BACKGROUND:To build up an effective method of isolating and culturing granule cels is a pivotal step to enhance fertilization-embryo transfer rate. Current studies mainly focus on the isolation methods of human ovarian granulosa cels rather than cel counting, purity and subsequent growth. OBJECTIVE: To establish the effective methods of isolating, purifying and culturing human ovarian granulosa cels in vitro. METHODS: Folicular fluid was harvested from women undergoing fertilization-embryo transfer procedures. Human ovarian granulosa cels were obtained from the folicular fluid by lysis treatment, precipitation method or density gradient centrifugation. Granulosa cel mucus masses were digested with type I colagen enzyme or hyaluronidase and then cultured in the culture medium with or without autologous folicular fluid. RESULTS AND CONCLUSION: Lysis treatment yielded the largest amount of granulosa cels compared to the precipitation method and density gradient centrifugation (P > 0.05,P < 0.05, respectively). Cels prepared by the three methods showed the same cel viability. After 24 hours of culture, the precipitation method obtained the largest amount of adherent granulosa cels (P < 0.05); and the density gradient centrifugation obtained the least amount of cels (P < 0.05). Compared with type I colagen enzyme, hyaluronidase took less time to digest the cels thoroughly. Autologous folicular fluid could promote the growth and survival of granulosa cels. These findings indicate that the precipitation method, though time-consuming, can obtain the highest cel viability and harvested the largest amount of granulosa cels after culture; hyaluronidase is more suitable for digesting granulosa cel mucus mass than type I colagen enzyme; autologous folicular fluid added into the culture medium is more conducive to granulosa cel growth.

Objective To evaluate inspiratory and expiratory high resolution CT (HRCT) lung findings in normal adults and set up inspiratory and expiratory HRCT criterion for further clinical diagnosis and differentiating diagnosis. Methods Sixty-four healthy adults underwent inspiratory and expiratory lung HRCT scanning. The lung density and slice area in upper, middle and lower fields were measured in inspiratory and expiratory phases respectively. Results The lung density in inspiratory phase was (-795?58)HU, the lung density in expiratory phase was (-706?59)HU, the inspiratory expiratory value difference was (90?43)HU, and the expiratory inspiratory area ratio was 0.72? 0.14. Conclusion HRCT will be a new method for lung function testing in future.

Objective To investigate the improvement of postpartum pelvic floor by rehabilitation training assessed with three-dimensional transperineal ultrasound . Methods One hundred cases of healthy postpartum women were randomly divided into two groups :control group and training group .The control group received the customary education ,and the training group received pelvic floor rehabilitation training . At 6 and 12 weeks postpartum ,levator hiatus area ,thickness of the levator ani muscle ,bladder neck mobility ,and bladder posterior horn were measured with three-dimensional transperineal ultrasound in all the subjects . Meanwhile ,the muscle strength situations were tested . Results At 12 weeks postpartum ,the anal levator hiatal area ,bladder neck mobility and bladder posterior horn in the training group were lower than those of the control group[ ( 21 .6 ± 3 .2) cm 2 vs ( 25 .6 ± 2 .4 ) cm 2 ,( 27 .9 ± 5 .3) mm vs ( 31 .5 ± 5 .9) mm ,( 126 .3 ± 21 .2)° vs (135 .3 ± 11 .6)°] ( P < 0 .05) . Compared with control group ,the thickness of the levator ani muscle increased in training group [ ( 13 .6 ± 2 .3) mm vs ( 15 .3 ± 2 .5) mm ] ( P < 0 .05) . The incidence of stress urinary incontinence in the training group ( 5% ) was significantly lower than the control group ( 12 .5% ) at 12 weeks postpartum ( χ2 = 5 .487 , P = 0 .025) . The muscle strength had no significant difference at 6 weeks postpartum . At 12 weeks postpartum ,the pass rate of class Ⅰ muscle fiber was 78 .5% ,and that of class Ⅱ muscle fiber was 83 .3% in the training group ;the pass rate of class Ⅰ muscle fiber was 28 .5% ,and class Ⅱ muscle fiber was 37 .3% in the control group , the improvement was significant at 12 weeks postpartum . Conclusions The result of the transperineal real-time ultrasonographic evaluation of post-natal pelvic floor rehabilitation training has high consistency with the measurement of muscle strength . The ultrasound examination is simple and accurate ,and has highly applicable value in evaluating the effect of post-pelvic rehabilitation training .

