AIM:To establish and evaluate a colonoids model of intestinal barrier dysfunction with irritable bowel syndrome(IBS).METHODS:The colonic recess of 20～22 g male C57BL/6 mice were isolated and cultured in ma-trix glue to proliferate and differentiate into 3D hollow spheres with colonic epithelioid structure.The following experi-ments were carried out:(1)Colonoids and colonic tissues of mice were detected by immunofluorescence to identify colo-noids.(2)Fluorescein isothiocyanate dextran 4(FD4)evaluated the epithelial barrier function of colonoids.(3)To ex-plore the changes in the epithelial barrier of colonoids induced by interferon-γ(IFN-γ)at different concentrations and time points.FD4 and HE staining were used to evaluate the barrier function.RT-qPCR was used to detect the mRNA expres-sion of occludin and zonula occludens-1(ZO-1)in tight junctions of colonoids.Immunofluorescence was used to detect the distribution and localization of occludin and ZO-1 proteins.RESULTS:(1)The expression of EdU proliferation and in-testinal epithelial cell lineage markers in colonoids was consistent with that in mouse colonic tissues.(2)In the control group,FD4 did not infiltrate the colonoids lumen,but FD4 significantly infiltrated the colonoids lumen induced by ethyl-ene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA).(3)From 18 h,the IFN-γ at 60,100,200 and 240 ng/mL could significantly infiltrate into the cavity of colonoids(0.033,0.032,0.042 and 0.001),and the barri-er injury of colonoids could be seen by HE staining.After 18 h,all concentrations of IFN-γ could significantly decrease the mRNA expression of occludin and ZO-1,and the fluorescence of occludin and ZO-1 decreased significantly(P<0.05).CONCLUSION:(1)The cultured organoids are colonoids with complete epithelial barrier.(2)IFN-γ could in-duce the decrease of the transcriptional levels of occludin and ZO-1 in the tight junction of colonoids,the decrease of the expression of corresponding proteins,and the change of localization and distribution,thus increasing the epithelial perme-ability of colonoids.This model is highly consistent with the pathophysiological state of IBS colonic mucosal barrier dys-function,which provides a new tool and method for studying the direction of colonic mucosal barrier dysfunction in IBS.

Purpose: Cavernous nerve injury induced erectile dysfunction (ED) is a refractory complication with high incidence in person under radical prostatectomy. Studies have shown that relaxin-2 (RLX-2) plays a vital role of endothelial protection, vasodilation, anti-fibrosis and neuroprotection in a variety of diseases. However, whether penile cavernous erection can benefit from RLX-2 remains unknown. The purpose of the experiment was to explore the effects of RLX-2 on ED in the rat suffering with bilateral cavernous nerve injury (BCNI). Materials and Methods: The rats were divided into three groups: Sham group was underwent sham operation, BCNI+RLX group or BCNI group was underwent bilateral cavernous nerve crush and then randomly treated with RLX-2 (0.4 mg/kg/d) or saline by continuous administration using a subcutaneously implanted micro pump for 4 weeks respectively. Then, erectile function was evaluated by electrical stimulation of cavernous nerves. Cavernous nerves and penile tissues and were collected for histological evaluation. Results: Erectile function of rats with BCNI was partially improved after RLX-2 treatment. The BCNI group had lower expression of relaxin family peptide receptor (RXFP) 1, p-AKT/AKT, p-eNOS/eNOS ratios than sham operation rats, but RLX-2 could partially reversed these changes. Histologically, the BCNI+RLX group had a significant effect on preservation of neurofilament, neuronal glial antigen 2 of penile tissue and nNOS of cavernous nerves when compared with BCNI group. RLX-2 could inhibited the lever of BCNI induced corporal fibrosis and apoptosis via regulating TGFβ1-Smad2/3-CTGF pathway and the expression of Bax/Bcl-2 ratio, caspase3. Conclusions RLX-2 could improve erectile function of BCNI rats by protecting cavernous nerve and endothelial function and suppressing corporal fibrosis and apoptosis via RXFP1 and AKT/eNOS pathway. Our findings may provide a promising treatment for refractory BCNI induced ED.

