The earthworm fibrinolytic enzymes (EFE) were separated by affinity ch romatography using soybean trypsin inhibitor as a matrix. The enzymes were furth er separated and purified into 12 components after DEAE-32 chromatography and p reparative electrophoresis. The pI of these components gradually decreased f rom pH 4.0 according to electrophoresis mobility from higher to lower on PAGE. The molecular weights were in the range of 22～34 ku. 6.5 and 7 w ere glycoproteins proved by staining with the shiff reagent and thymol/sulfuric acid. The fibrinolytic activity of 7 was highest as determined usi ng chromzym UK and chromzym PL as specific substrates.

Objective Investigating activity variance of PKC.AchE and HSP70 in different tissues of rabbits caused by dying from suspension of anterior limbs.in order to study how factors above contribute in the mechanism of death.Methods Suspend rabbit by its anterior limbs until it dies.and coilect tissue from its diaphragmatic muscle,intercostal muscle,myocardium,gastrocnemius,cerebrum,brain stem,liver,kidney and lung.In the control groups,collect same tissues of rabbits dying from strangulation,craniocerebral injury and diseases as contrast.Detect activity of PKC,AchE and HSP70 in tissues of those groups mentioned above by SP stain of immunohistochemistry.and statistically analyze result of detection by rank SUm test of independent samples of multi-groups.Results Compared with data from other control groups,there are more obvious expressions in following tissues of group of suspension:PKC in disphragmatic muscle,intercostal muscles and cerebrum(P＜0.05);AchE in disphragmatic muscles,intercostal muscles,myocardium,cerebrum,liver and nerve tract ofmuscles(P＜0.05);HSP70 in cerebrum,myocardium and lung(P＜0.05).Conclusion Through different mechanisms,PKC,AchE and HSP70 could take some certain contributions in death pmcess of suspension by anterior limbs,and the results above could offer some experimental evidences for related studies and medicolegal investigations.

TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.

Objective This article aims to explore the role of HSP70 in the developing mechanism of SMDS(sudden manhood death syndrome,SMDS)and to investigate the value of it in the early diagnosis of SMDS.Methods By way of immunohistochemieal staining of left,right ventricular musles,sino atrial node and atrioventricular node from 30 cases of SMDS group and 20 cases of control group,we observed and measured the expression of HSP70 and its variance of intensity with graphical analysis software.Results Compared with the control group cases,in cardiac conduction system tissue of SMDS victims.we saw an obviously higher expression and intensity of HSP70(P<0.05).Conclusion As a kind of mediator in stress reaction,HSP70 is involved in the process of protecting the heart from impairment in SMDS,and it can be considerated as a reference index to identify the SMDS.

BACKGROUND: Tissue engineering is increasingly viewed as a promising avenue for functional cartilage reconstruction.However, chondrocyte dedifferentiation during in vitro culture remains an obstacle for clinical translation of tissue engineered cartilage. Re-differentiated induction have been employed to induce dedifferentiated chondrocytes back to their original phenotype. Regrettably, these strategies have been proven to be only moderately effective. METHODS: To explore underlying mechanism, RNA transcriptome sequencing was conducted on primary chondrocytes (P0), dedifferentiated chondrocytes (P5), and redifferentiated chondrocytes (redifferentiation-induction of P5, P5.R). Based on multiple bioinformatics analysis, LGR5 was identified as a target gene. Subsequently, stable cell lines with LGR5 knocking-down and overexpression were established using P0 chondrocytes. The phenotypic changes in P1 and P5 chondrocytes with either LGR5 knockdown or overexpression were assessed to ascertain the potential influence of LGR5 dysregulation on chondrocyte phenotypes. Regulatory mechanism was then investigated using bioinformatic analysis, protein–protein docking, immunofluorescence co-localization and immunoprecipitation. RESULTS: The current study found that dysregulation of LGR5 can significantly impact the dedifferentiated phenotypes of chondrocytes (P5). Upregulation of LGR5 appears to activate the PI3K/AKT signal via increasing the phosphorylation levels of AKT (p-AKT1). Moreover, the increase of p-AKT1 may stabilize b-catenin and enhance the intensity of Wnt/b-catenin signal, and help to restore the dedifferentated phenotype of chondrocytes. CONCLUSION LGR5 can modulate the phenotypes of chondrocytes in P5 passage through PI3K/AKT signaling pathway.