Objective:To study the anti-inflammatory effect of the ethanol extract of Coptis chinensis Franch.in vitro.Methods: An inflammatory cell model was established by stimulating the mouse peritoneal macrophages in vitro with lipopolysaccharide(LPS).LPS stimulated of RAW264.7 cells for a long time after administration of intervention.Effect of ethanol extract of Coptis chinensis Franch.on RAW264.7 cell growth activity was analyzed by MTT assay.The production of tumor necrosis factor-α(TNF-α),IL-1β,IL-6,NO,prostaglandin E2(PGE2) was determined by ELISA.The expression of mRNA of TNF-α,induced nitric oxide synthase(iNos) and HO-1 was detected by real-time fluorescent quantitative PCR(RT-PCR).Results: The ethanol extract of Coptis chinensis had no inhibition effect on the scope of RAW 264.7 cells the scope of 5-80 mg/L.Each treatment group concentration of interleukin-6 (IL-6),interleukin-1β (IL-1β),TNF-α,NO,prostaglandin E2 (PGE2) content of LPS stimulation model group were significantly (P<0.01),and content was not related to concentration.Real-time quantitative (RT-PCR) showed,the concentration of each treatment group were significantly lower iNos,HO-1 and TNF-α mRNA expression (P<0.05,P<0.05,P<0.01),and content was not related to concentration.Conclusion: Coptis chinensis Franch.ethanol extract has anti-inflammatory effects in vitro,the mechanism may be related to inhibition of TNF-α,NO and other inflammatory factors and the impact of the activation of arachidonic acid (AA) metabolism.

Objective To summary the experience of laparoscopic cystectomy ileal conduit (Bricker) and orthotopic ileal neo‐bladder (Hautmann) and compare the short‐term efficacy of the two types of urinary diversion for invasive bladder cancer . Methods Retorspective analysis of the patients in our hospital who accepted laparoscopic radical cystectomy from 2010 to 2014 was performed ,74 of them accepted ileal conduit ,and 30 of them accepted orthotopic ileal neobladder .The general clinical data ,postop‐erative recovery ,postoperative complications and Oncology feature were analyzed and compared between the two groups .Results There was no demonstrable difference was found in operation time ,blood loss ,intraoperative blood transfusion rate ,the number of removed lymph node ,average hospital stay ,specimens positive margin rate and postoperative pathology results between the two groups (P>0 .05) .But there were significant difference in postoperative intestinal function recovery time[(4 .2 ± 1 .4)d ,(5 .3 ± 2 .2)d] ,(P=0 .002) ,and the complication rates 31 .9% (23 cases)vs .53 .3% (16 case) ,P=0 .043 .After 6 months ,the daytime and nighttime urinary control were 76 .9% ,57 .7% ,after 12 months ,the daytime and nighttime urinary control increased to 90 .9% , 81 .8% .2 cases(7 .7% ) were diagnosed with recurrence or metastasis during follow‐up in Hautmann group ,while 9 cases(14 .1% ) were diagnosed with recurrence or metastasis in Bricker group .Conclusion Two kinds of surgical procedures both have the similar therapeutic effect ,but the postoperative quality of life is better for Hautmann orthotopic neobladder patients .

