Objective To detect the optimum reaction temperature,pH,Km of N-V proteinase and determine its enzymological classification.Methods The fibrin plate method was used to determine the optimum reaction temperature and pH of N-V proteinase.The Km of N-V proteinase was detected by using Leu-pNA as substrate.E64,PMSF,zymofren and 1,10-phenanthroline were used as inhibitors to determine proteinase classification of N-V proteinase.Results The optimum reaction temperature was 50℃,pH was 8-9,Km was 1.97?10-3mol?L-1.PSMF inhibited the fibrinolytic activity of N-V proteinase(P0.05).Conclusion N-V proteinase belongs to serine proteinase.

Objective To investigate effects of heroin on prolactin(PRL) mRNA in female rats.Methods Fifty female Wistar rats were randomly divided into five groups(10 rats each group): control,heroin-treated-3 d,heroin-treated-9 d,heroin-withdrawal-3 d and heroin-withdrawal-9 d groups.PRL mRNA of pituitary was assessed by(RT-PCR) and PRL concentration in rat plasma was determined by radioimmunoassay(RIA).Results The expression levels of PRL mRNA of pituitary were declined significantly in heroin-treated-3 d,9 d and heroin-withdrawal-3 d groups compared with control group(P0.05)).Conclusion The PRL mRNA in the pituitary and the PRL concentration in plasma in female rats are decreased by heroin and the effect can not be reversed in a short term after heroin withdrawal.

Objective To investigate the application of PKH26 and molecular light imaging system in cartilage en?gineering. Methods Canine chondrocyte was labeled by fluorescent dye PKH26 and seeded into the porous cartilage acel?lular matrix scaffold. The cells/scaffold constructs were cultured in vitro for 1 week. Then the constructs were implanted into the dorsal pocket of nude mice. We utilized a molecular light imaging system to macroscopically observe cells/scaffold con?structs in vivo with fluorescence at the 4th weeks, and compared with X-rays taken at the same position. The fluorescence im?ages were compared with the immunohistochemical and immunofluorescent results of cartilage-like tissue in vivo. Results Luminescent images were acquired at the 4th weeks, a red color enhanced overlay of the luminescent image over X-ray photo?graphic image demonstrated the location of the implants and the cell viability and cell growth on porous CACM scaffold in vivo were very well. Histological results show that the safranin O, anti-collagenⅡimmunohistochemistry and toluidine blue stain of cartilage-like tissue is positive. Immunofluorescence examination demonstrated chondrocytes in the constructs whitch is showen red fluorescence, and anti-collagenⅡimmunofluorescent staining was showen in green while the overlap?ping image is showen in yellow. Conclusion This study outlines an applicable non-destructive method to evaluate cell growth in tissue engineering constructs in vivo using PKH26 and molecular light imaging system.

Objective To establish an animal model of annulus fibrosus (AF) partial defect for the repairing of interver?tebral disc (IVD) defect. Methods Image J 1.46r software was used to measure the T12/L1-L6/S1 intervertebral height in ovine lumbar spine X-ray films. AF thickness was measured by axial split disc. A 11 blade was used to make a trapezoid de?fect of upper bottom 3 mm, lower bottom 5 mm, height 5 mm and thickness 3 mm, whose lower bottom toward the nucleus pulposus (NP) in the left front of ovine lumbar IVD in vitro. The minimally invasive lateral approach was used to make the same type of trapezoid defect in the left front of the ovine lumbar IVD in vivo. The trapezoidal defect length of the axial divid?ing disc was measured, AF and a small amount of NP from trapezoidal defect in IVD were weighed, and the production of trapezoidal defect in IVD was evaluated. Results The lumbar intervertebral space height of ovine was (4.45 ± 0.28) mm. There were significant differences in the thickness of AF (4.08±0.50) mm , thickness (3 mm) and height (5 mm) of trapezoidal defect (P<0.05), respectively. There were no significant differences in trapezoidal defects in ovine lumbar IVD in vitro on the upper bottom (3.03 ± 0.09)mm, the lower bottom (5.03 ± 0.09) mm, the height (4.97 ± 0.10) mm, the thickness(3.02 ± 0.06) mm and the trapezoidal defect predetermined value on the upper bottom 3 mm, the lower bottom 5 mm, the height 5 mm and the thickness 3 mm (P>0. 05). The weights of the AF and NP taken out from ovine lumbar IVD in vitro and in vivo were (0.162 ± 0.011) g and (0.166 ± 0.014) g, and there was no significant difference between them (P > 0.05). Conclusion Through the operation of minimally invasive lateral approach, the method of making a trapezoidal defect in the experiments can establish animal model of AF partial defect, which meets the requirements for the repairing of IVD defect, and is simple, safe and reliable.

