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*

Objective To explore the relationship between preoperative coronary angiography and postoperative acute kidney injury (AKI) in cardiac surgery. Methods The clinical data of patients who underwent coronary angiography within 30 days before cardiac surgery in the First Affiliated Hospital of Xi’an Jiaotong University from January 2015 to April 2019 were retrospectively analyzed. Univariate analysis and multivariate logistic regression analyses were used to explore the relationship between the interval from preoperative coronary angiography to cardiac surgery and postoperative AKI. Results Finally 1 112 patients were collected, including 700 males and 412 females, with a median age of 61 (55, 66) years. The incidence of postoperative AKI was 40.8% (454/1 112), of which grade 2-3 AKI accounted for 11.9%. Multivariate analysis showed that age (OR=1.049, 95%CI 1.022-1.077, P<0.001), body mass index (OR=1.065, 95%CI 1.010-1.123, P=0.020) and time interval between preoperative coronary angiography and cardiac surgery within 24 hours (OR=1.625, 95%CI 1.116-2.364, P=0.011) were independent predictors of postoperative AKI. Patients who underwent coronary angiography within 24 hours before surgery had a 10.6% higher incidence of postoperative AKI compared to those who underwent angiography ≥24 hours before surgery (P=0.004). Patients who underwent valve surgery with or without coronary artery bypass grafting (CABG) had a higher risk of AKI than those who only underwent CABG. The in-hospital stay of patients who developed AKI was 2 days longer than those without AKI. However, undergoing coronary angiography within 24 hours before cardiac surgery did not prolong the length of ICU stay or hospital stay, nor did it increase the risk of death or renal failure after the operation. Conclusion Undergoing coronary angiography within 24 hours before cardiac surgery increases the risk of postoperative AKI.

Objective To investigate the therapeutic effect and underlying mechanism of Qishishenshu Capsule on renal fibrosis in mice with early diabetic nephropathy(DN).Methods A DN mouse model was established by multiple injections of streptozotocin.The mice were randomly divided into a normal group(NC),model group(DN),and Qishi group(QS)(0.9 g/(kg·d)),with eight mice in each group.Mice were gavaged continuously for 4 weeks,and fasting blood glucose(FBG)was measured weekly.Four weeks later,urinary albumin/creatinine(UACR),serum creatinine,and blood urea nitrogen were measured.Hematoxylin-eosin,periodicacid-Schiff,and Sirius red staining were used to analyze renal pathological changes.Real-time fluorescence quantitative reverse-transcription polymerase chain reaction was used to detect the mRNA levels of fibronectin(FN),collagen type Ⅰ alpha 1(Col1a1),and α-smooth muscle actin(α-SMA).Immunohistochemistry and Western blot were performed to detect FN,collagen type Ⅰ(Collagen Ⅰ),collagen typeⅢ(Collagen Ⅲ),α-SMA,Podocin,Nephrin,and transforming growth factor-β1/SMAD family member2/3(TGF-β1/Smad2/3)pathway-related proteins.Results Compared with mice in the NC group,those in the DN group showed significantly higher levels of FBG and UACR(P＜0.001),and mesangial hyperplasia,basement membrane thickening,and collagen deposition in the renal tissue.The mRNA levels of FN,Col1a1,and α-SMA were increased(P＜0.05).Protein levels of Podocin and Nephrin were decreased(P＜0.05).The levels of FN,Collagen I,Collagen Ⅲ,α-SMA,and TGF-β1/Smad2/3 pathway proteins were increased(P＜0.05).Compared with the DN group,the QS group's level of UACR was decreased(P＜0.05),their renal pathological injury was alleviated,and mRNA levels of FN,Collagen Ⅰ,andα-SMA were attenuated(P＜0.05);whereas their protein levels of Podocin and Nephrin were elevated(P＜0.05).The levels of FN,Collagen Ⅰ,Collagen Ⅲ,α-SMA,and TGF-β1/Smad2/3 pathway proteins were also decreased(P＜0.05).Conclusions Qishishenshu Capsule improved renal fibrosis in DN mice,probably through the inhibition of the TGF-β1/Smad2/3 signaling pathway.

