Objective To observe the effect of different conserve time in ultra deep cryopreservation (-196 ℃)on physiological characteristics of allograft nerve transplantation .Methods A total of 80 female Wistar rats were divide into three groups by random principle:derived group which 20 rats were sacrificed to get both sides of femoral nerves ;control groups which each group had 10 rats include group A for fresh autologous nerve transplantation ,group B for fresh allograft nerve transplantation ;experiment groups which each group had 10 rats including groups C ,D ,E ,F that transplanted after femoral nerves conserve in the -196 ℃ for 3 ,6 ,9 , 12 weeks respectively ,and the results of exterior appearance ,light microscope ,electron microscope were observed and electrophysi-ological test was conducted after transplantation .Results After 9 weeks transplantation :the physiological characteristics of group B was most affected ,followed by group C ,D ,E ,F ,group A was with minimal impact ;the result of electrophysiological test showed that groups A&B ,A&E ,A&F had significant difference(P<0 .05) .Conclusion The physiological characteristics of allograft nerve transplantation relate to the freezing time .

BACKGROUND: The precartilaginous stern cells are limited regarding in vitro proliferative capacity, but the immortalized cell lines can provide a large number of stable immortalized cells, and simian virus 40 large T antigen gene (SV40Tag) is one of gene fragments which are commonly used and effective in vitro immortalized ceils. OBJECTIVE: To construct human immortalized precartilaginous stem cells (IPSCs) using human precartilaginous stem calls induced by SV40LTAg gane. METHODS: The human immortalized precartilaginous stem calls were isolated from aborted fetus and purified with enzyme digestion and immunomagnetic beads screening method. By using liposome-mediated gene transfection technology, plasmid pCMVSV40T/PUR containing SV40Tag was transfected in primary embryonic precartilaginous stem cells, while non-transfected cells sewed as negative controls. Positive clones were cultured to observe the cell morphology and the passage recovery, to calculate cell survival rata and population doubling time, to drew call growth curve. Immunofluorescence cytochemistry was used to detect the expression of IPSCs fibroblast growth factor receptor 3, the expressions of SV40Tag and fibroblast growth factor receptor 3 in the human precartilaginous stem cells were determined by RT-PCR. RESULTS AND CONCLUSION: Morphology of human IPSCs seemed coincidence with primary human precartilaginous stem cells. The survival rate of human IPSCs was not influenced by subculture, freezing and recovery, but the survival rate was descended in the human precartilaginous stem cells at the 6~(th) and 10~(th) passages (P < 0.01). Compared with cells at the 6~(th) and 10~(th) passages, the proliferation of human IPSCs was greater, with short population doubling time and high growth rate (P < 0.01). The immunofluorescence showed that fibroblast growth factor receptor 3 was positive in human IPSCs at the second passage, and the RT-PCR results of fibroblast growth factor receptor 3 revealed a specific amplification band at 400 bp,.while that of SV40Tag revealed at 560 bp. No band was seen in the primary cells. It is indicated that SV40Tag human IPSCs can be constructed successfully using immunomagnatic bead screening technology and liposome transfection technique.

Objective To evaluate the effects of dexmedetomidine on the expression of tumor necrosis factor-α(TNF-α) and intercellular adhesion molecule-1 (ICAM-1 ) during cerebral ischemia-reperfusion (I/R ) in rats .Methods Thirty-six male Sprague-Dawley rats , aged 8-12 weeks ,weighing 280-320 g ,were randomly divided into 3 groups ( n= 12 each ):sham operation group (group S ) , cerebral I/R group (group I/R ) and dexmedetomidine group (group D ) .The animals were anesthetized with 10% chloral hydrate 3.5 ml/kg .The focal cerebral ischemia was induced by middle cerebral artery occlusion using a nylon thread with rounded tip inserted into internal carotid artery and advanced cranially until resistance was met .In group D ,dexmedetomidine 3μg/kg was injected intravenously immediately after beginning of ischemia ,followed by infusion at 3μg·kg-1 ·h-1 for 120 min ,while the equal volume of normal saline was given in group I/R .Four rats from each group were chosen at 24 ,48 and 72 h of reperfusion , and blood samples were taken from the left ventricle to determine the concentrations of serum S100B protein .The rats were then sacrificed and the brains were removed to determine the expression of TNF-αand ICAM-1 in brain tissues .Results Compared with group S ,the concentrations of serum S100B protein were significantly increased , and the expression of TNF-α and ICAM-1 in brain tissues was up-regulated in I/R and D groups ( P<0.05) .Compared with group I/R ,the concentrations of serum S100B protein were significantly decreased , and the expression of TNF-α and ICAM-1 in brain tissues was down-regulated in group D ( P<0.05 ) .Conclusion The mechanism by which dexmedetomidine reduces the cerebral I/R injury is related to down-regulation of TNF-αand ICAM-1 expression in rats .