Objective To explore the clinical effect of Masquelet membrane induction technique combined with antibiotic coated intramedullary nail fixation in the treatment of lower limb large segment infected bone defects.Methods From June 2009 to August 2015,53 patients who have lower limb large segment infected bone defects were analyzed retrospectively,including 40 males and 13 females,aged from 23 to 61 years,with an average age of 36.2±8.4 years.37 cases were secondary to infection after fracture surgery,and 16 cases were caused by open fractures.There were 17 cases of femoral shaft defects and 36 cases of tibia diaphysis defects.All 53 cases were treated with Masquelet technique.The first stage was infection debridement,then bone defect was filled by bone cement mixed with sensitive or broad-spectrum antibiotics,and then temporary fixation was given.When the infection was controlled,debridement was given again and sensitive antibiotic bone cement was replaced to induce membrane,and antibiotic coated intramedullary nail was used for internal fixation.In the second stage,after intramedullary nailing internal fixation for 4-6 weeks,the bone cement occupying device was taken out and the autologous cancellous bone was planted in the induced membrane.Then the membrane was covered and sutured.The cure rate of infection,the time of bone healing and the related complications were observed.Results 53 patients were followed up for 24 to 63 months (with an average of 39±4.7 months).The length of tibia bone defect after debridement was 6-15 cm (average 8.7±4.9 cm).49 patients' infection were cured in 12 months after operation,and the bone defects were healed,with healing time of 5.3-9.7 months (mean 7.4±3.2 months).No refracture occurred.The healing time of tibia was 7.8±2.1 months,while the healing time of the femur was 7.2±3.9 months.1 case of femoral shaft defect had recurrence of infection 4 months after membrane induced bone grafting,and the first stage treatment was restarted which were debridement and implantation of sensitive antibiotic bone cement occupying device.After 6 weeks,the infection was controlled and the second stages continued.3 cases' s (2 cases of femoral shaft,1 case of tibial shaft) autologous cancellous bone were absorbed 3 to 6 months after operation,and no bone density increased in the bone defect area.The autologous cancellous bone was reimplanted and the bone defect was cured in 8 months.Conclusion Masquelet technology combined with antibiotic coated intramedullary nailing can effectively control infection and create a good biological and mechanical environment for bone defect repair.It has good clinical efficacy.

Objective:To explore the curative effect of surgical treatment for Crohn′s disease (CD), to investigate the timing of surgical intervention and the choice of surgical methods.Methods:From January 1, 2016 to August 31, 2020, at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, the clinical data of 123 patients with CD and receiving surgical treatment were retrospectively analyzed, which included the type of lesion, the location of lesion, clinical manifestation, surgical method, preoperative inflammatory and nutritional indicators, postoperative recovery of digestive tract function, and the development and treatment of postoperative complications. CD was diagnosed according to Consensus opinion on diagnosis and treatment of inflammatory bowel disease ( Beijing 2018). Patient was classitied according to the Montreal Classification. Postoperative complications were graded according to the Clavien-Dindo Criteria. Mann-Whitney U test was used for statistical analysis. Results:Among 123 patients, according to the Montreal classification, two cases (1.6%) were diagnosed at ≤16 years old (type A1), 66 cases (53.7%) were diagnosed at 17 to 40 years old (type A2), and 55 cases (44.7%) were diagnosed at >40 years old (type A3). The lesions were 52 cases (42.3%) of terminal ileum (L1) type, 20 cases (16.3%) of colon (L2) type, and 51 cases (41.5%) of ileocolon (L3) type. Four cases (3.2%) were non-stenosis and non-penetrating (B1) type, 87 cases (70.7%) were stenosis (B2) type, and 32 cases (26.0%) were penetrating (B3) type. Eighteen patients (14.6%) underwent emergency surgery due to complete intestinal obstruction (10 cases), gastrointestinal perforation (five cases), gastrointestinal bleeding (two cases), and rectovesical fistula complicated with septic shock (one case). One hundred and five patients (85.4%) received selective surgery due to poor conservative treatment effects. 51 cases (41.5%) underwent traditional open surgery and 72 cases (58.5%) underwent laparoscopic surgery. Nineteen patients (15.4%) received temporary or permanent ostomy. The preoperative C reactive protein level of patients with emergency surgery was higher than that of patients undergoing selective surgery ((39.23±24.13) mg/L vs. (11.48±2.68) mg/L), while the levels of plasma albumin (ALB) and pre-ALB were lower than those of patients receiving selective surgery ((29.90±10.60) g/L vs. (38.38±8.30) g/L, (146.00±125.49) mg/L vs. (209.06±61.19) mg/L), and the differences were statistically significant ( Z=9.603, 8.754 and 7.111, all P<0.01). During the follow-up, a total of 23 cases (18.7%) developed postoperative complications, including one case of postoperative intra-abdominal hemorrhage and underwent re-operation (Clavien-Dindo grade Ⅲ complication); four cases of anastomotic leakage after operation; six cases of postoperative paralytic ileus; 11 cases of surgical site infection, all of which were Clavien-Dindo grade Ⅱ complications, and one case of deep venous thrombosis of lower extremity. No patient with severe intraoperative complication was observed, and no patients died during the operation or hospitalization. The postoperative exhaust time of patients was (3.2±1.4) d, the time of open fluid diet was (5.8±0.8) d, the length of hospital stay was (18.0±14.1) d, and the length of postoperative hospital stay was (11.2±8.8) d. Conclusions:The concept of multidisciplinary collaboration should be emphasized in the treatment of CD. Surgical treatment can effectively control the complications and improve the quality of life of patients, but the timing of operation and the choice of surgical methods should be decided prudently after perioperative treatment, multi-disciplinary participated and regulation of the internal environment. The standardized and targeted treatments for the surgical difficulties of inflammatory bowel disease should be conducted.

