Sepsis,a complex clinical syndrome resulting from a harmful and damaging host response to infection, is the leading cause of mortality in intensive care units.Early diagnosis and appropriate intervention can improve the prognosis of patients with sepsis.Many studies have confirmed that sepsis at different stages is in different immune status.Priority used to be given to systemic inflammatory response, but immune-suppression has become the focus of study. Immune-suppression and secondary infection are the major causes of death of patients with sepsis.Study of sepsis is shifting to immune-suppression and its regulation mechanisms.

Objective To compare the phenotype of myeloid derived suppressor cells (MDSCs) separated from the bone marrow of mice 3 d and 7 d after cecal ligation and puncture ( CLP) and to elucidate its potential role in the accumulation and immuno-function of MDSCs by determining the expression of microRNA-146a(miR-146a)in order to explore the effect of miR-146 a on immonosuppression of MDSCs in sepsis .Methods A septic model was prepareol by CLP in adult male C57BL/6J mice.MDSCs(expressing cell-surface CD11b and GR-1 antigens )from bone marrow were harvested 3 and 7 days after CLP and were separated with magnetic bead sorting technique .Then,cytokines secretion and arginase-I activity were detected and the T cell proliferation in vitro and the expression of miR-146a of MDSCs (3 d and 7 d after CLP)were observed.Results MDSCs secreted mostly such promoting inflammatory factors as TNF-α, IL-6 3 days after CLP, but 7 days after CLP , they primarily secreted IL-10 and TGF-βwhich were anti-inflammatory factors . MDSCs had potent immunosuppressive properties by increasing T cell suppression in a late anti-inflammatory phase ( CLP3 d vs CLP7 d, P<0.05).In the meantime,miR-146a of the MDSCs in bone marrow was overexpressed in septic mice at 7 days(P<0.05). Moreover,the expression of miR-146a of the MDSCs in bone marrow of septic mice was higher at 7 days than at 3 days after CLP(P<0.05).Conclusion The data indicate that the phenotype of MDSCs evolves through early pro -inflammatory phase into the late anti-inflammatory phase .MDSCs have potent immunosuppressive properties in the late phase of sepsis . miR-146 a might play a crucial role in the regulation of immunosuppressive activity of MDSCs in late sepsis .

Objective To evaluate the effects of sevoflurane preconditioning on caveolin-3 expression during myocardial ischemia-reperfusion (I/R) in rats.Methods Healthy male Wistar rats,aged 6-8 weeks,weighing 250-300 g,were anesthetized with intraperitoneal pentobarbital 30 mg/kg and heparin 1 000 IU/kg.Their hearts were excised and perfused with K-H solution in a Langendorff apparatus.Thirty isolated rat hearts were randomly assigned into 3 groups (n =10 each) using a random number table:control group (group C),I/R group and sevoflurane preconditioning group (group SP).After 30 min of equilibration,group C was continuously perfused with K-H solution for 90 min,group I/R underwent 30 min of ischemia followed by 60 min of reperfusion,and group SP was perfused with K-H solution saturated with 2％ sevoflurane for 10 min followed by 5 min washout with K-H solution,then underwent 30 min of ischemia followed by 60 min of reperfusion.HR,left ventricular enddiastolic pressure (LVEDP),+ dp/dtmax and-dp/dtmax were recorded at the end of equilibration,immediately before ischemia and at 30 and 60 min of reperfusion.Myocardial specimens were obtained from the cardiac apex for microscopic examination.Myocardial specimens were obtained from the left ventricle for determination of caveolin3 expression.Results Compared with group C,+ dp/dtmax and-dp/dtmax were significantly decreased immediately before ischemia,and HR,LVDEP,+ dp/dtmax and-dp/dtmax were decreased at 30 and 60 min of reperfusion in groups I/R and SP,and caveolin-3 expression was down-regulated in group I/R and up-regulated in group SP (P ＜ 0.05).Compared with group I/R,HR,LVDEP,+ dp/dtmax and-dp/dtmax were significantly decreased immediately before ischemia and increased at 30 and 60 min of reperfusion,and caveolin-3 expression was up-regulated in group SP (P ＜ 0.05).The pathological changes were significantly attenuated in group SP as compared with group I/R.Conclusion The mechanism by which sevoflurane preconditioning attenuates myocardial I/R injury in rats may be related to up-regulation of myocardial caveolin-3 expression.