Objective:To evaluate the efficacy and side effects of PD-1 monoclonal antibody in the treatment of advanced metastatic renal cell carcinoma in China.Methods:The clinical data of 117 patients with advanced metastatic renal cell carcinoma (mRCC) treated with PD-1 monoclonal antibody from October 2016 to February 2022 were retrospectively analyzed. There were 87 males (74.4%) and 30 females (25.6%), with an average age of (57.9±10.9) years old, BMI of (23.6±3.4) kg/m 2and smoking history of 79 (67.5%). There were 44 cases (37.6%) with hypertension, 19 (16.2%) cases of diabetes. The ECOG score of 59.8% (70/117) patients was 0, 33.3% (39/117) was 1, 4.3% (5/117) was 2, and 2.5% (3/117) was 3. The pathological type of 104 cases were renal clear cell carcinoma (ccRCC), 8 cases of papillary renal cell carcinoma, 2 cases of chromophobe cell carcinoma, 2 cases of collecting duct carcinoma and 1 case of eosinophilic cell carcinoma. The general condition of the overall population and the overall survival (OS) of relevant subgroups were analyzed. Secondary goals included progression free survival (PFS), objective response rate (ORR), adverse reactions, overall survival (OS), and progression free survival (PFS). Results:65.8% (77 / 117) of the patients chose targeted combined with PD-1 monoclonal antibody in the first-line treatment. The main targeted drugs were acitinib (81.8%, 63 / 77), tirelizumab (37.6%, 29 / 77) and cindilimab (25.9%, 20 / 77). After first-line treatment, 19.6.1% (23 / 117) patients needed to be converted to second-line treatment, and 15 patients changed the type of PD-1 antibody during treatment. In addition, the targeted drug of combined therapy was replaced by acitinib in 8 patients. The main causes of drug withdrawal were disease progression (70.7%, 29 / 41) and death (29.2%, 12 / 41). The median OS of the overall population was 35.6 (19-60) months and PFS was 12.1 (1-60) months. The ORR of the overall population was 47.8% (56 / 117). 4.2% (5/117) patients had complete remission, another 17.0% (20/117) patients were in stable condition, and 43.5% (51 / 117) patients were in partial remission. In the first-line treatment, the median PFS time of targeted combined with PD-1 monoclonal antibody was 12.6 (1-30) months, the median PFS time of PD-1 single drug immunotherapy was 10.5 (1-60) months. In the second-line treatment, the PFS of patients treated with PD-1 monoclonal antibody was 10.1 (4-19) months, and that of patients treated with PD-1 monoclonal antibody combined with targeted therapy was 11.7 (1-25) months. The most common adverse reactions were elevated blood pressure (18.5%, 23 / 124), followed by hypothyroidism (15.3%%, 19/124), rash (14.5%, 18 / 124), elevated transaminase (10.5%, 13 / 124) and bone marrow suppression (9.7%, 12/124). 9.4% (11 / 117) patients needed to reduce the related adverse reactions by interrupting the treatment control of PD-1 monoclonal antibody.Conclusions:The safety and efficacy of PD-1 monoclonal antibody in domestic patients are better, and the side effects are less. The efficacy and safety of PD-1 monoclonal antibody combined with targeted therapy in the real world population are consistent with many key clinical trials abroad. PD-1 monoclonal antibody combined with targeted drugs can be popularized in the domestic MRCC population.

OBJECTIVE: To carry out genetic testing for a pedigree affected with carotid body tumor (CBT). METHODS: Members of the pedigree were enrolled and underwent physical examination, ultrasonography and CT scan. Genomic DNA of the proband was extracted from peripheral blood sample and subjected to exome sequencing. Candidate variants were predicted using bioinformatic tools and verified among members from his pedigree. RESULTS: A c.170-1G>T splicing variant of the SDHD gene was detected in 15 individuals from the pedigree. Physical examination and imaging confirmed that 9 of them had CBT and hypertension, while the remaining 6 died of cardiovascular and cerebrovascular diseases. CONCLUSION The c.170-1G>T variant of the SDHD gene probably underlies the CBT in this pedigree. Genetic testing should be considered for CBT patients with CBT in addition to conventional clinical examination.

