AIM: To examined the effects of hypoxic preconditioning ( HPC) on oxygen-glucose deprivation ( OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy .METHODS: Cultured PC12 cells were randomly divided into control group , HPC group, 3-methyladenine (3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group .CCK-8 assay was used to detect the cell viability .The caspase-3 activity was also tested . TUNEL staining and flow cytometry were used to detect the cell apoptosis .The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot .RESULTS:Compared with control group, the viability of PC12 cells was significantly reduced , and the activity of caspase-3 was significantly increased in OGD group.Compared with 3-MA+HPC+OGD group and OGD group , the viability of PC12 cells was significantly in-creased, and the activity of caspase-3 was significantly reduced in HPC +OGD group (P<0.05).The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05).Compared with OGD group, the apoptotic rate significantly decreased in HPC +OGD group ( P<0.05 ) .Compared with control group , the protein level of cleaved caspase-3 was significantly increased in OGD group ( P<0.05) .Compared with OGD group , the protein level of cleaved caspase-3 was significantly decreased , and the levels of LC3-2 and beclin-1 were significantly increased in HPC +OGD group (P<0.05).CONCLUSION:OGD decreases cell survival and induces apoptosis .Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC 12 cells from OGD induced injury .

Aim To investigate the protective effects of hydrogen sulfide ( H2 S) on focal cerebral ischemia/reperfusion injury in rats and the possible mechanisms. Methods Male Sprague Dawley rats were divided into three groups randomly: sham-operated group, cerebral ischemia/ reperfusion ( I/R) group and sodium hydro-sulfide ( NaHS ) + I/R group. The left temporary middle cerebral artery occlusion ( MCAO ) model was established by the line-embolism method. After rats were suffered 2h/24h ischemia/reperfusion stress, the mortality rate was evaluated, and the nervous function-al defect degree was evaluated by Longe scoring, the volumes of cerebral infarction was evaluated by 2 ,3 ,5-triphenyltetrazolium chloride ( TTC) staining, and the expression of P2X7 receptor protein in brain tissue was detected by the immunofluorescence method. Results The mortality rate in NaHS + I/R rats ( 29.41%) was obviously lower than those of I/R group ( 42 . 86%) . The nervous defect scores in NaHS + I/R rats were significant lower than those of I/R group ( P <0.05 ) . The volumes of cerebral infarction in NaHS +I/R group (21.88% ±3.53%) were significant lower than those of I/R group ( 36.71% ±3.73%) ( P <0.01 ) . The results of immunofluorescence showed that the positive expression cells of P2X7 receptor protein in cerebral cortex and hippocampal CA1 area of I/R group were significantly higher than those of sham-op-erated group(P<0. 01). However, compared with I/R group, the positive expression cells of P2X7 receptor protein in cerebral cortex and hippocampal CA1 area of NaHS + I/R group were significantly decreased ( P<0. 01). Conclusions H2S exerts the neuroprotective effect on focal cerebral ischemia/reperfusion injury in rats, and the protective mechanism might be associated with down-regulating the expression of P2X7 receptor protein in brain tissue.

[ABSTRACT]AIM:ToexaminetheeffectsofhighconcentrationofextracellularATPonhumanneuroblastoma SH-SY5Y cell injury.METHODS: Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time.The cell viability was detected by CCK-8 assay.The variation of autophagic vacuoles was observed with monodansylcadaverine staining .The cell apoptosis was analyzed by Hoechst 33258 staining.Meanwhile, apoptotic rate was detected by flow cytometry .The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ ( LC3-Ⅱ) were determined by Western blotting .RESULTS:Compared with control group , the survival rate of SH-SY5Y cells was signifi-cantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h).The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment , trended to decrease over time and returned to control level at 6 h.The protein expression of LC3-Ⅱwas significantly increased at 1 h with ATP treatment , which was consistent with the time points of increasing auto-phagic vacuoles .LC3-Ⅱexpression level gradually decreased at 2~3 h with ATP treatment , and returned to control level at 6 h.Compared with control group , the apoptotic rate and the expression level of caspase-3 were enhanced synchronously . The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION:High concentration of extracellular ATP induces the autophagy and apoptosis of SH -SY5Y cells.The in-creased autophagy shows up , followed by the climax of apoptosis until 6 h.With the prolonged duration of ATP , apoptosis is the main process in the cells .

Objective To investigate the different biochemical testing system inter laboratory comparability of results,provide reference for promoting inter laboratory test results of the recognition.Methods Five patients with laboratory detection of fresh mixed serum,20 consecutive determination of 10 biochemical items,precision analysis.According to America clinical and Laboratory Standards Institute (CLSI)Document EP9-A2,the Panzhihua Iron and Steel Group General Hospital detec-tion system as the reference system,the remaining four hospital detection system as the detection system,with a fresh mixed serum,determination of five biochemical items (Urea,Cr),(AST,ALT),(TP,ALB),(TG,TC)and (HDL-C,LDL-C),the determination results were compared and analyzed,calculated reference system and the correlation coefficient,linear regres-sion equation between the system and the various medical decision level relative deviation (SE%),and to America Clinical Laboratory Improvement Amendment ability test (CLIA’88)allowed total error of 1/2 as the standard,to the assessment system and the reference system between the comparability and clinical acceptability.Results In Urea,Cr determination for example,CV of five laboratories on Urea and Cr two project was less than CLIA’88 allowed total error of 1/3,the precision could meet the clinical requirements.The detection results significantly correlated (r2 >0.975).The evaluation of clinical ac-ceptability,in Urea low at medical decision level,there were two laboratory determination results that could not be accepted for clinical.In Urea high at medical decision level,there was a laboratory measurement result that could not be accepted for clinical.In the low Cr at medical decision level,there were two laboratory determination results that could not be accepted for clinical.The rest of the system Urea,Cr projects in various medical decision level compared with the system,the SE% was less than CLIA’88 allowed total error of 1/2,for clinical acceptable.Conclusion Laboratory determination results between different biochemical testing system had bias in different degrees,bias part of the project exceeds the allowed error range.