Objective:To analyze locoregional recurrence (LRR) pattern of patients with pT 1-2N 1 breast cancer after modified radical mastectomy, with and without adjuvant radiotherapy (RT). Methods:A total of 5442 eligible patients with breast cancer from 12 Chinese centers were included. The LRR sites and the effect of RT at different sites on recurrence in patients with and without RT were analyzed. The Kaplan-Meier method was used to calculate the cumulative LRR rate, and the difference was compared by the log-rank test.Results:With a median follow-up time of 63.8 months for the entire cohort, 395 patients developed LRR. The chest wall and supraclavicular fossa were the most common LRR sites, regardless of RT or molecular subtypes. The 5-year chest wall recurrence rates for patients with and without chest wall irradiation were 2.5% and 3.8%( P=0.003); the 5-year supraclavicular lymph nodal recurrence rates for patients with and without supraclavicular fossa irradiation were 1.3% and 4.1%( P<0.001); the 5-year axillary recurrence-free rates for patients with and without axillary irradiation were 0.8% and 1.5%( HR=0.31, 95% CI: 0.04-2.23, P=0.219); and the 5-year internal mammary nodal recurrence-free rates for patients with and without internal mammary nodal irradiation were 0.8% and 1.5%( HR=0.45, 95% CI: 0.11-1.90, P=0.268). Conclusions:The chest wall and supraclavicular fossa are the most common LRR sites of patients with pT 1-2N 1 breast cancer after modified radical mastectomy, which is not affected by adjuvant RT or molecular subtypes. The chest wall and supraclavicular fossa irradiation significantly reduce the risk of recurrence in the corresponding area. However, axillary and internal mammary nodal irradiation has no impact on the risk of recurrence in the corresponding area.

Objective To evaluate the effect of surgery-radiotherapy interval (SRI) on clinical prognosis of locally advanced stage c Ⅱ-Ⅲ breast cancer patients treated with neoadjuvant chemtherapy and modified radical mastectomy.Methods Clinical data of 1 087 breast cancer patients treated with neoadjuvant chemotherapy and modified radical mastectomy from 11 hospitals in China were retrospectively analyzed.The optimal threshold value of SRI upon clinical prognosis was determined by maxstat method.The effect of SRI on clinical prognosis was evaluated by using multivariate Cox regression analysis and propensity score matching (PSM).Results The median follow-up time was 72.9 months.The 5-year disease-free survival (DFS) and overall survival (OS) rates were 68.1％ and 81.8％.All patients were divided into SRI ≤18 weeks (n=917) and SRI＞ 18 weeks groups (n=170).Multivariate Cox regression analysis demonstrated that hormone receptor status (P＜0.001),pathological T stage (P＜0.001),pathological N stage (P＜0.001) and SRI (P=0.023) were independent influencing factors of DFS.Hormone receptor status (P=0.013),pathological T stage (P=0.006),pathological N stage (P＜0.001),endocrine therapy (P=0.013) and SRI (P=0.001) were significantly associated with OS.After balancing the clinical and pathological factors with PSM,patients with SRI＜ 18 weeks had superior DFS and OS to those with SRI＞ 18 weeks.Conclusions SRI affects the clinical prognosis of locally advanced breast cancer patients treated with neoadjuvant chemotherapy and modified radical mastectomy.Radiotherapy should be performed within 18 weeks after mastectomy.

