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 the applicant effect of improved flushing suction method for SpyClass choledochoscope operation. Methods Seventy-three patients who underwent SpyGlass choledocho-scopy from February 2013 to January 2015 were enrolled. Negative pressure biopsy channel was used in group A(n= 34)during conducting SpyGlass choledochoscopy with the tube directly connected to the conveying pipe and combined with duodenoscope suction.In group B(n= 39),the biopsy channel of disposable syringe jacket was first connected to a delivery catheter,then the negative pressure pipe was put in the injector coat, combined with duodenoscope suction. Adverse reactions,theⅠ image acquisition time after the placement of SpyGlass delivery catheter to pancreaticobiliary duct,the total operation time,and the occurrence of compli-cations after operation were recorded. Results Sinus bradycardia,arrhythmia,agitation occurred in 7 cases (20. 6%),5 cases( 14. 7%),and 10 cases( 29. 4%) respectively in group A,those were 1 case (2. 6%),0 case(0/ 39),2 cases(5. 1%)respectively in group B with significant difference(P<0. 05). The time to acquire a clear image and the total operation time in group A were 6. 2 min and 24. 9 min,and 3. 3 min and 16. 8 minutes in B group with significant difference(P<0. 05),postoperative cholangitis oc-curred in 5 cases in group A(14. 7%),1 case(2. 6%)in group B with significant difference(P<0. 05). Conclusion New irrigation and suction method has obvious advantages in the SpyGlass system.

Objective To evaluate accuracy,safety and clinical practicality of three measurements for the length of biliary stent.Methods A total of 120 patients with indwelling biliary stents were randomly divided into group A,B and C by the admission time,who then received endoscopic guide wire measurement (Group A),cutting knife measurement (Group B) and formula measurement (Group C) for biliary stent length.Results There were no statistical significance in sex,age,type of primary disease and biliary stent materials (P＞0.05).Operation time in group C (9.4 ± 2.47 min) was shorter than that in group A (15.8±1.71 min) and B (16.2 ± 2.22 min) with significant difference (P＜0.05).There was no significant difference in the incidence of postoperative cholangitis [7.5％ (3/40) VS 5.0％ (2/40) VS 5.0％ (2/40)]or the measurement accuracy of biliary stent length [100.0％ (40/40) VS 95.0％ (38/40) VS 97.5％(39/40)] (P＞0.05).Conclusion Three stent length measurements are accurate and safe,but formula measurement has more advantage in clinical practicality.

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 explore the application effect of critical ultrasound combined with extravascular lung water (EVLW) and intrathoracic blood volume (ITBV) on volume management of mechanically ventilated patients.Methods:From May 2017 to January 2020, 98 patients treated with mechanically ventilated admitted to Hebei Petro China Central Hospital were selected and divided into two groups by random number table method, with 49 cases in each group. Both groups were treated with fluid resuscitation, the control group was guided by central venous pressure (CVP) and the observation group was guided by critical ultrasound combined with EVLW and ITBV. The acute physiology and chronic health score Ⅱ(APACHE Ⅱ) and sequential organ failure assessment (SOFA) scores, hemodynamic indexes, respiratory system indexes , urine output and fluid intake at 6 h and 24 h after resuscitation and mechanical ventilation time, ICU stay, complications and prognosis were compared between the two groups.Results:The scores of APACHE Ⅱ and SOFA in the observation group at 6 h and 24 h after resuscitation were lower than those in the control group: 6 h after resuscitation: (22.02 ± 4.29) scores vs. (23.94 ± 3.56) scores, (10.02 ± 3.11) scores vs. (11.64 ± 2.30) scores; 24 h after resuscitation: (19.66 ± 2.85) scores vs. (21.78 ± 3.60) scores, (7.64 ± 2.15) scores vs. (9.83 ± 2.07) scores, the differences were statistically significant ( P<0.05). The mean arterial pressure (MAP) and CVP in the observation group at 6 h and 24 h after resuscitation were higher than those in the control group: 6 h after resuscitation: (69.44 ± 5.25) mmHg(1 mmHg=0.133 kPa) vs. (65.98 ± 4.33) mmHg, (13.64 ± 2.30) mmHg vs. (11.89 ± 3.07) mmHg; 24 h after resuscitation: (72.89 ± 4.69) mmHg vs. (69.26 ± 5.53) mmHg, (13.07 ± 2.15) mmHg vs. (11.89 ± 3.07) mmHg; the heart rate was lower than those in the control group: 6 h after resuscitation: (98.58 ± 9.32) bpm vs. (105.03 ± 8.76) bpm; 24 h after resuscitation: (94.97 ± 8.46) bpm vs.(101.44 ± 7.34) bpm, the differences were statistically significant ( P<0.05). The central venous oxygen saturation (ScvO 2) and oxygenation index (OI) in the observation group at 6 h and 24 h after resuscitation were higher than those in the control group: 6 h after resuscitation: 0.749 ± 0.043 vs. 0.711 ± 0.047, (258.18 ± 20.75) mmHg vs. (234.66 ± 25.42) mmHg; 24 h after resuscitation: (77.68 ± 4.09)% vs. (73.54 ± 4.23)%, (376.29 ± 22.39) mmHg vs. (234.66 ± 25.42) mmHg; the blood lactic acid was lower than that in the control group: 6 h after resuscitation: (3.04 ± 0.52) mmol/L vs. (4.22 ± 0.39) mmol/L; 24 h after resuscitation: (1.01 ± 0.34) mmol/L vs. (1.87 ± 0.41) mmol/L, the differences were statistically significant( P<0.05). The urine output at 6 h and 24 h in the observation group was higher than that in the control group: 6 h after resuscitation: (0.49 ± 0.08) ml/(kg·h) vs. (0.35 ± 0.06) ml/(kg·h); 24 h after resuscitation:(0.54 ± 0.05) ml/(kg·h) vs. (0.42 ± 0.07) ml/(kg·h); the fluid intake was lower than that in the control group: 6 h after resuscitation: (1 230.2 ± 562.3) ml vs. (1 782.4 ± 534.7) ml; 24 h after resuscitation: (3 065.5 ± 521.2) ml vs. (3 642.0 ± 507.8) ml; the mechanical ventilation time, and ICU stay in the observation group were lower than those in the control group: (3.3 ± 0.9) d vs. (5.0 ± 0.7) d, (9.7 ± 2.1) d vs. (10.9 ± 1.8) d, the differences were statistically significant ( P<0.05). There was no significant differences in complication rate and 28-day survival curve between the two groups ( P>0.05). Conclusions:Critical ultrasound combined with EVLW and ITBV has a good application effect on volume management of patients with mechanical ventilation, which can help maintain hemodynamic stability, improve oxygenation status.

