Objectives:To investigate the mechanism underlying the protective effects of polydatin(PD)a-gainst oxygen-glucose deprivation(OGD)-induced neuronal injury and acute spinal cord injury(SCI).Methods:The mouse hippocampal neuronal cell line HT22 was used in in vitro experiments.The cytotoxicity of PD was assessed using the CCK-8 assay:cells were treated with 0,1,3,10,30 and 100μM PD for 24h to de-termine the safe concentration range.After OGD modeling(24h),cells were treated with different concentrations of PD(0,3,10,and 30μM),and cell viability was measured via CCK-8 to identify the optimal protective concentration.Autophagy markers(LC3 and P62)were detected by immunofluorescence and Western blot(WB);Autophagosome formation was observed using transmission electron microscopy;And the apoptosis rate was e valuated by TUNEL staining.For in vivo studies,an acute SCI model was established in C57BL/6 mice using Allen's impact method.Animals were divided into sham,SCI model,and PD treatment(20mg/kg)groups,with n=10 per group.Tissues were collected on 7d and 14d post-injury.Spinal cord pathology was examined by HE staining on 7d and 14d.Immunofluorescence was performed on 14d to evaluate glial scar formation(GFAP+),neuronal survival(MAP2+),cell proliferation(BrdU+),and autophagy level(LC3 Ⅱ).Results:In vitro results showed that 1-100μM PD did not exhibit significant cytotoxicity toward HT22 cells.The optimal concentration of PD was determined to be 10μ M after OGD induction.Compared with the OGD group,10μM PD significantly enhanced cell viability after OGD[OGD group:(54.63±3.90)％vs OGD+PD(1 0μ M)group:(84.35±1.38)％,P＜0.05]and effectively attenuated autophagy activation,as evidenced by a decreased LC3Ⅱ/Ⅰratio(OGD group:11.0±0.57 vs OGD+PD group:3.50±0.28,P＜0.05),increased P62 accumulation(OGD group:0.55±0.04 vs OGD+PD group:0.93±0.06,P＜0.05),reduced number of autolysosomes,and significantly lower apoptosis rate[OGD group:(35.33±2.6)％vs OGD+PD group:(19.67±1.76)％,P＜0.05].In vivo,HE staining confirmed that pathological damage in spinal cord tissue was markedly alleviated in the PD-treated group compared with the SCI model group.Immunofluorescence results indicated that PD treatment inhibited fibrous scar formation(SCI group:2.32±0.19mm2 vs PD group:1.07±0.24mm2,P＜0.05),reduced neuronal damage(SCI group:1.72±0.28mm vs PD group:0.93±0.12mm,P＜0.05)and promoted cell proliferation[SCI group:(16.14±2.24)％vs PD group:(39.09±3.16)％,P＜0.05],and downregulated LC3 Ⅱ expression(SCI group:62.81±5.25 vs PD group:34.09±3.98,P＜0.05).Conclusions:PD ameliorates neural damage by concurrently suppressing autophagy and apoptosis,providing a dual-pathway therapeutic strategy for SCI.

Objective:To develop a double-antibody sandwich ELISA for detecting soluble advanced oxidation protein products(AOPPs).Methods:BALB/c mice were immunized with sodium hypochlorite-oxidized mouse albumin to generate AOPPs-specific monoclonal antibodies(mAbs).Specificity of the mAbs was assessed using indirect ELISA and Western blot.Competitive ELISA was employed to determine if the epitope recognized by newly prepared mAb was consistent with that recognized by mAb 3F2,which developed in our previous work.The sandwich ELISA was then established,and its specificity and sensitivity were compared with the chloramine-T method,the repeatability of double-mAb sandwich ELISA was verified.Results:A mAb AP-4C5 with specific AOPPs recognition was obtained,two sandwich ELISA were developed for the specific detection of soluble AOPPs.Double-mAb sandwich ELISA,using mAb 3F2 as the capture antibody and mAb AP-4C5 as the detection antibody,detected AOPPs in range of 0.25～2 μg/ml(R2=0.991 80).PcAb-mAb sandwich ELISA,using goat anti-HSA polyclonal antibody as the capture antibody and AP-4C5 as detection antibody,detected AOPPs in range of 1.5～25 μg/ml(R2=0.968 75).The double-mAb sandwich ELISA was found to be more sensitive and specific compared to chloramine-T method.Double-mAb sandwich ELISA has good reproducibility(intra-assay CV:3.23％～4.51％,inter-assay CV:3.08％～5.29％).Conclusion:Two kinds of sandwich ELISA for detecting soluble AOPPs have been estab-lished,which hold promise for the detection of clinical samples and understanding of the pathogenic mechanisms of AOPPs.