Objective To investigate the preventive effects of aspirin on liver metastases of colorectal cancer in mice and study the mechanisms.Methods Twenty BALB/c mice were divided into the control group and the experimental group according to the random number table with 10 mice in each group.Mice in the control group were fed with saline each day at a concentration of 0.2 mL/d for 60 days,while mice in the aspirin group were fed with aspirin each day at a concentration of 30 μg/(g · d) for 60 days.Then C26 colon cancer cells were injected into the spleen and then the spleen was cut to establish mice model of colon cancer liver metastasis.The C26 colon cancer cells were divided into 2 groups.C26 colon cancer cells in the control group remained untreated,and C26 colon cancer cells in the experimental group were treated with aspirin at a concentration of 10 mmol/L for 24 hours.The scratches and transwell assays were conducted to observe the effects of aspirin on the invasion and metastasis of C26 colon cancer cells.The expressions of epithelial-mesenchymal transition (EMT)-related genes were detected using RT-PCR and Western blot.All data were analyzed using the Student t test.The survival curve was drawn by Kaplan-Meier method,and the survival analysis was done by Log-rank test.Results The numbers and weights of hepatic metastatic tumors were 4.8 ± 1.9 and (504 ± 107) mg in the control group and 2.6 ± 1.6 and (362 ± 67) mg in the experimental group,with significant difference between the 2 groups (t =2.840,3.584,P ＜ 0.05).The 1-month survival rate was 80％ in the experimental group,which was significantly higher than 40％ of the control group (x2=4.418,P ＜ 0.05).The results of pathological examination showed that tumor cell heteromorphism was reduced by aspirin.The results of scratches experiment showed an obvious migration of C26 colon cancer cells in the control group at 24 hours later,while no C26 colon cancer cells migrated in the experimental group.The numbers of C26 colon cancer cells penetrated the Watrige were 253 ± 21 in the control group and 148 ± 13 in the experimental group,with significant difference between the 2 groups (t =5.101,P ＜0.05).The relative mRNA expression of the E-cadherin and the Vimentin were 0.002 ±0.001 and 1.005 ±0.286 in the control group and 0.005 ± 0.001 and 0.270 ± 0.168 in the experimental group,with significant difference between the 2 groups (t =-4.606,4.942,P ＜ 0.05).The relative protein expressions of the E-cadherin and the Vimentin were 0.473 ±0.179 and 0.787 ± 0.118 in the control group and 1.585 ± 0.410 and 0.280 ± 0.133 in the experimental group,with significant difference between the 2 groups (t =-5.542,6.355,P ＜ 0.05).Conclusion Aspirin inhibits liver metastasis of colon cancer and promote the survival ratio of mice.Aspirin can up-regulate the expression of E-cadherin and down-regulate the expression of Vimentin,which inhibits EMT and reduces the invasion and metastasis of tumor cells.

Objective To construct quality evaluation table ( QET) for assessing the effect of ICU nursing quality care objectively.Methods The study utilized Delphi method to construct frame, clause and weight of QET.Results To construct QET included 2 dimensions:evaluation of the content (6 clauses-attendance, daily life of nursing quality, nursing quality of pressure ulcer, collaborative quality, order implementation quality and quality of communicated with families ) and evaluation methods ( 3 clauses-department assessment, inner-team assessment and self-assessment) .After survey, the nursing quality of pressure ulcer and quality of communicated with families achieved relative lower score (88.20 and 87.68).Conclusions Through quantitative research methods,we establish a suitable QET which could provide an objective standard for nursing quality assessment.

Objective This study investigates the protective effect of bicyclol on liver function in patients after liver resection.Methods One hundred and twenty patients undergoing liver resection with Pringle's maneuver were randomly divided into groups A,B,and C,and given bicyclol (50 mg),diammonium glycyrrhizinate (150 mg),and silybum marianum (77 mg),respectively.The medication was orally given preoperatively for 5 days and postoperatively for days 3 to 7.The fasting serum ALT,AST,TB,ALP,and PAB levels were determined before operation and on days 1,3,5,and 7 after operation.Results ALT levels in the A group were significantly lower than those in the B and C group on post operative days 1,3,5,and 7 (P＜0.01).On postoperative day 7,the ratio of serum ALT returned to normal was significantly higher than the B and C group ratios (P＜0.05).Conclusion Therefore,oral bicyclol given before and after liver resection can significantly inhibit the rapid increase and promote the normalization of serum ALT levels.