Objective: To evaluate Kangli hollow screws with sliding compression locking plate system (KHS) in the treatment of femoral neck fractures. Methods: From February 2015 to October 2016, 47 femoral neck fractures were treated at Department of Orthopaedics, Wuhan Fourth Hospital, Wuhan Puai Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. They were 30 men and 17 women, aged from 26 to 68 years (average, 43.5 years). According to the Pauwels classification, there were 24 cases of type Ⅰ, 16 cases of type Ⅱ and 7 cases of type Ⅲ; according to the Garden classification, there were 23 cases of type Ⅱ, 19 cases of type Ⅲ and 5 cases of type Ⅳ. All the fractures were immobilized with KHS after closed reduction or open reduction (3 cases). The fracture union time, femoral head necrosis and femoral neck shortening were observed after operation. The Harris scores were used to evaluate therapeutic effects at the final follow-up. Results: All the patients were followed up for an average of 29.7 months (from 22 to 39 months). All the fractures obtained solid bony union after 9 to 15 weeks(average, 12.2 weeks). Femoral head necrosis occurred in one case (2.1%). Femoral neck shortening ≤ 5 mm was observed in 6 cases and femoral neck shortening >5 mm <10 mm in 2 cases, giving a total shortening rate of 17.0%. The Harris scores at the final follow-up ranged from 77 to 98 points, averaging 92.2 points. There were 42 excellent, 3 good and 2 moderate cases, giving an excellent and good rate of 95.7%. Conclusion KHS can lead to excellent therapeutic effects in the treatment of femoral neck fractures.

OBJECTIVE To screen for mutations in a Chinese pedigree affected with hypokalemic periodic paralysis. METHODS The proband and nine family members were enrolled for the analysis of CACNA1S and SCN4A gene mutations. Genomic DNA was extracted from peripheral blood samples. The coding regions of the two genes were amplified with PCR and subjected to Sanger sequencing. Potential impact of suspected mutations was predicted with Bioinformatics software. The mutations were also verified among 100 healthy controls. RESULTS The proband and 5 family members (including 5 males and 1 female) had presented with episodes of flaccid paralysis accompanied by low serum potassium. Genetic testing has identified a c.664C>T (p.Arg222Trp) mutation in the proband, which has been reported previously. The same mutation was identified in other 5 affected members from the family. No mutation of the CACNA1S gene was detected. CONCLUSION The c.664C>T mutation of the SCN4A gene probably underlies the hypokalemic periodic paralysis in this family. All patients from the family have shown a complete penetrance of the disease.

Objective To investigate the causes of bleeding due to arterial injury after minimally invasive percutaneous nephrolithotomy (mPCNL).Methods We retrospectively analyzed the clinical data of 2 980 patients who underwent ultrasound-guided mPCNL between January 2012,and January 2017,in our hospital.Among them,1 853 were male and 1 127 were female.Age from17 to 76 years old,average age was (45.7 ± 24.1) years.The calculi size was from 1.5 to 4.2 cm,average of (2.62 ± 1.08) cm.There were 2 478 kidney stones and 502 cases of ureteral calculi.Besides,727 cases with mild hydronephrosis,1 971 cases with moderate hydronephrosis,282 cases with severe hydronephrosis;480 patients with urinary tract infection;103 patients with renal empyema or acute renal failure;63 patients with cardiovascular and cerebrovascular stent implantation;214 patients with diabetes mellitus.Results In this study,all of the patients were established percutaneous renal tract successfully,indwelling drainage tube (6 ± 2)d,and postoperative hemorrhage in 117 cases,of which 29 patients suffered from bleeding due to arterial injury and DSA showed 20 cases with acute arterial injury,5 cases with pseudo-aneurysm,and 4 cases with arteriovenous fistula.However,all these cases were rehabilitated after the treatment of super-selective renal artery embolization.The rates of arterial injury of renal calyx access and pelvic access were 0.39％ (10/2 535) and 4.27％ (19/445),respectively.The rate of arterial injury in renal pelvic access was significantly higher than renal calyx access of PCNL (P ＜ 0.05).The rates of arterial injury in lower,middle,upper calyx access were 0.38％ (1/267),0.40％ (6/1 516),0.40％ (3/752),respectively.There was no significant effect of different renal calyx on postoperative arterial injury rate of mPCNL(P ＞0.05).All the percutaneous renal accesses were dilated with Amplatz sheaths,and the arterial injury rate of F16-18 and F20-22 tracts in the calyx access were 0.35％ (5/1 446),0.46％ (5/1 089),respectively,with no significant difference in size of calyx access associated with rate of arterial injury after mPCNL(P ＞0.05).The arterial injury rates of F16-18 and F20-22 tracts in the pelvic access were 1.98％ (5/253) and 7.29％ (14/192),respectively.There was significant difference in the size of the pelvic access in the rate of arterial injury after mPCNL(P ＜ 0.05).In addition,the arterial injury rate of single tract PCNL was 0.94％ (25/2 653) as compared to 1.22％ (4/327) in multi-tracts PCNL.There was no significant difference in the rate of arterial injury between single and multi-tracts PCNL(P ＞ 0.05).Conclusions The puncture of the renal pelvis and size of renal pelvis tract significantly increased the probability of postoperative bleeding due to arterial injury.