BACKGROUND: Total hip arthroplasty is an effective measure to treat hip involvement in ankylosing spondylitis.Ankylosing spondylitis patients have different degrees of anemia after total hip arthroplasty. The hidden blood loss accounts for a large proportion of perioperative blood loss in total hip arthroplasty, and can affect the recovery of joint function.OBJECTIVE: To investigate risk factors of hidden blood loss after total hip arthroplasty in patients with hip involvement in ankylosing spondylitis.METHODS: We studied a consecutive series of 70 hips in 60 patients with ankylosing spondylitis hip involvement who were converted to cementless total hip arthroplasty. The average age of surgery was 35.12 years. The hidden blood loss was calculated according to Cross formula linear equation. The effects of operation time, erythrocyte sedimentation rate,C-reactive protein, body mass index, Bath ankylosing spondylitis radiology index, allogenic blood transfusion, and osteoporosis on hidden blood loss after total hip arthroplasty in patients with ankylosing spondylitis were analyzed. The patients were divided into the high blood loss group (≥ 480 mL) and the low blood loss group (< 480 mL) according to the high blood loss. Risk factors of high hidden blood loss after total hip arthroplasty in patients with ankylosing spondylitis were analyzed by single factor analysis and multivariate Logistic regression analysis (SPSS 17.0).RESULTS AND CONCLUSION: (1) The hidden blood loss after primary total hip arthroplasty in patients with ankylosing spondylitis was (737.76±419.18) mL, and the total blood loss was (1312.83±487.41) mL, and the percentage of hidden blood loss was 51.48%. The high blood loss group included 41 hips, and the low blood loss group included 29 hips; and the ratio was 41:29. (2) Single factor analysis showed that the operation time, Bath ankylosing spondylitis radiology index and osteoporosis, allogenic blood transfusion, decrease of hemoglobin were significantly associated with high hidden blood loss. (3) Multivariate Logistic regression analysis showed that Bath ankylosing spondylitis radiology index,allogeneic blood transfusion, and decrease of hemoglobin were significantly associated with high hidden blood loss. (4)Hidden blood loss is an important portion of total blood loss after primary total hip arthroplasty in patients with ankylosing spondylitis. Bath ankylosing spondylitis radiology index, allogeneic blood transfusion and decrease of hemoglobin are risk factors for high hidden blood loss.

Objective To observe the inhibitory effect of matrine on K-ras gene mutation colon cancer, and to clarify the inhibitory mechanism. Methods SW480 cells were treated with different concentrations of matrine. MTS method was used to detect the proliferation of SW480 cell lines. The apoptosis of SW480 cells was measured by flow cytometry. The migration of SW480 cells was examined by the scratch test. The expression of MEK1/2 protein was detected by Western blotting method. Results Compared with the blank control group, matrine (0.125-1 mg/mL) could inhibit the growth and proliferation of human colorectal cancer SW480 cell lines, promote the apoptosis, restrain the migration of SW480 cells, and inhibit the expression of MEK1/2 protein(P < 0.05), the effect showing a dose-dependent trend. Conclusion Matrine can effectively inhibit the proliferation and migration of SW480 cells, and promote SW480 cell apoptosis through the down-regulation of MEK1/2 protein expression in MAPK signal pathway system.