PURPOSE: The Pipeline embolization device (PED) is approved in the USA for treating giant and large aneurysms arising from the petrous to superior hypophyseal segments of the internal carotid artery in patients older than 21 years of age. This study investigates off-label PED results in a large cohort. MATERIALS AND METHODS: Retrospective, single-center review of all patients who had off-label PED surgery. RESULTS: Sixty-two aneurysms (48 patients) underwent off-label PED treatment from 2012–2017. There were 44 females and four males (age 21 to 75 years; mean/median, 54.3/55.0 years). The most common presenting symptom was headache (47/62, 75.8%). All aneurysms were in the anterior circulation. Aneurysm size ranged from 1.4 to 25.0 mm (mean/median, 7.6/6.9 mm). Fifty-two aneurysms had post-operative imaging with total/near-complete occlusion of 84.6% (44/52). Aneurysm-based operative near-term complication rate was 9.7% while there were no permanent complications. For aneurysms and headache, 86.7% improved/resolved after embo-surgery, and were four times more likely to have a better clinical outcome (resolved or improved symptoms) after surgery (odds ratio [OR], 4.333; P=0.0325). Left-sided aneurysms had a higher occlusion rate (OR, 20; P=0.0073). Hypertension (OR, 4.2; P=0.0332) and smoking (OR, 7; P=0.0155) were more prone towards aneurysm occlusion. Patients without a family history were 14 times more likely to have favorable imaging outcome (P=0.0405). There is no difference of occlusion rates between untreated and previously treated aneurysms (P=0.6894). Overall, occlusion rate decreased by 14% with an increase of aneurysm size by 1 mm (P=0.0283). CONCLUSION: For anterior circulation aneurysms, the off-label application of PED is as effective and safe as reported for on-label intracranial aneurysms.
Aneurysm ; Carotid Artery, Internal ; Cohort Studies ; Female ; Headache ; Humans ; Hypertension ; Intracranial Aneurysm ; Male ; Retrospective Studies ; Smoke ; Smoking

OBJECTIVETo establish a zebrafish model of liver fibrosis via diethylnitrosamine (DEN)-induced liver injury.

METHODSA total of 120 wild-type 3-month-old zebrafish were randomly divided into DEN-treated group and control group. The survival rate and behavioral changes of each group were observed. After treatment with DEN for 2, 4, and 6 weeks, liver index was measured, and liver fibrosis was evaluated with HE staining, Gomori staining and Sirius red staining.

RESULTSNo obvious behavioral change was observed in DEN-treated group during the experiment. Compared with that in control group, the liver index of zebrafish in DEN-treated group showed no significantly changes at the time points of observation. Proliferation of reticulate fibers was found in 30% of zebrafish treated with DEN for 4 weeks, and the rate increased to 80% at 6 weeks when reticulate fibers and collagen fibers actively proliferated to result in fiber collapse and formation of fibrotic nodules.

CONCLUSIONA stable zebrafish liver fibrosis model was successfully established by inducing liver damage to facilitate studies of the pathogenesis of liver fibrosis and screening therapeutic drugs.

Animals ; Diethylnitrosamine ; Disease Models, Animal ; Liver Cirrhosis ; chemically induced ; Zebrafish

Objective:To compare the effect and safety of continuous veno-venous hemofiltration (CVVH)+double plasma molecular absorption (DPMA)+hemoperfusion (HP), CVVH+HP, and CVVH+plasma exchange (PE) in treatment of patient with severe wasp stings injury.Methods:Multicenter, historical cohort study and superiority test were used. From July 2020 to October 2022, patients with wasp sting injury and multiple organ damage admitted to the intensive care units (ICU) of five hospitals were consecutively screened and recruited into the CVVH+DPMA+HP group (intervention group). Propensity score matching was used to establish historical cohorts. Patients with severe wasp sting injury who hospitalized from January 2016 to June 2020 in each ICU were collected and matched 1∶1 with the intervention group, and divided into CVVH+HP group and CVVH+PE group according to their actual hemopurification protocols (historical control groups). The primary outcome was the acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score on days 3 and 7 after initiation of treatment. Secondary outcomes included complications, length of ICU and hospital stays, and all-cause mortality. Multivariate Cox proportional risk regression was used to analyze the prognosis of patients.Results:After propensity score matching, 56 patients in intervention group and each of the two historical control groups were matched successfully. There were no significant differences in age, gender, comorbidities, biochemical test indices and critical illness scores among the groups. After treatment, APACHE Ⅱ score markedly declined in all groups, and the decrease was faster in the intervention group; treatment with DPMA [hazard ratio ( HR) = 1.04, 95% confidence interval (95% CI) was 1.02-1.08, P = 0.00], the decreased levels of body temperature ( HR = 1.02, 95% CI was 1.00-1.03, P = 0.02), serum creatine kinase (CK; HR = 0.98, 95% CI was 0.96-1.00, P = 0.05) and myoglobin (MYO; HR = 2.88, 95% CI was 1.24-6.69, P = 0.01) were independent risk factors for APACHE Ⅱ score decline to the target value (15 scores). There were no significant differences in the incidence of bleeding complications, filter or perfusion thrombosis, blood pressure reduction, catheter-related infection and anaphylaxis among the groups. Conclusion:CVVH+DPMA+HP regimen can significantly reduce the APACHE Ⅱ score of patients with severe wasp sting injury, and the efficacy is superior to CVVH+HP and CVVH+PE regimens, with safety.