Objective To review the clinical characteristics of prostate mucinous adenocarcinoma cases and update literatures,and recommend the corresponding clinical treatment strategy.Methods From October 2010 to March 2018,36 cases of prostate mucinous adenocarcinoma were involved from 5 urinary centers in China,including 9 cases from Shanghai Changhai Hospital,4 cases from Wuhan Tongji Hospital,13 cases from Shanghai Renji Hospitals,8 cases from the First Affiliated Hospital of Nanjing Medical University,and 2 cases from Sichuan West China Hospitals.The patients' age were (66.8 ±7.2) years (53-83 years) and the median PSA was 22.89 ng/ ml (2.67-1786 ng/ ml).Prostate biopsy confirmed Gleason score 3 + 3 points in 6 cases,3 + 4 points in 9 cases,4 + 3 points in 5 cases,8 points in 11 cases,and 9 to 10 points in 5 cases.According to D'Amico risk stratification,2 patients were in the low-risk group,9 in the intermediate-risk group,and 25 in the high-risk group.Eight cases underwent radical retroperitoneal prostatectomy,13 cases underwent laparoscopic radical prostatectomy,and 12 cases underwent robotic laparoscopic radical prostatectomy.Twenty-three cases underwent pelvic lymphadenectomy,including 12 cases of bilateral obturator lymph node dissection,and 11 cases of bilateral obturator + intraorbital + para-vascular para-aortic lymphadenectomy.Results All 36 operations were completed successfully.Twenty-three cases underwent pelvic lymphadenectomy,including 12 of bilateral obturator lymph node dissection,and 11 of bilateral obturator,intraorbital,and para-aortic lymphadenectomy.Pathological examination showed 9 cases of prostate mucinous adenocarcinoma,26 cases of mucinous adenocarcinoma with acinar adenocarcinoma,and 1 case of mucinous adenocarcinoma with neuroendocrine and immunohistochemical positive of MUC2 (+).Among 33 cases undergoing radical surgery,the pathological stage of ≤T2b in 12 cases (36.3％),T2c in 7 cases (21.2％),T3a in 7 cases (21.2％),T3b in 6 cases (18.2％),and T4 in 1 case (3.0％).Four cases had positive pelvic lymph nodes and 9 cases had positive margin.The median follow-up period was 26 months (6-48 months).The biochemical recurrence occurred in 6 patients one year after surgery,including 3 cases in the intermediaterisk group and 3 cases in the high-risk group.Six cases with postoperative biochemical recurrence and 19 cases with PSA ＞ 0.2 ng/ml after radical or palliative resection underwent adjuvant androgen deprivation therapy(ADT),no postoperative adjuvant radiotherapy or chemotherapy was administered,and 4 cases progressed to castration-resistant prostate cancer.Four cases with CRPC were in the high-risk group and had underwent radical surgery,and the median period progressed to CRPC was 26 months(3-37months)with 2 cases of death.However,there was no significant difference in the rate of biochemical recurrence and the incidence of CRPC in the low-risk group,the intermediate-risk group and the high-risk group.In addition,2 cases had metastases,with pelvic MRI presenting pelvic multiple nodular mass in one case which was consistent with recurrence and metastasis at the 5th month after radical surgery,and pathological examination presenting the mucinous adenocarcinoma being neurosecretory in another case and mestastasis being detected on glans at the 3rd months after radical surgery.The recovery rate of urinary continience at 6 and 12 months after radical surgery was 86.2％ (31/36) and 89.7％ (32/36) respectively.Conclusions Prostate mucinous adenocarcinoma is a variant of acinar adenocarcinoma.This study clarifies prostate mucinous adenocarcinoma of Chinese patients with high Gleason scores,advanced pathological stage,variant in prognosis,and prone to recurrence and metastasis.For treatment strategy,the low-risk and intermediate-risk mucinous adenocarcinoma is recommended undergoing radical surgery,and the prognosis maybe good.High-risk mucinous adenocarcinoma could treated with radical surgery or palliative surgery with adjuvant ADT,and most high-risk patients can benefite,with a small number of poor prognosis.