Objective To investigate the effect of transplantation of CXC receptor 4 (CXCR4)gene-modified bone marrow mesenchymal stem cells (BMSCs) on repairment of carotid injure in rats.Methods BMSCs were cultured and transfected with lentivirus vector carrying CXCR4 gene to generate CXCR4 gene-modified BMSCs (CXCR4-BMSCs).CXCR4 expression was detected by Western blot.Rat model of carotid artery balloon injury was established.Rats were randomly divided into the PBS control group (n=12),CXCR4-BMSCs group (n=12) and BMSCs group (n=12).Two weeks after transplantation,the injured arteries were obtained.The homing of BMSCs was detected by immunofluorescence with green fluorescent protein (GFP).Platelet endothelial cell adhesion molecule (CD31) expression was detected by immunofluorescence staining.At 4 weeks after transplantation,proliferating cell nuclear antigen (PCNA) expression was determined by immunohistochemical staining,and the vascular morphological changes were observed by hematoxylineosin staining (HE).Results Compared with the control and BMSCs groups,the protein level of CXCR4 was increased in CXCR4-BMSCs group (both P＜0.05).The percentage of GFP-positive cells homing were much more in CXCR4-BMSCs group than in BMSCs group [(58.8±4.4)％ vs.(36.2±5.0) ％,P＜0.05].The CD31 expression were higher in CXCR4-BMSCs group than in BMSCs group [(58.8±4.3)％ vs.(28.8±4.2)％,P＜0.05].Compared to the control group,the PCNA expression was decreased in CXCR4-BMSCs and BMSCs groups [(21.0±4.2) ％,(36.5±4.9) ％vs.(78.3±3.5) ％,both P＜0.05].There was a significant difference in PCNA expression between the CXCR4-BMSCsgroupandBMSCs group [(21.0±4.2)％vs.(36.5±4.9)％,P＜0.05].The neointimal area and the ratio of neointimal/medial area were decreased in CXCR4 BMSCs and BMSCs group as compared with the control group [(0.205±0.018) mm2,(0.323±0.071) mm2 vs.(0.536 ± ±0.054) mm2; (1.039±0.123),(1.660±0.404) vs.(2.460±0.328); all P＜0.05],and there were significant differences in neointimal area and the ratio of neointimal/medial area in CXCR4-BMSCs group and BMSCs group [[(0.205±0.018) mm2 vs.(0.323±0.071) mm2,(1.039±0.123)vs.(1.660±0.404),both P＜0.05].Conclusions CXCR4 gene-modified BMSCs may increase the CXCR4 expression in BMSCs.CXCR4-BMSCs transplantation is more effective than BMSCs transplantation in increasing BMSCs homing capacity,reducing the reendothelialization and vascular restenosis.

Objective To evaluate the efficacy and safety of continuous erythropoietin receptor activator (C.E.R.A.) once every 4 weeks by subcutaneous administration on hemoglobin (Hb)maintenance in dialytic patients with chronic renal anemia who had been treated with stable dose of erythropoietin (EPO).Methods This was an open,randomized,controlled,multi-center trial.All the hemodialysis or peritoneal dialytic patients in EPO maintenance treatment received subcutaneous EPO-β during the 6-week pre-treatment period to maintain Hb level between 100 g/L and 120 g/L.Eligible patients were randomized (2∶1 ) to accept either C.E.R.A.once every 4 weeks by subcutaneous administration ( C.E.R.A.group,n =187 ) or subcutaneous EPO-β 1-3 times weekly ( EPO group,n =94) for 28 weeks (including 20-week dose titration period and 8-week efficacy evaluation period ). The starting dose of C.E.R.A.was converted according to the dose of EPO-β administered in the week preceding the first study drug administration.The primary outcome was the change of Hb level between the baseline and that in the efficacy evaluation period.Results Totally 253 patients completed the whole 28-week treatment.The change of baseline-adjusted mean Hb was +2.57 g/L for C.E.R.A.group and + 1.23 g/L for EPO group,resulting in a treatment difference of 1.34 g/L (95％ CI - 1.11-3.78 g/L).Since the lower limit of 95％ CI was greater than the pre-defined non-inferiority margin -7.5 g/L( P ＜ 0.0001 ),C.E.R.A.once every 4 weeks by subcutaneous administration was clinically non-inferior to EPO regarding the maintenance of stable Hb level.The proportion of patients maintaining Hb level within the range of 100-120 g/L through efficacy evaluation period was similar between the two groups ( 69.0％ for C.E.R.A.group vs 68.9％ for EPO group,P ＞0.05 ).The overall incidence of adverse events was similar between the C.E.R.A.(41.7％)and EPO (46.2％ ) groups ( P ＞ 0.05 ).The safety findings were in accordance with the patients' primary diseases rather than the administration.Conclusions Conversion from EPO to C.E.R.A.once every 4 weeks by subcutaneous injection could maintain the Hb in target level in dialytic patients with renal anemia,and it was non-inferior to EPO.In general,subcutaneous administration of C.E.R.A.is well tolerated in dialytic patients with chronic renal anemia.