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 investigate the roles of a epoprostenol(PGI2) analog (Iloprost) in regulating the differentiation of CD4+ T cells to Treg cells and the possible mechanism.Methods Naǐve CD4+ T cells were isolated from human peripheral blood samples by using the magnetic-activated cell sorting (MACS) and then cultured under Treg-polarizing condition.The percentages of Treg cells and the expression of Foxp3 at mRNA level were respectively measured by using flow cytometry and RT-PCR for evaluation the effects of Iloprost on the differentiation of CD4+ T cells to Treg cells.The cAMP accumulation assay was used to detect the level of intracellular cAMP.Flow cytometry analysis was performed to detect the phosphorylation of signal transducer and activator of transcription 5 (STAT5).Results lloprost decreased the percentage of Treg cells and inhibited the expression of Foxp3 at mRNA level in a dose dependent manner (P＜0.05).However,the inhibitory effects of Iloprost were weakened when IP receptors were blocked by IP antagonist (CAY10449).A six-fold increase in the levels of intracellular cAMP in Treg cells was induced by Iloprost (P＜0.05) and a similar effect could be achieved by using a cAMP agonist,db-cAMP (P＞0.05).H-89,a protein kinase A inhibitor,inhibited the Iloprost-induced expression of cAMP in Treg cells.Moreover,Iloprost inhibited the IL-2 mediated phosphorylation of STAT5 (P＜0.05) and a similar effect could be achieved by using db-cAMP (P＞0.05).The Iloprost-mediated down-regulation of pSTAT5 was blocked by using H-89.Conclusion PGI2 could activate the cAMP-PKA signaling pathway by binding to the IP receptor,resulting in inhibited phosphorylation of STAT5 and suppressed differentiation of naǐve CD4+ T cells to Treg cells.

Objective To evaluate the effects between video endoscopic inguinal lymphadenectomy (VEIL ) and open inguinal lymphadenectomy(OIL) to provide the evidence-based basis for the selection of the clinical therapy schemes .Methods The related clinical controlled trial literature on the effective comparison of VEIL and OIL were retrieved from the databases of PubMed ,Co-chrane library ,Elsevier ,CNKI and Wanfang database .The screening was independently performed by 2 reviewers according to the including and excluding criteria .The related data were extracted and performed the meta analysis by the RevMan 5 .2 software .Re-sults A total of 4 trials were included .There were 146 cases of inguinal lymphadenectomies ,in which 61 cases were VEIL and 85 cases were OIL .The meta-analysis results showed that there were no statistical differences between the two operation modes in terms of the operative time(WMD=32 .33 ,95% CI -25 .70-90 .36 ,P=0 .27) ,intraoperative blood loss(WMD=9 .10 ,95% CI -76 .03-94 .23 ,P=0 .83) ,number of removed lymph nodes(WMD=0 .77 ,95% CI -1 .66-3 .20 ,P=0 .53) ,number of positive re-moved lymph nodes(WMD=0 .08 ,95% CI -0 .23-0 .40 ,P=0 .61) ,postoperative drainage time(WMD= -1 .30 ,95% CI -6 .40 -3 .80 ,P=0 .62) ,postoperative hospital stay (WMD= -4 .02 ,95% CI -10 .19-2 .15 ,P=0 .20) ,but the difference between VEIL and OIL in term of surgical complications had statistical significance (OR=0 .08 ,95% CI 0 .03-0 .26 ,P<0 .01) .Conclusion VEIL has equivalent efficacy to OIL ,but has less surgical complications .

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 .

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.

