[Objective]To study the expression of Cathepsin B,Caspase-3 and to explore the significance of their expression initially after acute spinal cord injury in rats.[Method]The spinal cord injury of the healthy adult SD rats (78) was induced with Nystrom’s way by the moderate compression at the level of T8 and T9 spinal cord. HE methods were used to detect the pathologic change of spinal cord. The expression of Cathepsin B and Caspase-3 were detected by immunohistochemical study. Besides,using the terminal deoxynucleotidyl transferase mediated DUTP nick and labeling (TUNEL) methods to detect the level of the apoptosis of neural cells.[Result]There were few expressions of Cathepsin B, Caspase-3 and TUNEL positive cells in normal and sham operated spinal cord as detected by immunohistochemical study. The number of positive cells of Cathepsin B increased clearly at 3 days,reached a peak at 5 days,and was constant at 7 days after injury. While the expression of Caspase-3 increased obviously at 8 hours,got to a peak at 3 days,and was lowest at 7 days. And the number of positive cells of TUNEL also increased obviously at 8 hours,got to a peak at 3 days,and was lowest at 7 days. There were significance differences of morphological and position of positive cells between Cathepsin B and Caspase-3 or Tunel. [Conclusion]Caspase-3 protein expressions are enhanced in combination with neuronal apoptosis,while Cathepsin B may involve in the secondary injury by inflammatory cells after acute spinal cord injury.

Objective To explore the effect of general anesthesia on Wuzhishan miniature pigs induced by a mixture of ketamine, Sumianxin II and midazolam, and maintained by ketamine and propofol in surgery lasting up to 8 hours. Methods A total of 18 Wuzhishan miniature pigs (body weight (20. 3 ± 1. 9) kg, 14 male and 4 female) were used in this study. The induction of anesthesia was performed with intramuscular injection of ketamine (8 -10 mg/kg) Sumianxin II (1. 5 mL) and midazolam (10 mg) behind the ear, and the general anesthesia was maintained with a mixture containing 0. 9％ sodium chloride 8 mL, ketamine 100 mg/2 mL and propofol 200 mg/40 mL, continuously injected through the marginal ear vein through a syringe infusion pump. The time spent for anesthesia induction and the duration time of anesthesia were recorded. Physiological indexes including body temperature, blood pressure, heart rate and respiratory rate, the reflex activities, and the effects of analgesia, sedation and muscular relaxation of the miniature pigs under anesthesia at 0, 0. 5, 1, 1. 5, 2, 4, 6, 8 h were observed. Results All the 18 pigs were successfully anaesthetized, but 4 pigs died during surgery due to hypovolemic shock, anesthesia accident, left main coronary thrombosis and reperfusion arrhythmia, respectively. During anesthesia, the analgesia, sedation and muscular relaxation effects on the pigs were obvious. The average time spent for anesthesia induction was (4. 8 ± 1. 2) min and the duration time of anesthesia was (54. 1 ± 5. 8) min. The eyelid reflex, corneal reflex and anal reflex in the pigs were weak or disappeared during 1 -8 h after the anesthesia was induced. The body temperature of the pigs was decreased gradually, with a significant difference between 1 h and 0 h (P＜ 0. 05), reaching the lowest point at 4 h, and then maintained stable. The blood pressure was gradually decreased, reaching the lowest level at 2 h (P ＜ 0. 05), then somehow increased, and maintained at a stable level until the end of surgery. The respiratory rate fluctuated during the anesthesia, with no significant difference. Conclusions The anesthesia induced by a combination of ketamine, Sumianxin II and midazolam and maintained with a combination of ketamine and propofol is simple to operate, shows effects fast, and has good effects of analgesia, sedation and muscular relaxation, keeping the circulatory system and respiratory system relatively stable throughout the anesthesia. Thus it is suitable for general anesthesia for miniature pigs.

