Objective To investigate the method to isolate and culture hepatic stellate cells ( HSCs) for studying the cellular mechanisms of hepatic frbrosis.Methods HSCs were isolated by nycodenz density gradient centrifugation after the hepatocytes obtained from adult male gebils were digested with pronase, collagenase and DNase, infused via portal vein.The cell viability was determined by trypan blue exclusion test.The purity of HSCs was identified by detectingα-SMA, desmin immunohistochemical staining.Results The yield rate of HSCs was 0.5~1 ×107 per gerbil liver, and the cell viability was more than 90%.The percentage ofα-SMA-positive cells was more than 75%after 3 days primary culture and almost 100% cells were α-SMA and desmin positive in passage culture.Conclusion The successful protocol of primary culture of Mongolian gerbil HSC provide a technical support for research of relevant liver diseases and drug development in the future.

OBJECTIVETo explore the stability of seminal plasma miR-122-3p and miR-141-5p and their diagnostic value for idiopathic asthenospermia.

METHODSSeminal plasma miR-122-3p and miR-141-5p with various incubation time, freeze-thaw cycles and incubation time were analyzed. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the content of miR-122-3p and miR-141-5p, with U6 snRNA as the reference. The idiopathic asthenospermia group was further divided into grade I and II based on the progressive motility percent, and the content of miR-122-3p and miR-141-5p in the two grades were compared.

RESULTSNo difference was detected after incubation at room temperature and 4 degrees Celsius. But seminal plasma miR-122-3p and miR-141-5p have decreased along with the increase in freeze-thaw cycles increases. RT-PCR assayed showed that the miR-122-3p content in the idiopathic asthenospermia group was significantly lower than the control group, while miR-141-5p was significantly higher. The difference in both miR-122-3p and miR-141-5p content was statistically significant (P < 0.05). A significant difference in miR-122-3p and miR-141-5p content between a and b groups was also detected (P < 0.05). The AUC-ROC of miR-122-3p was 0.88. At the cutoff value of 1.02, the sensitivity and specificity was 83% and 84%, respectively. At the cutoff of 2.95, the AUC-ROC of miR-141-5p was 0.88. The sensitivity and specificity was 84% and 70%, respectively.

CONCLUSIONMiR-122-3p and miR-141-5p in seminal plasma are stable and have certain value for the diagnosis of idiopathic asthenospermia.

Adult ; Asthenozoospermia ; diagnosis ; genetics ; Humans ; Male ; MicroRNAs ; analysis ; chemistry ; Middle Aged ; ROC Curve ; Real-Time Polymerase Chain Reaction ; Semen ; chemistry

Objectives: To propose a novel clinical classification system of gallbladder cancer, and to investigate the differences of clinicopathological characteristics and prognosis based on patients who underwent radical resection with different types of gallbladder cancer. Methods: The clinical data of 1 059 patients with gallbladder cancer underwent radical resection in 12 institutions in China from January 2013 to December 2017 were retrospectively collected and analyzed.There were 389 males and 670 females, aged (62.0±10.5)years(range:22-88 years).According to the location of tumor and the mode of invasion,the tumors were divided into peritoneal type, hepatic type, hepatic hilum type and mixed type, the surgical procedures were divided into regional radical resection and extended radical resection.The correlation between different types and T stage, N stage, vascular invasion, neural invasion, median survival time and surgical procedures were analyzed.Rates were compared by χ² test, survival analysis was carried by Kaplan-Meier and Log-rank test. Results: Regional radical resection was performed in 940 cases,including 81 cases in T1 stage,859 cases in T2-T4 stage,119 cases underwent extended radical resection;R0 resection was achieved in 990 cases(93.5%).The overall median survival time was 28 months.There were 81 patients in Tis-T1 stage and 978 patients in T2-T4 stage.The classification of gallbladder cancer in patients with T2-T4 stage: 345 cases(35.3%)of peritoneal type, 331 cases(33.8%) of hepatic type, 122 cases(12.5%) of hepatic hilum type and 180 cases(18.4%) of mixed type.T stage(χ²=288.60,P<0.01),N stage(χ²=68.10, P<0.01), vascular invasion(χ²=128.70, P<0.01)and neural invasion(χ²=54.30, P<0.01)were significantly correlated with the classification.The median survival time of peritoneal type,hepatic type,hepatic hilum type and mixed type was 48 months,21 months,16 months and 11 months,respectively(χ²=80.60,P<0.01).There was no significant difference in median survival time between regional radical resection and extended radical resection in the peritoneal type,hepatic type,hepatic hilum type and mixed type(all P>0.05). Conclusion With application of new clinical classification, different types of gallbladder cancer are proved to be correlated with TNM stage, malignant biological behavior and prognosis, which will facilitate us in preoperative evaluation,surgical planning and prognosis evaluation.