AIM:To investigate the formation of membrane pore in PC 12 cells induced by exogenous adenosine triphosphate ( ATP) and to identify the key molecular targets .METHODS:PC12 cells were treated with different concen-trations of ATP to establish the injury model .The morphological change was observed under an inverted phase -contrast mi-croscope.The viability of the PC12 cells was measured by CCK-8 assay.Fluorescent dye YO-PRO-1 was used to detect the membrane permeability.The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was as-sessed by real-time PCR and Western blotting .RESULTS:After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous , and the number of adherent cells decreased gradually in a dose-dependent man-ner .The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with con-trol group (P<0.05).YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner .The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G ( a P2X7 receptor inhibitor ) pre-treatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0. 05).The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expres-sion of Panx1 was not changed ( P>0.05) when PC12 cells were exposed to ATP for 3 h.CONCLUSION:Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P 2X7 receptor in PC12 cells.

Objective To research the effect of exogenous estradiol(E-2) on transcriptional activity of regenerating hepatocytes in rats. Methods The E-2 levels in serum were measured using RIA and the numbers of argyrophil nucleolar organizer regions(AgNORs) were investigated. Results Rat serum E-2 level gradually declined after ovariectomy,and no E-2 could be measured after the 15th day.Exogenous E-2 could promote transcriptional activity of regenerating rat hepatocytes,particularly during the first 24h after PH,there was a dose-dependent increase in numbers of AgNORs 24-36h after PH,the exogenous E-2 cause a dose-dependent decrease in numbers of AgNORs,but all of the values were higher than that of control group.Conclusion E-2 can regulate the transcriptional activity of regenerating hepatocytes in rats.

OBJECTIVE: To investigate the expression and concentration of lipopolysaccharide (LPS) and matrix metalloproteinase-9 (MMP-9) in middle ear cholesteatoma and discuss their relations. METHOD: Twenty-nine cases of middle ear cholesteatoma tissue, 18 cases of external auditory canal tissue were detected by limulus amebocyte lysate assay (LAL-assay), and expression of MMP-9 protein in formalin-fixed, paraffin-embedded tissues was detected by immunohistochemical method. RESULT: The concentrations of LPS in cholesteatoma were higher than that in external auditory canal tissues. In group of cholesteatoma: M = 0.739 0, IQR = 0.6203, and in group of external auditory canal tissues: M = -0.2538, IQR = 1.1692 (P < 0.01). In cholesteatoma groups, in extensive type: M = 0.8403, IQR = 0.5254; in localized type: M = 0.4048, IQR = 0.6139, the concentrations of LPS were higher in extensive cholesteatoma in comparison with localized cholesteatoma (P < 00.05). In cholesteatoma epithelium samples, MMP-9 were 79.3%. Compared with external auditory canal epithelium, the expression of MMP-9 was higher in middle ear cholesteatoma epithelium (P < 0.05). There was no significant difference in the expression of MMP-9 between two types of cholesteatoma epithelium (P > 0.05). LPS, MMP-9 weren't significantly correlated by Spearman test. CONCLUSION LPS was responsible for middle ear cholesteatoma and its related bone erosion. MMP-9 was related to the development of middle ear cholesteatoma. There's no correlation between LPS and MMP-9.
Adult ; Cholesteatoma, Middle Ear ; metabolism ; pathology ; Female ; Humans ; Lipopolysaccharides ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Receptors, Calcitriol ; metabolism

OBJECTIVE: To evaluate the effect of the combined application of dissociate skin flap and vacuum sealing drainage (VSD) for the repairing for defect after surgical management of huge neck neoplasms. METHOD: Nineteen patients with huge neck malignant tumor involving the skin of the neck were given radical operation, making use of VSD covering the wound surface. After giving 6.65-7.98 mm Hg continuous negative pressure drainage for 72 h, the patients turned to be treated by intermittent negative pressure therapy with 2 min free interval after each treatment period for 5 min. After dismantling the VSD at 7th to 10th day postoperatively, the good wounds covered by granulation tissue were treated by the skin graft operation with dissociate skin flap from thighs; as for the wounds of which the granulation tissue didn't grow well and important cervical tissues was not fully covered by the granulation tissue, VSD was applied again for 1 week, followed by the skin graft operation. RESULT: Nineteen patients have received a total of 23 times of VSD wound treatment, one-stage operation time was significantly shortened. The granulation tissue grew faster on the wound after VSD treatment, and the important cervical tissues such as great vessels could be well covered. The infection and tumor recurrence were observed directly after dismantling the VSD. The skin graft transplantation would be performed after 1-3 weeks. CONCLUSION The treatment by vacuum sealing drainage combined with skin graft for surgical wounds of huge neck tumor postoperatively has the advantages of simple operation, little injury and promotion of the wound healing, which is an effective way for treatment of neck skin defect by surgical operation for the huge tumor.
Adult ; Aged ; Female ; Head and Neck Neoplasms ; surgery ; Humans ; Male ; Middle Aged ; Negative-Pressure Wound Therapy ; Skin Transplantation ; methods ; Soft Tissue Injuries ; etiology ; surgery ; Surgical Flaps