The American Association for the Study of Liver Diseases (AASLD) updated and published the Practice Guidance for the Diagnosis and Management of Nonalcoholic Fatty Liver Disease (NAFLD) in July 2017, which provides recommendations for the accurate diagnosis, treatment, and effective prevention of NAFLD. Related metabolic diseases should be considered during the initial evaluation of patients suspected of NAFLD. Noninvasive diagnostic techniques including transient elastography, magnetic resonance elastography, and serum biochemical models should be used to evaluate the development and progression of liver fibrosis in patients with NAFLD. Clinical liver pathology report should clearly differentiate between nonalcoholic fatty liver (NAFL), NAFL with inflammation, and nonalcoholic steatohepatitis (NASH) and identify the presence or absence of liver fibrosis and its degree. Early medication for NAFLD can only be used in patients with pathologically confirmed NASH and liver fibrosis, and it is not recommended to use pioglitazone and vitamin E as the first-line drugs for patients with NASH which has not been proven by biopsy or non-diabetic NASH patients. Foregut bariatric surgery can be considered for obese patients with NAFLD/NASH who meet related indications. It is emphasized that the risk factors for cardiovascular disease should be eliminated for NAFLD patients. Statins can be used for the treatment of dyslipidemia in patients with NAFLD/NASH, but they cannot be used in patients with decompensated liver cirrhosis. Routine screening or hepatocellular carcinoma surveillance is not recommended for NASH patients without liver cirrhosis. Cardiovascular disease should be taken seriously during liver transplantation evaluation. There is still no adequate clinical evidence for the treatment of NAFLD in children and adolescents, and intensive lifestyle intervention is recommended as the first-line therapy for such patients.

Objective To investigate the effects of gemstone energy spectrum CT atomic number method and infrared spec-troscopy for analyzing the composition of urinary calculi and to compare their values in qualitative diagnosis of urinary calculi .Meth-ods Two hundreds and sixty cases of urinary tract stones were performed the gemstone spectrum CT urinary scanning and the stone composition was identified by atomic number method .After removing stone ,the stone composition analyzed by infrared spec-troscopy served as the gold standard .Then the consistency identified by the two methods was analyzed .Results The Kappa consis-tency test results showed that the two kinds of method for identifying stone type had good consistency (Kappa=0 .787 ,P<0 .01) . The paired chi square test results showed that the difference of the two methods for identifying the stone type had no statistical sig-nificance(χ2 =6 .581 ,P=0 .254) .The stone crystal composition types measured by gemstone energy spectrum CT atomic number method were less than those measured by infrared spectroscopy .The precise quantification of the stones with different crystal struc-tures was not as accurate as that of infrared spectroscopy (calcium oxalate monohydrate and calcium oxalate dihydrate ) .Conclusion The two methods for analyzing theurinary stone composition all have clinical significance ,the stone analysis method should be se-lected according to the actual situation .

Objective:To investigate the anti-fatigue phenomenon induced by forced swimming in the mice,and to explore the anti-fatigue effect of argininyl fructoyl glucose (AFG) from red ginseng in the mice and its mechanism.Methods:The AFG was extracted from red ginseng.The ICR mice were divided into blank control group,low dose of AFG group (100 mg · kg-1),middle dose of AFG group (200 mg · kg-1) and high dose of AFG group (400 mg · kg-1) (n=20).The mice mere given a forced swimming test after continuous gavage for 28 d.The weights,organ indexes,time of forced swimming,contents of lactic acid (LD),blood urea nitrogen (BUN),hepatic glycogen (Gly) and expressing levels of PGC-1α in gastrocnemius of the mice in various groups were detected.Results:Compared with blank control group,the weights and organ indexes of the mice in low,middle and high doses of AFG groups had no significant differences (P＞ 0.05).Compared with blank control group,the time of forced swimming,contents of Gly and expressing levels of PGC-1α of the mice in low,middle and high doses of AFG groups were significantly increased (P＜0.05 or P＜0.01) in a dose-dependent manner.Compared with blank control group,The contents of LD and BUN in serum of the mice in low,middle and high doses of AFG groups were significantly decreased (P＜0.01).Conclusion:AFG has anti-fatigue effect in mice,and its mechanism may be related to energy metabolism.