OBJECTIVETo establish a diet-induced obesity model in zebrafish larvae.

METHODSAt 7 days post-fertilization (dpf), 200 zebrafish larvae with normal development were randomly allocated to two groups with the feeding quantity of 30 mg per day (normal feeding group) or 180 mg per day (overfed group) for 20 days. The weight, length, BMI, triglyceride (TG) and total cholesterol (TCH) of each group were measured. Whole-mount Oil Red O staining, frozen Oil Red O staining and hematoxylin-eosin (HE) staining were used to estimate the rate of hepatic steatosis and liver histology of the zebrafish. The dynamic change of hepatic lipid droplets and distribution of adipose tissue were observed with Nile Red staining in overfed zebrafish in vivo.

RESULTSThe weight, length, BMI and TG of overfed zebrafish were significantly increased compared with those in normal feeding group. Whole-mount Oil Red O staining showed that the percent of hepatic steatosis in overfed group (89.4%) was markedly higher than that in normal feeding group (20.7%). Macrovesicular steatosis was observed in the liver of the overfed larvae. Nile Red staining visualized hepatic lipid droplets and the distribution of larval adipose tissue, which increased with feeding time in the overfed zebrafish. Starving larvae showed depletion of fat and hepatic lipid, and adipose tissue was induced after refeeding.

CONCLUSIONSWe successfully established an diet-induced obesity model in zebrafish larva, in which Nile Red staining allows in vivo observation of the adipocytes and hepatic lipid droplets.

Adipose Tissue ; Animals ; Cholesterol ; Diet ; adverse effects ; Disease Models, Animal ; Fatty Liver ; Larva ; Lipids ; Obesity ; pathology ; Triglycerides ; Zebrafish

Objective:To analyze independent influencing factors of surgical textbook outcome (TO) in patients with gallbladder carcinoma, and to establish a nomogram for predicting TO and evaluated the predictive ability.Methods:Patients with gallbladder carcinoma who underwent surgery in Department of Hepatobiliary and Pancreatic Surgery at Dongfang Hospital Affiliated to Shanghai Tongji University and Department of Biliary Tract Surgery Ⅰ, Third Affiliated Hospital of Naval Medical University (Shanghai Eastern Hepatobiliary Surgery Hospital) from January 2013 to December 2018 were included and the clinical features were retrospectively analyzed. A total of 232 patients were included, including 114 males and 118 females, aged (61.0±9.8) years. According to whether TO reached or not, they were divided into TO group ( n=86) and non-TO group ( n=146). Univariate and multivariate logistic regression were used to analyze the independent influencing factors of TO. The predictive nomogram model of TO was constructed. The receiver operating characteristic (ROC) curve was drawn to evaluate the predictive ability of the model, and the consistency of the predictive model was evaluated by the consistency curve graph and the Hosmer-Lemeshow test. Results:The 1-year and 3-years cumulative survival rates of patients with gallbladder carcinoma in the TO group (86.0% and 62.8%) were better than those in the non-TO group (46.6% and 27.3%), and the difference was statistically significant (χ 2=60.74, P<0.001). In multivariate analysis, higher T stage ( OR=0.16, 95% CI: 0.03-0.79, P<0.001) and cervical gallbladder cancer ( OR=0.14, 95% CI: 0.02-0.94, P=0.004) had the greatest negative association with a TO, and the higher the degree of tumor differentiation ( OR=7.08, 95% CI: 1.34-37.56, P=0.001), the easier it is to achieve TO. The ROC curve showed that the area under the curve of the predictive model was 0.84 (95% CI: 0.79-0.90), suggesting that the model had good predictive performance. A nomogram to assess the probability of TO was developed and had good accuracy in both the consistency curve and Hosmer-Lemeshow test (χ 2=5.77, P=0.673). Conclusion:Tumor T stage, tumor differentiation degree and tumor location are independent influencing factors for achieving TO in patients with gallbladder carcinoma after surgery. The nomogram model constructed according to the above conclusions could accurately predict the probability of reaching TO.