Objective To assess the prospect of integrated biphasic silk fibroin scaffold made by annulus fibrosus-nu?cleus pulposus tissue engineering in application as integrated intervertebral disc(IVD). Methods An integrated annulus fi brosus-nucleus pulposus(AF-NP)biphasic scaffold was made by silk fi broin using two different uncomplicated methods which were paraffin spheres-leaching method(outer AF phase)and phase separation method(inner NP phase). The scaf?fold was investigated by general observation, stereomicroscope and scanning electron microscopy(SEM). Its pore size, poros?ity, and compressive elastic modulus were determined. AF and NP cells were isolated from rabbit IVD and seeded into the corresponding phase of the scaffold respectively. The cell-scaffold complex was cultured for 48 hours. The biocompatibility of the scaffold was evaluated by SEM, live/dead staining while CCK-8 assay was used to assess cell proliferation. Results Stereomicroscope and SEM showed that AF phase and NP phase integrated perfectly without cross-linking. Both phases pos?sessed highly interconnected porous structure [pore size of AF and NP phase were(220.0±23.1)μm and(90.0±17.8)μm, re?spectively] and highly porosity(AF and NP phase were respectively 91%and 93%). In addition, this silk biphasic scaffold had impressive mechanical properties(150.7 ± 6.8)kPa. SEM revealed that disc cells attached to regions of pore walls, dis?tributed uniformly and secreted extracellular matrix. Live/Dead staining and cell count kit-8(CCK-8)analysis showed that the silk composite scaffold was non-cytotoxic to disc cells. Conclusion This silk biphasic AF-NP scaffold has satisfied pore size, porosity, biomechanical properties and biocompatibility, so it is ideal candidate for IVD tissue engineering.

Objective To investigate the influence of dynamic mechanical stimulation on the annulus fibrosus (AF) cells seeded on silk scaffolds.Methods AF cells were isolated from rabbits and were seeded on the scaffold,then cultured for 3,7,14 days with different range of dynamic compression.Stereomicroscope and scanning electron microscope (SEM) was used to observe the surface morphology of tissue engineering annulus fibrosus cells (TE-AFs).After fixation,samples were harvested for histological staining.AF cells related extracellular matrix (ECM) was evaluated by the quantitative analysis of total DNA,proteoglycan and collagen I.The mechanical properties were compared within different groups.Results Stereomicroscope and SEM results showed that the colors of TE-AFs in all groups were deepening with time going.SEM showed cell adhesion on the scaffold and the secretion of extracellular matrix.Histological,immunohistochemical staining,biochemical quantitative analysis and total DNA content showed that the AF cells inside scaffolds could support AF cell attachment,proliferation and secretion.As a result,the compressive properties were enhanced with increasing culture time.Stereomicroscope showed that the colors of TE-AFs in all groups were deepening with time going after dynamic compression.HE staining,Safranin O staining and Type Ⅰ collagen staining showed that cell proliferation and secretion,GAG secretion and collagen secretion were increased with time going within different groups.Quantitation of GAG achieved maximum in 15％ strain group,and quantitation of collagen achieved maximum in 10％ strain group.The total DNA content achieved maximum in 5％ strain group,and compression elastic modulus achieved maximum in 15％strain goup.The height of TE-AFs did not change after mechanical stimulation for 14 days.Conclusion Suitable mechanical stimulation is a positive factor for new AF tissue engineering that will tend to the nature tissue.Excessive compression can accelerate the progress of cell apoptosis.

Objective:To explore the current status of surgery for portal hypertension to grasp current status and future development of surgery in China.Methods:This study is jointly sponsored by China Hepatobiliary & Pancreatic Specialist Alliance & Portal Hypertension Alliance in China (CHESS).Comprehensive surveying is conducted for basic domestic situations of surgery for portal hypertension, including case load, surgical approaches, management of postoperative complications, primary effects, existing confusion and obstacles, liver transplantation(LT), laparoscopic procedures and transjugular intrahepatic portosystemic shunt(TIPS), etc.Results:A total of 8 512 cases of portal hypertension surgery are performed at 378 hospitals nationwide in 2021.Splenectomy plus devascularization predominated(53.0%)and laparoscopy accounted for 76.1%.Primary goal is preventing rebleeding(67.0%) and 72.8% of hospitals used preventive anticoagulants after conventional surgery.And 80.7% of teams believe that the formation of postoperative portal vein thrombosis is a surgical dilemma and 65.3% of hospitals practiced both laparoscopy and TIPS.The major reasons for patients with portal hypertension not receiving LT are due to a lack of qualifications for LT(69.3%)and economic factors(69.0%).Conclusions:Surgery is an integral part of management of portal hypertension in China.However, it is imperative to further standardize the grasp of surgical indications, the handling of surgical operation and the management of postoperative complications.Moreover, prospective, multi-center randomized controlled clinical studies should be performed.