purinergic 2X7 receptor(P2X7R)and the NLRP3 inflammasome in benign prostatic hyperplasia(BPH)tissues and to analyze the clinical correlations.Methods Twelve patients undergoing surgery for BPH were enrolled.Based on the presence or absence of inflammatory cell infiltration in HE-stained tissue sections,the patients were divided into inflammation group and non-inflammation group.Preoperative routine ex-aminations excluded surgical contraindications,and International Prostate Symptom Score(IPSS)questionnaires,urinary flow rate measurements,expressed prostatic secretions(EPS)analysis,and prostate-specific antigen(PSA)tests were conducted.Prostate tissues obtained during surgery were subjected to HE staining,immunofluorescence/Western blot to detect the expression of NLRP3,P2X7R,Caspase-1,Cleaved-Caspase-1,IL-1 β,and TNF-α,and immunofluorescence to assess lymphocyte infiltra-tion.SPSS 26.0 software was used to analyze correlations between the expression levels of NLRP3 and P2X7R in prostate tissues and indicators including Caspase-1,Cleaved-Caspase-1,IL-1 β,TNF-α,lymphocyte count,IPSS score,urinary flow rate,EPS,and PSA.Results In BPH tissues,the expression levels of NLRP3 and P2X7R were positively correlated(P＜0.05).The ex-pression levels of NLRP3 and P2X7R were positively correlated with Caspase-1,Cleaved-Caspase-1,IL-1 β,TNF-α,and lym-phocyte count(P＜0.05).NLRP3 and P2X7R expression levels were positively correlated with white blood cell count in EPS,but showed no correlation with IPSS score,lecithin body count in EPS,maximum urinary flow rate and PSA(P＜0.05).Conclusion P2X7R and NLRP3 in prostate tissues exacerbate local inflammatory responses,which may be an important mecha-nism in BPH development.However,they are not correlated with IPSS score,lecithin body count in EPS,maximum urinary flow rate and PSA.

This paper elaborated the structural principles,structures and working principles of the A5 type passive training system for hand function through discussed the clinical application of passive training system for hand function in the rehabilitation of patients with stroke.It analyzed a series of occurring common faults during system operation,such as system errors caused by missing components,improper sensitivity settings for spasticity,abnormal connections for equipment,motor failures,jam and pause of robotic hand,and faults of lifting table,and conducted fault analysis for them.After that,this paper proposed corresponding handling strategies and solutions for faults,which included software configuration,calibration and detection of robotic hand sensors,repair of connection fault,and regular maintenance and replacement of system's components.It provided theoretical basis for the maintenance and optimization of the passive training system for hand function.Thus,it can rapid remove troubles,and reduce the equipment's failure rate of passive training system for hand function,and improve the equipment's normal utilization rate,and ensure favorable performance of equipment in long-term use.

Objective To evaluate the risk of right heart failure after heart transplantation by establishing nomogram based on preoperative pulmonary artery pressure.Methods A total of 184 patients undergoing heart transplantation were retrospectively collected and divided into the training group(126 cases)and the verification group(58 cases).Patients in the training set were divided into the right heart failure group(60 cases)and the non-right heart failure group(66 cases)according to whether right heart failure occurred after operation.The differences of clinical data between the two groups were compared,and the influencing factors of right heart failure occurred after operation in the training set were screened by Lasso-Logistic regression.According to the screened influencing factors,nomograms were drawn,and the predictive efficiency of the model was evaluated by using the receiver's operating characteristic(ROC)curve,calibration curve,receiver's operating characteristic(ROC)curve and clinical decision curve.Further vertification of the clinical application effect of centralized evaluation model was conducted.Results The Lasso-Logistic regression analysis identified the following independent risk factors for right heart failure after heart transplantation:elevated total bilirubin(OR=2.649,95％CI:1.339-5.239),increased mean pulmonary artery pressure(OR=3.082,95％CI:1.608-5.910),elevated pulmonary artery resistance(OR=3.171,95％CI:1.710-5.879),and widened right ventricular outflow tract diameter(OR=2.681,95％CI:1.361-5.281),all of which demonstrated statistical significance(P＜0.05).The nomogram model was constructed accordingly.The AUC of the nomogram model was 0.846(95％CI:0.813-0.947).The calibration curve demonstrated good fit via the goodness-of-fit test(Hosmer-Lemeshow x2=0.862,P=0.361).Clinical decision curve analysis revealed that the net benefit rate remained＞0 when the high-risk threshold probability ranged from 1％to 95％,indicating favorable clinical utility of this nomogram model.Based on the model predictions,among 58 heart transplant patients in the validation cohort,34 were classified as high-risk for right heart failure and 24 as low-risk.Actual diagnosis results showed 29 cases with right heart failure and 29 without.The Kappa coefficient reached 0.483(95％CI:0.261-0.705),demonstrating high consistency between model predictions and actual clinical outcomes.Conclusion Preoperative pulmonary systolic pressure increase is an independent risk factor for right heart failure after heart transplantation.A nomogram prediction model for right heart failure after heart transplantation is established by combining other clinical risk factors,and it has good prediction efficiency.