Objective To investigate the expression and influence to tumor angiogenesis of urokinase-type plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) in esophageal carcinoma. Methods The expression of uPA and VEGF in the tissue of normal (18 cases) and esophageal carcinoma (68 cases) were evaluated by SP immunohistochemistry, CD34 was detected as marking tumor microvessel density (MVD). uPA and VEGF expression were assessed as to the pathologically biological features of esophageal cancer and to the influence to tumor angiogenesis. Results The positive rates of uPA were 27.8 % (5/18) and 70.6 % (48/68) in the tissue of normal and esophageal carcinoma, respectively, there was significant difference in two tissues (x2 =11.63, P <0.05). The positive rates of VEGF were 22.2 % (4/18)and 63.2 % (43/68) in the tissue of normal and esophageal carcinoma, respectively, there was significant difference in two eissues (x2 =9.78, P <0.05). The expressions of uPA and VEGF in esophageal carcinoma were uniformity (x2 =9.72, P <0.05). The mean of MVD was 42.38±11.62. The positive rates of uPA and VEGF were higher in the high MVD group than those in the low MVD group (x2 =6.13, P <0.05, x2 =10.12, P <0.05,respectively). uPA and VEGF expressions in malignant tumors weren' t associated with age, gender and pathological types (P >0.05), but associated with clinical stage, histologic grading and lymph node metastasis (P <0.05). Conclusion Rising expression levels of uPA and VEGF are common in esophageal carcinoma. Altered expression of uPA and VEGF may contribute to tumor angiogenesis of esophageal carcinoma, whose overexpression indicate worse prognosis.

Objective To investigate the effect of amiloride on the invasion capacity of esophageal carcinoma EC9706 cell line in vitro and to elucidate its possible mechanism.Methods The invasion capacities of EC9706 cells pretreated with amiloride were measured by transwell chamber assay. The urokinase-type plasminogen activator (uPA) transcription were determined by RT-PCR.The protein expression of uPA were assessed by Western blot.Results After the EC9706 cells were pretreated with amiloride at different concentrations,the number of invaded cells was obviously less than those of control group with obvious dosage dependent pattern (96±7,78±6,57±6,33±4,15±3,F =43.46,P ＜ 0.01).The transcription levels of uPA mRNA and the protein expression levels of uPA in EC9706 cells decreased significantly compared with the control (mRNA:0.623±0.065,0.526±0.054,0.389±0.041,0.312±0.038,0.247±0.025,F =6.71,P ＜0.01; protein:0.732±0.064,0.644±0.057,0.533±0.058,0.391±0.036,0.267±0.043,F =6.71,P ＜0.01).Conclusion Amiloride inhibits the invasion capacity of esophageal carcinoma EC9706 cells.The mechanism might be associated with down-regulation of the expression of uPA.

BACKGROUND:Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells.It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line(SH-SY5Y)was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells. OBJECTIVE:To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemic-hypoxic SH-SY5Y and their associated epigenetic mechanisms. METHODS:Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury,and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture,respectively,with mesenchymal stem cells,as well as lentiviral-transfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture.Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification,and joint analysis was conducted with RNA-sequencing.Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells.qRT-PCR and western blot assay were performed to detect the expression level of REST.The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression difference of H3K36me3 group proteins was detected by western blot assay,and transcriptome sequencing was performed to analyze the differentially expressed genes. RESULTS AND CONCLUSION:(1)Compared with the control group,monocyte chemotactic protein 1,interleukin-6,interleukin-18,and interleukin-1 beta,interferon α2,and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy(P<0.01).(2)After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia,the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced.(3)Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1,interleukin-6 related regulatory proteins CXCL1 and BGN.(4)Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated,and functional enrichment of histone modifications,and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group.(5)Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3.(6)Stable knockdown REST in SH-SY5Y cells was successfully constructed;the transcript levels of REST mRNA and protein expression were both decreased.(7)After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells,apoptosis was significantly increased and H3K36me3 expression was significantly decreased.Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level(P<0.01).(8)It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3,which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2,and facilitated neuronal survival.