Objective To study the effects of a synthetic miR3619-5p mimics on bladder carcinoma cell lines of EJ and T24 in vitro.Methods EJ and T24 cells were cultured in vitro and treated with three different processing:negative control group(tinfection with dsControl),positive control group(infection with dsP21-322) and the experimental group(infection with miR-3619-5p)during October 2015 to March 2016.Real-time fluorescent quantitative PCR (qPCR) was performed to detect the expression of p21 mRNA,cell cycle protein D1 (CyclinD1) and cell cycle-dependent kinase (CDK4 and CDK6) mRNA.Western Blot method was conducted to evaluate the expression of p21,CyclinD1 and CDK4 and CDK6 proteins;the change of cell cycle was displayed by flow cytometric analysis.Colony formation assay was used to test the ability of single cancer cell clone proliferation.Cell proliferation assay(MTS) was implemented to observed the inhibitive effect of cell proliferative potential.Results qPCR results showed that miR-3619-5p upregulated p21 mRNA expression (P ＜ 0.05),while the expression of CyclinD1,CDK4 and CDK6 were a little lower(P ＜ 0.05) in EJ and T24cells,respectively.Western Blot analysis testified that the expressions of p21,CyclinD1,CDK4 and CDK6 were difference among groups.Flow cytometry displayed that,the G0/ G1 phase increased significantly after transfected with miR-3619-5p and dsP21-322,compared with dsControl group(P ＜ 0.05),indicating that the cell cycle block in G0/G1 phase.Cell colony formation assay certified that the colony formation rates were less in the groups of miR-3619-5p and dsP21-322 than in that of dsControl group(P ＜ 0.05).Cell proliferation assay demonstrated that,cell proliferation ability decreased obviously when transfected with miR-3619-5p and dsP21-322 (P ＜0.05),compared with dsControl group.Conclusions miR-3619-5p could up-regulate the expression of p21 by RNA activation pathway and remarkably induced cell cycle arrest in G0/G1 phase,inhibiting the proliferation of bladder cancer cells.

BACKGROUND:Rat models of complete spinal cord transection are common models for neural tissue engineering. After transecting the spinal cord by the previous methods, gap length of broken end cannot keep relatively uniform, so we cannot objectively evaluate effects of various treatments or tissue engineering materials. OBJECTIVE:The spinal cord transection models were established by using double edged micro scissors, andthe feasibility of this new model was explored by comparing with the conventional method. METHODS: A total of 42 adult female Sprague-Dawley rats were divided randomly into group A (n=6), group B (n=18) and group C (n=18). Group A only received laminectomy. In the group B, the spinal cord was transected with a sharp-pointed knife. Knife point should touch anterior wal of spinal canal and sidewal bone surface. Complete spinal cord transection models were prepared by repeated cutting. In group C, complete spinal cord transection models were established by using self-made double edged micro scissors. RESULTS AND CONCLUSION: (1) At 1 week after model establishment, in the groups B and C, complete paralysis of the hind limbs was found, and BBB scores were similar. However, significant differences in the spacing of broken end were detected. (2) At 4 weeks after model establishment, hind limb functions could restore to different degrees in both groups, but no significant difference in BBB scores was found. (3) At 8 weeks after model establishment, significant differences in hindlimb motor function scores were detectable between both groups. Biotin glucosamine tracer display: In group B, a few labeled axon fibers were observed at the caudal side of the injured spinal cord. In group C, spinal cord was completely transected, and labeled axon fibers cannot be found at the caudal side. (4) Results suggested that the modeling method of self-made double edged micro scissors could effectively eliminate individual differences, contribute to quantitative analysis and comparative study of therapeutic effects.