Objective To establish a simple , practical , highly-effective and stable method for the isolation and cultivation of rat hair follicle cells. Methods Under sterile condition, single hair follicle was taken out after the skin around the barbel of SD neonatal rats was sheared off. And then the hair follicles were digested with two-step method with Type Ⅰcollagenase and trypsin. The obtained cell suspension was planted into the culture plate which was covered with extracellular matrix, and then was cultivated with K-SFM culture medium containing fetal bovine serum with volume fraction of 1%. On the next day, K-SFM culture medium was replaced with serum-free culture medium. The remaining tissues were cut into pieces and spread out in the culture flask, and then were cultivated with HG-DMEM culture medium containing serum. Two kinds of cells were harvested and then were identified by immunofluorescence. The hair follicle epithelial cells were tested by flow cytometer. Results The hair follicle epithelial cells obtained through the above methods showed rapid adherence, and were round or polygon-like , with typical cobblestone-like morphology. The long spindle-shaped cells were seen around the tissues cultivated, having many protrusions on the surface of the cells, and they were interconnected into reticular structure. The expression of cytokeratin 15, cytokeratin 19 and β1 integrin in epithelial cells were positive. Most of the epithelial cells were in the G1 phase, accounting for 75.6%. The expression of laminin ( LN), fibronectin ( FN) and vimentin in the connective tissue sheath cells were also positive. Conclusion The cells harvested by modified two-step enzyme digestion method have confirmed as hair follicle cells and fibroblasts, and the obtained cells are of rapid adherence, good homogeneity, and active proliferation.

Objective To explore the feasibility of repairing skin defect with Pelnac tissue engineering skin and cultured epidermal stem cells ( ESCs) and fibroblasts. Methods ESCs and fibroblasts in SD rats were isolated and cultured in vitro, and then were detected by immunohistochemistry. Fibroblasts were seeded in Pelnac material to construct dermis, and ESCs were seeded on the surface of dermis to construct tissue engineering skin. Thirty BALC/c nude mice were randomly divided into groups A, B and C. After mouse back skin defect model was established, group A was transplanted with Pelnac material, group B was transplanted with compound of Pelnac and fibroblasts, and group C was transplanted with Pelnac tissue engineering skin. The wound healing was observed after operation. The wound tissues were sampled for pathological histology test on the 5th and 7th day after transplantation. Results The expression of cytokeratin 15 and β1 integrin in ESCs was positive, and the expression of laminin ( LN) and fibronectin ( FN) in fibroblasts was positive. In group C, the wound healing of nude mice was the best, characterized by the proliferation of fibroblasts, obvious new capillaries and regularly arranged epidermal cells at the transplantation sites. Conclusion Pelnac tissue engineering skin has good effects on promoting repair of skin defect of nude mice.

Objective To investigate the expression changes of Ire1α and caspase-12 in rats with spinal cord ischemia-reperfusion injury.Methods Fifty-five adult SD rats(250-300 g)were randomly divided into control group(re=5)and operation group(n=50).The spinal cord ischemia-reperfusion models were established and the neuronal apoptosis was detected by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling(TUNEL).The expressions of Ire1α and caspase-12 in spinal cord tissue were detected by immunohistochemistry,immunofluorescence and Western blot analysis at 1,4,8,16and 24 hours after ischemia-reperfusion.Results TUNEL staining showed that the number of apoptotic cells was gradually elevated with time.The expressions of Ire 1α and caspase-12 were increased at 1 hour after reperfusion,and peaked at 16 hours,but began to decline at 24 hours after reperfusion.The number of neurons with positive expressions of Irelaand caspase-12 was significantly higher than that of control group(P＜0.05).Conclusion Ire 1α and caspase-12 synergistically participate in the neuronal apoptosis induced by the endoplasmic reticulum stress.

Objective To observe the effect ropivacaine mesylate combined with sufentanil on walking epidural laboranalgesia.Methods 360 puerperae who accepted 0.12％ ropivacaine mesylate combined with sufentanil (0.5 μg/ml) epidural analgesia were analgesic group;at the same time and under the similar conditions,the 356 cases of puerprtae unused analgesic were control group.With visual analogue scale(VAS) assessing the uterine contraction pain and improved Bromage scale assessing lower limb movement block,recording the analgesic effect,every labor time,delivery mode,and the condition of postpartum hemorrhage,fetal distress and neonatal Apgar score in every group.Results Labor pain of pain group significant relief,the active period shorten and the vaginal eutocia percentage were higher than those in the control group; Labor time of the secend and the third stage,vaginal birth rate and oxytocin utilization rate had no differences between the two groups.Conclusion The effect of epidural block labor analgesia on 0.12％ ropivacaine mesylate combined with sufentanil is obvious and security.There is no motor nerve block and do not affect the stage of labor and neonatal.It still can reduce the rate of cesarean section and improve maternal satisfaction.