Objective:To develop a double-antibody sandwich ELISA for detecting soluble advanced oxidation protein products(AOPPs).Methods:BALB/c mice were immunized with sodium hypochlorite-oxidized mouse albumin to generate AOPPs-specific monoclonal antibodies(mAbs).Specificity of the mAbs was assessed using indirect ELISA and Western blot.Competitive ELISA was employed to determine if the epitope recognized by newly prepared mAb was consistent with that recognized by mAb 3F2,which developed in our previous work.The sandwich ELISA was then established,and its specificity and sensitivity were compared with the chloramine-T method,the repeatability of double-mAb sandwich ELISA was verified.Results:A mAb AP-4C5 with specific AOPPs recognition was obtained,two sandwich ELISA were developed for the specific detection of soluble AOPPs.Double-mAb sandwich ELISA,using mAb 3F2 as the capture antibody and mAb AP-4C5 as the detection antibody,detected AOPPs in range of 0.25～2 μg/ml(R2=0.991 80).PcAb-mAb sandwich ELISA,using goat anti-HSA polyclonal antibody as the capture antibody and AP-4C5 as detection antibody,detected AOPPs in range of 1.5～25 μg/ml(R2=0.968 75).The double-mAb sandwich ELISA was found to be more sensitive and specific compared to chloramine-T method.Double-mAb sandwich ELISA has good reproducibility(intra-assay CV:3.23％～4.51％,inter-assay CV:3.08％～5.29％).Conclusion:Two kinds of sandwich ELISA for detecting soluble AOPPs have been estab-lished,which hold promise for the detection of clinical samples and understanding of the pathogenic mechanisms of AOPPs.

BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

Objectives:To investigate the mechanism underlying the protective effects of polydatin(PD)a-gainst oxygen-glucose deprivation(OGD)-induced neuronal injury and acute spinal cord injury(SCI).Methods:The mouse hippocampal neuronal cell line HT22 was used in in vitro experiments.The cytotoxicity of PD was assessed using the CCK-8 assay:cells were treated with 0,1,3,10,30 and 100μM PD for 24h to de-termine the safe concentration range.After OGD modeling(24h),cells were treated with different concentrations of PD(0,3,10,and 30μM),and cell viability was measured via CCK-8 to identify the optimal protective concentration.Autophagy markers(LC3 and P62)were detected by immunofluorescence and Western blot(WB);Autophagosome formation was observed using transmission electron microscopy;And the apoptosis rate was e valuated by TUNEL staining.For in vivo studies,an acute SCI model was established in C57BL/6 mice using Allen's impact method.Animals were divided into sham,SCI model,and PD treatment(20mg/kg)groups,with n=10 per group.Tissues were collected on 7d and 14d post-injury.Spinal cord pathology was examined by HE staining on 7d and 14d.Immunofluorescence was performed on 14d to evaluate glial scar formation(GFAP+),neuronal survival(MAP2+),cell proliferation(BrdU+),and autophagy level(LC3 Ⅱ).Results:In vitro results showed that 1-100μM PD did not exhibit significant cytotoxicity toward HT22 cells.The optimal concentration of PD was determined to be 10μ M after OGD induction.Compared with the OGD group,10μM PD significantly enhanced cell viability after OGD[OGD group:(54.63±3.90)％vs OGD+PD(1 0μ M)group:(84.35±1.38)％,P＜0.05]and effectively attenuated autophagy activation,as evidenced by a decreased LC3Ⅱ/Ⅰratio(OGD group:11.0±0.57 vs OGD+PD group:3.50±0.28,P＜0.05),increased P62 accumulation(OGD group:0.55±0.04 vs OGD+PD group:0.93±0.06,P＜0.05),reduced number of autolysosomes,and significantly lower apoptosis rate[OGD group:(35.33±2.6)％vs OGD+PD group:(19.67±1.76)％,P＜0.05].In vivo,HE staining confirmed that pathological damage in spinal cord tissue was markedly alleviated in the PD-treated group compared with the SCI model group.Immunofluorescence results indicated that PD treatment inhibited fibrous scar formation(SCI group:2.32±0.19mm2 vs PD group:1.07±0.24mm2,P＜0.05),reduced neuronal damage(SCI group:1.72±0.28mm vs PD group:0.93±0.12mm,P＜0.05)and promoted cell proliferation[SCI group:(16.14±2.24)％vs PD group:(39.09±3.16)％,P＜0.05],and downregulated LC3 Ⅱ expression(SCI group:62.81±5.25 vs PD group:34.09±3.98,P＜0.05).Conclusions:PD ameliorates neural damage by concurrently suppressing autophagy and apoptosis,providing a dual-pathway therapeutic strategy for SCI.

BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

BACKGROUND:Tauroursodeoxycholic acid is a hydrophilic bile acid derivative that has neuroprotective effects in a variety of neurological disease models.However,there are few reports on the effects of tauroursodeoxycholic acid on spinal cord injury. OBJECTIVE:To investigate the effect of tauroursodeoxycholic acid on apoptosis of spinal cord neurons under hypoglycemic and hypoxic conditions,as well as the effect on recovery of motor function in mice after spinal cord injury. METHODS:(1)In vitro experiment:Primary spinal cord neurons were isolated from C57 BL/6 mouse embryos at 13.5 days of gestation.After 72 hours of culture,the cells were divided into three groups.In the normal group,cells were cultured in Neurobasal complete medium that was incubated in a CO2 incubator(5%CO2 + 95%air)for 24 hours.In the oxyglucose-deprived group,sugar-free Neurobasal medium was added and incubated in a triple-gas incubator(94%N2+5%CO2+1%O2)for 12 hours,and then the medium was replaced with Neurobasal complete medium and incubated in a CO2 incubator for 12 hours.In the experimental group,the treatment procedure was approximately the same as that in the oxyglucose-deprived group,except that taurodeoxycholic acid was added along with the sugar-free Neurobasal medium.TUNEL staining was used to detect apoptosis,cell counting kit-8 assay was applied to detect cell activity,and immunofluorescence staining was performed to detect cellular β-microtubule protein expression.(2)Animal experiment:Sixty C57 BL/6 mice were randomly divided into sham-operated group,spinal cord injury group and experimental group,with 20 mice in each group.Animal models of T9-T10 spinal cord injury were established using Allen's percussion method in the spinal cord injury group and the experimental group.Starting from the 1st day after modeling,taurodeoxycholic acid solution was given by gavage in the experimental group and normal saline was given by gavage in the sham-operated and spinal cord injury groups once a day for 14 consecutive days.Spinal cord tissue repair was assessed using behavioral and histological methods. RESULTS AND CONCLUSION:In vitro experiment:TUNEL staining,cell counting kit-8 and immunofluorescence staining showed that compared with the normal group,the number of apoptotic cells was higher(P＜0.01),while cell activity and β-microtubule protein expression were lower in the oxyglucose-deprived group(P＜0.01);compared with the oxyglucose-deprived group,the number of apoptotic cells was lower(P＜0.01),while cell activity and β-microtubule protein expression were higher in the experimental group(P＜0.01).Animal experiment:The Basso-Beattie-Bresnahan scores in the open field test and hind limb footprint experiments showed that the mice in the experimental group had better recovery of walking and motor functions than those in the spinal cord injury group.Hematoxylin-eosin staining showed that significant deformities and cavities were observed at the site of spinal cord injury and the number of nerve cells was significantly reduced in the spinal cord injury group.Compared with the spinal cord injury group,the experimental group showed a significant reduction in the area of spinal cord injury,less spinal cord deformity,fewer cavities,and an increase in the number of nerve cells.Immunofluorescence staining showed that the number of neuronal nucleus-labeled neuronal cells in the spinal cord injury group was less than that in the sham-operated group(P＜0.01),and the number of neuronal nucleus-labeled neuronal cells in the experimental group was higher than that in the spinal cord injury group(P＜0.01).To conclude,tauroursodeoxycholic acid could effectively reduce glucose/oxygen deprivation-induced apoptosis of spinal cord neurons and axonal loss,and promote the recovery of motor function in mice with spinal cord injury.