" Two-way referral, and synergy between the tertiary hospitals and primary institutions" is the core measure for promoting hierarchical diagnosis and treatment in the region. The authors introduced the exploration and practice of constructing a two-way referral path in the medical alliance based on telemedicine in West China Second University Hospital of Sichuan University. As of May 2021, the hospital had established a two-way referral path with 52 member hospitals of the medical alliance. By clarifying the functionality and positioning of these institutions, optimizing work processes and IT support, building the mechanism of referral and liaison, and that of appraisal as support, these efforts had achieved progresses in such fields as innovative medical service modes, lean management of referral processes, and promotion of hierarchical diagnosis and treatment system building.

OBJECTIVE To evaluate the econo mical efficiency of Recombinant human thrombopoietin injection （called “rhTPO”for short ）versus Etrapopa ethanolamine tablets （called“Etrapopa”for short ）in the second-line treatment of primary immune thrombocytopenia （ITP）in the Chinese adult patients. METHODS Based on the decision tree-embedded Markov model with a 4-week cycle ，the cost and utility related to bleeding events and adverse events after the use of the two drugs were measured and compared from the perspective of Chinese health system. The horizon was 12 weeks，and the cost and health outcome were not discounted. RESULTS Compared with Etrapopa ，rhTPO improved the quality adjusted life year by 0.002 5 and reduced the cost by 1 824.36 yuan，which was the absolute advantage scheme. Univariate sensitivity analysis showed that the base results were greatly affected by the dosage of rhTPO and Etrapopa during maintainance period. In most cases ，rhTPO was economical. Probability sensitivity analysis showed that when willingness-to-pay threshold varied between 0 yuan and 250 000 yuan，the probability about that rhTPO was economical ranges from 99.90％ to 100％. CONCLUSIONS Based on the available evidence ，rhTPO is more economical in the short term than Etrapopa in the second-line treatment of ITP.

Objective: To establish a hydrogen peroxide (H₂O₂) induced injury model of pulmonary artery endothelial cells (PAECs) and explore the molecular mechanisms of oxidative stress on the structure and function of PAECs in this model. Methods: Human PAECs were treated with H₂O₂ at different concentrations (25, 50, 100, 200, 400, 800, 1 600, 3 200, 6 400 μmol/L) for 4 and 24 h, respectively. The PAECs survival curve was obtained according to the cell viability measured by CCK-8 assay. The cell apoptosis of PAECs was detected by flow cytometry. The reactive oxygen species (ROS) generation and mitochondrial activity were measured using small molecule fluorescent probes. Proteins were extracted and the phosphorylation levels of signal molecules in PAECs were detected by Western blot assays. Results: (1) The effect of H₂O₂ at various concentrations on cell viability of PAECs: cell viability of PAECs decreased in proportion to increasing concentration of H₂O₂ after incubation for 4 h. The half maximal inhibitory concentration (IC₅₀) of PAECs exposed to H₂O₂ for 4 and 24 h were 397.00 and 488.77 μmol/L, respectively. (2) The effect of H₂O₂ on cell apoptosis of PAECs: After H₂O₂ incubation for 4 h, proportions of PAECs at late-apoptosis ((22.58±3.69) %) and necrotic stage( (11.86±4.27)%) were significantly higher than those of control PAECs at late-apoptosis stage( (3.41±1.44)%, P<0.01) and at necrotic stage ((1.94±1.15) % , P<0.05). The survival rate of PAECs post H₂O₂ was dramatically lower than that of control PAECs ((7.98±3.21)% vs. (48.89±8.08)%, P<0.01). However, there is no statistical difference between both groups regarding to the early apoptosis. (3) The effect of H₂O₂ on mitochondrial activity and ROS production of PAECs: the mitochondrial activity and ROS generation of PAECs treated by H₂O₂ were significantly increased compared to those in control PAECs (P<0.01). (4) The effect of H₂O₂ on signaling molecules in PAECs: there was a significant increase in phosphorylation level of Akt in PAECs incubated with H₂O₂ for 30 minutes compared to that in control PAECs (P<0.01), while there was no significant difference in levels of Akt between H₂O₂ treated PAECs and control PAECs. Phosphorylation level of JNK as well as p38 were also significantly upregulated in H₂O₂ treated PAECs (P<0.01). Conclusion H₂O₂ at the concentration of 400 μmol/L could induce human PAECs injuries via the regulation of Akt and MAPK signaling pathways.