Gallbladder cancer is a high malignancy which is predisposed to invade adjacent organs and have lymph node metastasis. Gallbladder cancer is not sensitive to radiotherapy or chemotherapy with the worst prognosis among biliary tract cancers. At present, radical resection is the only possible method to cure gallbladder cancer. However, there are still many controversies about the surgical strategies, the extent of liver resection and lymph node dissection, and the treatment of incidental gallbladder cancer. In addition, under the background of the great success of immunotherapy and targeted therapy in a variety of solid tumors, it is also a question worthy of further considerations that whether the status of surgery in the treatment of advanced gallbladder cancer will be changed in the near future.

Objective To investigate the effect of alogliptin on albuminuria in patients with early type 2 diabetic kidney disease (DKD) and the related mechanism.Methods One hundred patients with early DKD admitted in our hospital from May 2016 to May 2017 were randomly divided into two groups with 50 cases in each group.Patients in the control group were given metformin and gliclazide,while those in study group were given metformin and alogliptin,the treatment lasted for 24 weeks.The changes of urinary albumin-to-creatinine ratio (UACR),stromal cell-derived factor-1α (SDF-1α) and the fasting plasma glucose (FPG),2-h postprandial plasma glucose (2 hPPG),glycosylated hemoglobin(HbA1c) were measured before and after the treatment in two groups.Results There were no significant differences in HbA1c [(8.17± 0.46)％ vs.(8.29±0.48)％],UACR[(109±53) vs.(105±48)mg/g],SDF-1α [(1.21±0.3 9) vs.(1.17±0.35)μg/L] levels before treatment between two groups (t=0.343,0.464,0.075,all P＞0.05).After treatment,the HbA1c levels were significantly decreased in both groups (t=2.293,2.302,all P=0.03) and there was no significant difference between two groups[(6.82±0.75)％ vs.(6.93 ±0.79)％,t=0.295,P=0.77];the UACR levels were significantly reduced in both groups,but the level of study group was significantly lower than that of control group [(82±38) vs.(94±47) mg/g,t=3.320,P＜0.01];the SDF-1α levels were significantly increased in both groups,but the level of study group was significantly higher than that of control group[(3.01 ±0.38) vs.(2.76±0.42)μg/L,t=5.474,P＜0.01].There was no significant difference in the incidence of adverse reactions between the two groups [13％ (6/46) vs.12％ (6/48),x2=0.002,P＞0.05].Conclusion Alogliptin can effectively control the blood glucose,reduce urine albumin excretion and protect renal function in patients with early type 2 diabetic nephropathy,which is associated with the increased SDF-1α levels.