Objective: To compare the efficacy and safety of Cardi-O-fix patent foramen ovale (PFO) occluder and Amplatzer PFO occluder for the treatment of patients with PFO. Methods: A total of 246 consecutive patients (105 males and 141 females) with PFO were prospectively enrolled from May 30, 2013 to March 30, 2015 in our hospital. PFO interventional closure was applied according to the anatomical structure of the disease and patients′ wishes.Cardi-O-fix PFO occluder was used in 180 cases (COF group), Amplatzer PFO occluder was used in the remaining 66 cases (Amp group). Post-procedure safety including recurrent stroke, transient ischemic attack, death, and complete closure rate, and efficacy including procedure related complications of different devices were compared during the 12 months follow-up. Results: (1) Rate of transient ischemic attack was similar between COF group and Amp group at 12 months after procedure(1.1%(2/180) vs. 1.5%(1/66), P=1.000). There was no recurrent stroke and death during the 12 months follow-up period.Complete closure rate was similar between COF group and Amp group at 12 months after the procedure(90.6%(163/180)vs. 86.4%(57/66), P=0.355). (2) Three cases(1.7%) of paroxysmal atrial fibrillation were observed in COF group during the 12 months follow-up period, 1 patient converted spontaneously to sinus rhythm and 2 patients received successful pharmacologic conversion and converted to sinus rhythm. One patient(1.5%)developed paroxysmal atrial fibrillation and was pharmacologically converted to sinus rhythm in the Amp group. There was no significant difference in rate of paroxysmal atrial fibrillation between the two groups(P=1.000). There was no complications such as occluder translocation, erosion, pericardial effusion and puncture site bleeding in the 2 groups during the 12 months follow-up. Conclusion Efficacy and safety are similar for PFO treatment with Cardi-O-fix PFO occluder or Amplatzer PFO occluder in this patient cohort.

Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl₄+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl₄+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl₄+PHx group. Compared with the CCl₄+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl₄+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl₄+PHx group. Compared with the CCl₄+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl₄+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl₄+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl₄+PHx+MSC-EV group was higher than that in the CCl₄+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.

【Objective】 To study the quality control of human albumin intermediates content in production process using High Performance Liquid chromatography (HPLC) method. 【Methods】 In accordance with the general principles of The Chinese Pharmacopoeia (2015 edition), 3121 human albumin polymer was used to determine the component V precipitation solution, human albumin stock solution, human albumin semi-finished products and human albumin products in the production process of human albumin. The chromatographic retention time and the chromatographic peak area percentage corresponding to the retention time were analyzed. 【Results】 The polymer content of component V precipitation solution, human albumin stock solution, human albumin semi-finished products, and human albumin products were was 0.2% vs 0.0% vs 1.9% vs 1.7%, 0.2% vs 0.0% vs1.8% vs1.5% and 0.1% vs 0.0% vs 1.6% vs 1.5%, respectively. 【Conclusion】 HPLC can be used as a monitoring method for human albumin production.

【Objective】 To establish gas chromatography for the determination of tributyl phosphate(TBP) residues in human prothrombin complex and then verify it. 【Methods】 Acid modified polyethylene glycol(PEG)(20M) capillary column was used with n-hexane as solvent. The chromatographic parameters were as follows: gasification chamber temperature at 220 ℃, column temperature at 155 ℃, detector temperature at 220 ℃, column flow rate at 2.0 mL/min, carrier gas as N2, detector as FID, and collection time for 10min. The accuracy, repeatability, linearity, specificity, intermediate precision, detection limit, quantitative limit, range and durability were verified. 【Results】 The verification results showed that the method had good specificity. The linear regression correlation coefficient of standard curve was 0.999 90. The recovery rate were 98.4%, 97.5% and 95.7% when the concentration at 50%, 100%(30μg/mL) and 150%, respectively, with an average recovery of 97.2% and a relative deviation of 2.15%. When the concentration was 100%, the repeatability was 2.08%, and the relative deviation of intermediate precision was 1.63%. The detection limit was 0.255 μg/mL, and the quantitative limit was 0.511 μg/mL. After changing capillary chromatographic columns with different batch numbers but the same types and manufacturer, the applicability test of the system met the requirements, and the method had good durability. 【Conclusion】 This method can be used for the determination of TBP residues of human prothrombin complex in laboratory.