Objective To observe the clinical effect and safety of subanesthetic doses of ketamine and fentanyl assisted regional anesthesia.Methods 90 children received abdominal operation or limbs operation were selected in our hospital.They were randomly divided into A,B and C group with 30 children in each group.The children in group A received sub-anesthetic doses of ketamine and fentanyl in nerve block anesthesia; the children in group B received sub-anesthetic doses of ketamine and fentanyl anesthesia aided;while the children in group C accepted traditional ketamine anesthesia.The indexes of respiratory frequency,mean arterial pressure,heart rate and oxygen saturation and other basic vital signs as well as anesthesia adverse events of situation were compared.Results The respiratory frequency,mean arterial pressure and heart rate in group A and B were lower than those in group C,the difference was statistically significant(all P ＜0.05),whereas no difference was observed on the oxygen saturation between two groups(P ＞0.05).The respiratory rate,mean arterial pressure and heart rate in group A and B showed no significant difference (all P ＞ 0.05).In adverse reactions,the muscle relaxants was in good condition in group A and B,no obvious body movement and gastrointestinal adverse events observed either.Group C with muscle relaxants in good condition,but body movement and some gastrointestinal adverse reaction can be observed occasionally.Conclusion Subanesthetic doses of ketamine and fentanyl has good anesthetic effect on regional anesthesia,and can effectively reduce the occurrence of adverse reactions,which is worthy of clinical application.

Objective To determine low-concentration clenbuterol in edible meat based on the solidphase extraction coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).Method The homogenated sample was acidized to remove proteins,and purified using the liquid-liquid extraction and MCX Oasis solid prepared extraction column,then further treated with gradient elution with the mobile phase of ammonium formate (10 mmol/L and pH 3.5) and acetonitrile.The clenbuterol was completely separated on Eclipse C18 (1.8 μm,4.6×100 mm) column and detected in multiple reaction monitoring (MRM) mode.Results A good linearity was achieved for clenbuterol in the arrange of 0.01～0.2 μg/kg based on the internal standard calibration of D9-clenbuterol,with the linearity correlation coefficient greater than 0.99 and the detection limit of 0.005 μg/kg.The relative recovery of target compounds spiked in blank sample at three levels ranging from 78.8 to 114.8％,with the relative standard deviations less than 10％.Conclusion The method in this research is simple,rapid,reliable and suitable to confirm low-concentration clenbuterol in edible meat.

30 open-chest rabbits were randomly divided into 3 groups. In group Ⅰ the left anterior descending coronary artery (LAD) was false ligated. Rabbits of group Ⅱ and Ⅲ underwent 30 minuts of LAD occlusion and 4h of reperfusion. After LAD ligated in group Ⅲ were intravenously administered nitroglycerin and in group Ⅰ and Ⅱ were sodium chloride. The level of plasme nitric oxide (NO), alpha-granule membrance protein (GMP-140) on the surface of platelet and thromboxane B_2 (TXB_2) were determined in some time points before and after operation. The results showed that the level of NO, GMP-140 and TXB_2 in group Ⅰ were not difference before and after operation. In group Ⅱ and Ⅲ the level of NO was significantly decreased and the level of the GMP-140 and TXB_2 were significantly increased after myocardial ischemia and reperfusion. Changes in group Ⅱ were more marked than that in group Ⅲ. There were close relation between the value of NO and the value of GMP- 140 and TXB_2. It suggested that NO inhibited platelet activation after myocardial ischemia-reperfusion in vivo.