Objective:To evaluate the efficacy and safety of clip-with-endoloop method during endoscopic submucosal dissection (ESD) in treatment of early gastric angle cancer and precancerous lesions.Methods:A total of 59 patients with early gastric angle cancer or precancerous lesions underwent ESD from January 2018 to December 2018 were randomly divided into the routine ESD group ( n=28) and the clip-with-endoloop group ( n=31). The frequency of supplementary submucosal injection, ESD procedure time, area of the resected specimen, dissection time, submucosal dissection speed, complete resection rate and complications were compared between the two groups. Results:The frequency of supplementary submucosal injection in the clip-with-endoloop group was less than that in the routine ESD group (2.3±1.1 VS 3.7±1.4, t=4.557, P<0.001). There was no significant difference in the area of the resected specimen between the two groups (12.7±2.6 cm 2 VS 11.7±2.7 cm 2,t=1.485, P=0.143). The ESD procedure time (72.4±24.7 min VS 93.6±28.9 min, t=3.043, P=0.004) and dissection time (67.7±23.3 min VS 88.2±28.3 min, t=3.054, P=0.003) in the clip-with-endoloop group were significantly shorter compared with those in the routine ESD group. The submucosal dissection speed in the clip-with-endoloop group was higher than that in the routine ESD group (20.2±3.2 mm 2/min VS 14.3±3.4 mm 2/min, t=6.879, P<0.001). The complete resection rate was 100.0% in the both groups. No perforation or postoperative bleeding occurred in the two groups. The incidence of intraoperative bleeding in the clip-with-endoloop group was lower than that in the routine ESD group [19.4% (6/31) VS 35.7% (10/28), χ2=1.992, P=0.158]. Conclusion:Clip-with-endoloop method makes ESD procedures easier and faster, with a lower possibility of intraoperative bleeding in treatment of early gastric angle cancer.

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.

Objective To investigate the characteristics of population and the changes of disease spectrum in patients treated by endoscopic retrograde cholangiopancreatography ( ERCP ) . Methods Data of 20170 patients, who underwent ERCP in the First People' s Hospital of Hangzhou from May 2004 to February 2018, were enrolled in the retrospective analysis. According to the year of diagnosis and treatment,patients were divided into 2004-2008, 2009-2013, and 2014-2018 group; according to the postoperative diagnosis of ERCP, they were divided into biliary tract diseases ( including calculus, benign stenosis, malignant stenosis, and other causes ) and pancreatic diseases ( including acute pancreatitis, chronic pancreatitis, pancreatic malignant tumor, and other causes ) group; and according to the age, they were divided into the younger age (0-18 years old), young and middle age (19-65 years old), old age (66-85 years old), and advanced age (>85 years old) group. Statistical analysis was performed in the different groups. Results Among the 20170 patients, there were 10260 males and 9910 females, with age of 62. 65± 17. 11 years. The proportion of the younger age group and the advanced age group was 1. 04％( 24/2308 ) , 1. 69％ ( 127/7520 ) , 2. 39％ ( 247/10342 ) , and 2. 95％ ( 68/2308 ) , 4. 19％( 315/7520 ) , 6. 15％ ( 636/10342 ) , respectively, in the 2004-2008, 2009-2013 and 2014-2018 subgroups, with a rising trend (P<0. 017). A total 20032 patients had a clear postoperative diagnosis, including 15618 ( 77. 97％) of biliary diseases and 4414 ( 22. 03％) of pancreatic diseases. Biliary stones accounted for the majority of biliary diseases (63. 2％, 9863/15618), and its proportion increased from 59. 9％ ( 1191/1987 ) in 2004-2008 to 64. 5％ ( 5118/7939 ) in 2014-2018 ( P= 0. 000 ) . Acute pancreatitis accounted for the majority of pancreatic diseases (67. 4％, 1973/4414), and its proportion increased from 52. 4％ ( 162/309) in 2004-2008 to 69. 9％ ( 1636/2340) in 2014-2018 ( P=0. 000) . The top 3 diseases in the younger group and the advanced age group were acute pancreatitis ( 32. 58％, 129/396) , biliary stones ( 25. 25％, 100/396 ) , chronic pancreatitis ( 22. 22％, 88/396 ) , and biliary stones (56. 46％, 568/1006), malignant biliary stenosis (12. 52％, 126/1006), benign biliary stricture ( 10. 34％, 104/1006) , respectively. Conclusion The main disease of patients receiving ERCP is biliary stone, and the proportion of acute pancreatitis is increased. The overall age of the patients is old, and the proportion of the elderly and underaged patients is gradually increasing. Biliary diseases and pancreatic diseases are the main diseases in elderly patients and younger patients, respectively.