[Objective] To explore whether diabetes mellitus (DM) can influence the early bacterial translocation (BT) and progression of acute necrotizing pancreatitis (ANP) for guiding the early clinical treatment.[Methods] 35 Wistar male rats were randomly allocated to 4 groups,Group ANP associatcd with DM (DM+ANP,n =10):DM underwent induction of ANP;Group DM (n =10):DM underwent laparotomy with only manipulation of the pancreas and duodenum;Group ANP (n =10):non-DM underwent induction of ANP;Group sham operation (SO,n =5):non-DM underwent SO.After 12 h of the induction of ANP or laparotomy,the following parameters were analyzed:bacterial culture and identification of portal vein blood,mesenteric lymph nodes (MLNs),pancreas and liver,and calculate the total incidence of BT;serum amylase and endotoxin levels of portal vein blood;histological assessment of pancreas and ileum lesions.[Results] All animals except 3 in group DM+ANP (mortality rates:30％) and 1 in group ANP (mortality rates:10％) survived the experiment.The total incidence of BT was 23/28 (82.1％) in group DM+ANP whereas 16/36 (44.4％) in group ANP (P =0.002).Gram-positive bacteria were 17/23 (73.9％),3/16 (18.8％) in group DM+ANP and group ANP,respectively (P =0.001).Amylase activity (2302 ± 346) U/L in group ANP increased significantly (P =0.000) compared with other groups.However,group DM+ANP (501 ± 142) U/L decreased significantly (P =0.001) in comparison to group SO.Regarding to endotoxin concentrations and the severity of pancreas and ileum lesions,group DM + ANP increased significantly compared with group ANP,group DM and group SO (P ＜ 0.05).[Conclusion] Gram-positive bacteria translocates more frequently than Gram-negative bacteria in the early period of DM+ANP rats.DM aggravates the progression of ANP and increases early bacterial translocation,endotoxemia and severity of pancreas and ileum lesions.

Objective To investigate the results of defect reconstruction using anterolateral thigh free flap in medial and lateral approaches.Methods We reviewed the soft tissue defect reconstruction in 200 patients from February 2010 to June 2014 with anterolateral thigh flap in medial or lateral approach,to compare the operative time and donor site morbidity.Results The mean time of flap raising in medial group was (45 ± 4.5) minutes,while in lateral group was (65 ± 3.5) minutes.In medial group,fascial lata was closed directly in 39 cases (39％),while fascial lata was closed directly in all the cases (100％) in lateral group.All the 200 flaps survived uneventfully.All the donor sites healed smoothly.No infection occurred.An 8 to 32 months follow-up revealed a high satisfactory rate from the patients.No sensory deficit or functional impairment was noted in the donor sites.Conclusions Both in medial and lateral approach can achieve safely harvesting free anterolateral thigh flap.

Objective To analyze the characteristics of blood loss for laboratory test of critically ill premature infants,and to seek feasible measures to reduce the blood loss.Methods Two hundred and forty-six cases of critically ill premature infants admitted to the neonatal intensive care unit from April 2012 to April 2013 were analyzed the blood loss for test during the hospitalization and the clinical features of blood loss with different gestational age,different weight within the first four weeks after admission.Then the application of blood loss according to test category was described.The blood volume demanded in theory was determined by the formula B =5 (∑ s + 0.1),then calculated the phlebotomy overdraw on the basis of the practical blood loss and analyzed the characteristics of overdraw per patients per day in first two weeks after admission.Results Among 246 patients,The median blood loss figure was 25.57 ml for each infants with the range between 7.10 ml ～ 119.20 ml,and the blood loss concentrated in first four weeks,which showed a decreasing trend with time.There was a statistically significant difference(P ＜0.05)that the smaller gestational age,the lower birth weight,the more daily blood loss per patient per day in first two weeks,but no significant differences(P ＞ 0.05) between the third and fourth week.The largest proportion of the blood samples was used for clinical chemical tests(31.49％),followed by blood gas analysis (19.03％),immunoassays (12.69％),blood cultures (12.63％),hematology (12.28％).The practical blood loss was about twice times of blood volume demanded for tests in theory,which the median was 7.8 times to the latter(25.57 ml vs 3.26 ml).It showed statistically significant difference(P ＜0.05) between blood overdraw per patients per day in the first week and the second week.Compared with different gestational age and birth weight,the difference of overdraw was also statistically significant(P ＜0.05).According to test category,blood culture was the most significant samples of phlebotomy overdraw,followed by biochemical,other,blood gas analysis,the percentage was 76％,64％,45％ and 41％ respectively.Conclusion The blood loss for laboratory test and the phenomenon of blood waste is serious in critically ill preterm infants.The smaller the gestational age is,the lower the weight is,the amount of blood loss and phlebotomy overdraw are more significantly.Biochemical and blood gas analysis are the main items of blood loss.