Objective To verify the feasibility of Cas9 microinjection in frozen-thawed pronuclear embryos, based on the model of pronuclear embryos of C57BL/6J mice by in vitro fertilization.Methods After fertilized mouse pronuclear embryos cultured in vitro, one-cell and 2-cell embryos were frozen using EFS20/40 cryopreservation tube.The next day recovered and then cultured.The recovery rate and survival rate of the one-cell and 2-cell embryos were compared.The frozen-thawed and fresh pronuclear embryos were injected with Cas9 mixture and injection buffer into the cytoplasm, and then cultivated to 2-cell embryos,and the survival rate and development rate of the 2-cell embryos were compared.Results The recovery rate of frozen-thawed one-cell embryos was 92.5%, the survival rate was 92.8%, the recovery rate of 2-cell embryos was 90.5% and the survival rate was 95.8%, showing no significant difference.Furthermore, the survival rate of fresh one-cell embryos after Cas9 injection was 92.7%, the survival rate of one-cell embryos of the blank group was 97.5%.While the survival rate of Cas9 injected frozen-thawed one-cell embryos was 82.6%, and that of the blank group was 92%,showing a significant difference between the frozen-thawed injected group and other groups(P < 0.05).The development rate of 2-cell embryos after Cas9 injection was not significantly different.Conclusions Frozen-thawed pronuclear embryos can be used for Cas9 microinjection.

Objective To investigate the effect of propofol on myocardial injury induced by hepatic ischemia/reperfusion (I/R) in rats and the role of PI3K/Akt signaling pathway. MethodsOne hundred and two male SD rats weighing 250-280 g were randomly divided into 5 groups:Ⅰ sham operation group (group S, n =6), ⅡI/R group ( n = 30), Ⅲ propofol group (group P, n = 30), Ⅳ propofol + LY294002 group (group P+ LY, n =18), and Ⅴ propofol + dimethylsulfoxide group (group P+ DMSO, n = 18). Hepatic I/R was produced by occlusion of hepatic pedicle for 30 min followed by reperfusion in group Ⅱ - Ⅴ. Propofol 12 mg/kg, propofol 12mg/kg + LY294002 (a specific PI3K inhibitor) 1.5 mg/kg, and propofol 12 mg/kg + DMSO 0.5 ml were injected I.v.via femoral vein at 10 min before ischemia in group Ⅲ -Ⅴ respectively, and then propofol was infused I.v. At a rate of 30 mg· kg- 1 · h - 1 and the administration was stopped before the rats were sacrificed in group Ⅲ - Ⅴ . At 0,30, 60, 120, and 240 min of reperfusion (T1-5) in group Ⅱ and Ⅲ , and at T3.5 in group Ⅳ and Ⅴ , six rata were sacrificed and myocardial tissues were taken for determination of the total Akt (t-Akt) and phosphorylated Akt (p-Akt) expression and Bcl-2 expression and apoptosis were detected at T3. The hepatic tissues were taken for microscopic examination. The rats were sacrificed at T1 and the parameters mentioned above were detected in group Ⅰ . ResultsCompared with group Ⅰ , p-Akt expression and apoptosis rate were significantly increased in the other4 groups, and Bcl-2 expression was up-regulated in group Ⅱ , Ⅲ and Ⅴ (P ＜ 0.05). Compared with group Ⅱ , p-Akt and Bcl-2 expression was up-regulated, and the apoptosis rate was significantly decreased in group Ⅲand Ⅴ ( P ＜ 0.05). Compared with group Ⅲ , p-Akt and Bcl-2 expression was down-regulated, and the apoptosis rate was significantly increased in group Ⅳ ( P ＜ 0.05). The microscopic examination showed that the injury to the hepatic tissues was less severer in group Ⅲ and Ⅴ than in group Ⅱ and Ⅳ. ConclusionPropofol can attenuate myocardial injury induced by hepatic I/R in rats by activation of PI3K/Akt signaling pathway.