Objective The aim of this study is to detect the expression of purinergic 2X7 receptor(P2X7R)and the NLRP3 inflammasome in benign prostatic hyperplasia(BPH)tissues and to analyze the clinical correlations.Methods Twelve patients undergoing surgery for BPH were enrolled.Based on the presence or absence of inflammatory cell infiltration in HE-stained tissue sections,the patients were divided into inflammation group and non-inflammation group.Preoperative routine ex-aminations excluded surgical contraindications,and International Prostate Symptom Score(IPSS)questionnaires,urinary flow rate measurements,expressed prostatic secretions(EPS)analysis,and prostate-specific antigen(PSA)tests were conducted.Prostate tissues obtained during surgery were subjected to HE staining,immunofluorescence/Western blot to detect the expression of NLRP3,P2X7R,Caspase-1,Cleaved-Caspase-1,IL-1 β,and TNF-α,and immunofluorescence to assess lymphocyte infiltra-tion.SPSS 26.0 software was used to analyze correlations between the expression levels of NLRP3 and P2X7R in prostate tissues and indicators including Caspase-1,Cleaved-Caspase-1,IL-1 β,TNF-α,lymphocyte count,IPSS score,urinary flow rate,EPS,and PSA.Results In BPH tissues,the expression levels of NLRP3 and P2X7R were positively correlated(P＜0.05).The ex-pression levels of NLRP3 and P2X7R were positively correlated with Caspase-1,Cleaved-Caspase-1,IL-1 β,TNF-α,and lym-phocyte count(P＜0.05).NLRP3 and P2X7R expression levels were positively correlated with white blood cell count in EPS,but showed no correlation with IPSS score,lecithin body count in EPS,maximum urinary flow rate and PSA(P＜0.05).Conclusion P2X7R and NLRP3 in prostate tissues exacerbate local inflammatory responses,which may be an important mecha-nism in BPH development.However,they are not correlated with IPSS score,lecithin body count in EPS,maximum urinary flow rate and PSA.

Objective Establish a standardized evaluation system for medical faults,and provide theoretical basis for medical institutions and related industries to evaluate the illegality of medical behaviors.Methods Based on a litera-ture review,the medical fault assessment system was initially constructed,and then a research group was estab-lished to use Delphi method to invite 31 experts to evaluate the importance and feasibility of each article of the medi-cal fault assessment system and put forward suggestions for modification.Results The effective recovery rates of the two rounds of expert consultation were 83.9％and 96.8％,the expert authority coefficient was 0.902 and 0.887,and the Kendall's W test of all levels differences were statistically significant(P＜0.001).The medical fault assess-ment system finally constructed includes 5 first-level items including practicing medicine according to law,informed notification,diagnosis and treatment technology,medical record documents and hospital management,as well as 10 second-level items,20 third-level items and 47 fourth-level items.The mean values of importance and feasibili-ty scores of all articles were greater than 4,standard deviations were less than 1,and coefficients of variation were less than 0.2.Conclusion The medical fault standardized evaluation system is scientific,reliable,innovative and appli-cable.

Objective Establish a standardized evaluation system for medical faults,and provide theoretical basis for medical institutions and related industries to evaluate the illegality of medical behaviors.Methods Based on a litera-ture review,the medical fault assessment system was initially constructed,and then a research group was estab-lished to use Delphi method to invite 31 experts to evaluate the importance and feasibility of each article of the medi-cal fault assessment system and put forward suggestions for modification.Results The effective recovery rates of the two rounds of expert consultation were 83.9％and 96.8％,the expert authority coefficient was 0.902 and 0.887,and the Kendall's W test of all levels differences were statistically significant(P＜0.001).The medical fault assess-ment system finally constructed includes 5 first-level items including practicing medicine according to law,informed notification,diagnosis and treatment technology,medical record documents and hospital management,as well as 10 second-level items,20 third-level items and 47 fourth-level items.The mean values of importance and feasibili-ty scores of all articles were greater than 4,standard deviations were less than 1,and coefficients of variation were less than 0.2.Conclusion The medical fault standardized evaluation system is scientific,reliable,innovative and appli-cable.

Objective Establish a standardized evaluation system for medical faults,and provide theoretical basis for medical institutions and related industries to evaluate the illegality of medical behaviors.Methods Based on a litera-ture review,the medical fault assessment system was initially constructed,and then a research group was estab-lished to use Delphi method to invite 31 experts to evaluate the importance and feasibility of each article of the medi-cal fault assessment system and put forward suggestions for modification.Results The effective recovery rates of the two rounds of expert consultation were 83.9％and 96.8％,the expert authority coefficient was 0.902 and 0.887,and the Kendall's W test of all levels differences were statistically significant(P＜0.001).The medical fault assess-ment system finally constructed includes 5 first-level items including practicing medicine according to law,informed notification,diagnosis and treatment technology,medical record documents and hospital management,as well as 10 second-level items,20 third-level items and 47 fourth-level items.The mean values of importance and feasibili-ty scores of all articles were greater than 4,standard deviations were less than 1,and coefficients of variation were less than 0.2.Conclusion The medical fault standardized evaluation system is scientific,reliable,innovative and appli-cable.