Objective To investigate the effect of preconditioning with different concentrations of sevoflurane on hypoxia-reoxygenation(H/R)-induced apoptosis in rat hippocampal neurons and the role of mitochondrial KATP(mito-KATP)channels.Methods Primary cultured hippocampal neurons isolated from newborn SD rats(＜24h)of both sexes,weighing 5-6 g,were randomly divided into 7 groups with 48 wells and 12 dishes in each one:control group(C group),H/R group,preconditioning with 6%,4%and 2% sevoflurane groups(S1-3 groups),5-hydroxydecanoate(5-HD,mito-KATP channel blocker)100 μmol/L preconditioning group(5-HD group)and preconditioning with 5-HD 100 μmol/L+6% sevoflurane group(5-HD+S group).The neurons were exposed to 4 h hypoxia followed by 24 h reoxygenation. In S1-3 groups, preconditioning was performed with 6% , 4% and 2% sevoflurane respectively before H/R. In 5-HD group, preconditioning was performed with 5-HD (final concentration 100 μmol/L) before H/R. In 5-HD + S group, preconditioning was performed with 5-HD 100 μmol/L and 6% sevoflurane before H/R. The neuronal viability, apoptosis rate and expression of Bcl-2 and Bax were determined after 24 h reoxygenation.Results The neuronal viability was significantly lower,while the apoptosis rate and expression of Bcl-2 and Bax were significantly higher in the other 6 groups than in group C(P＜0.01).The neuronal viability and expression of Bcl-2 were significantly higher,while the apoptosis rate and Bax expression were lower in S1-3 groups than in group H/R. There was no significant difference in the parameters mentioned above between 5-HD and 5-HD + S groups(P＞0.05).The neuronal viability and expression of Bcl-2 were significantly lower, while the apoptosis rate and Bax expression were higher in S2, S3 and 5-HD + S groups than in group S1, and in group S3 than in group S2(P＜0.0l) .Conclusion Sevoflurane preconditioning can inhibit H/R-induced apoptosis in rat hippocampal neurons and reduce the injury to neurons in a concentration-dependent manner, and the underlying mechanism may be related to activation of mito-KATP channels, up-regulation of Bcl-2 expression and down-regulation of Bax expression.

Objective To study the biological effects of pulsed electromagnetic fields (PEMFs) on the pro-liferation of immunomagnetically separated human precartilaginous stem cells (PSCs) in vitro. Methods The cells from an aborted fetus's metaphysis were digested using collagenase. The PSCs were isolated by magnetic cell sorting (MACS), then subcultured and amplified. Flow cytometry, immunohistochemistry, immunofluorescence and RT-PCR analysis were performed to identify the purified PSCs. The PSCs were stimulated by PEMFs at 50 Hz frequency and 1 mT intensity. Cell proliferation was measured at different time points using methyl thiazolyl tetrazolium ( MTT), and the cell growth curve was plotted. Flow cytometry was applied to measure the cell cycle and apoptosis. Results The PSCs were successfully cultured. There was fibroblast growth factor receptor-3 (FGFR-3) on their sur-face. Cell proliferation was promoted after 4 and 6 days of PEMF stimulation. The percentage of cells at the S phase was higher than in a control group. Early, late and total rates of apoptosis in the experimental group decreased signifi-cantly. Conclusion PEMFs can enhance the proliferation and inhibit the apoptosis of human PSCs, and it is possi-ble to cultivate the high density human PSCs in vitro.

Objective To establish the strain of immortalized human precartilaginous stem cells (PSCs) which can be a stable cell resource for study of the molecular mechanism of gene targeting on differ-entiation of PSCs. Methods Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs using lipofeetin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Immunohistochemistry, RT-PCR and Southern blot were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. Results The positive colonies were isolated and subcultured, named as immortalized precartilaginous stem cells (IPSCs), which were confirmed as positive to fibrnblast growth factor receptor-3 (FGFR-3). The existence of SV40Tag cDNA was detected by Southern blot and the expression of SV40Tag mRNA and protein by RT-PCR and immunohistochemistry. Conclusion IPSCs strain with SV40Tag can be constructed successfully.

BACKGROUND: Heterotopic ossification (HO) is common following primary total hip arthroplasty (THA) in patients with ankylosing spondylitis (AS), which may cause certain influence on functional recovery.OBJECTIVE: To explore the risk factors for HO after primary THA in AS patients.METHODS: The clinical and radiological data from 87 patients (132 hips) with AS undergoing primary THA between June 2011 and December 2015 were retrospectively analyzed, and followed up for more than 6 months. The radiological information included preoperative and postoperative hip anteroposterior and lateral radiographs. The presence of HO surrounding the prosthesis was evaluated on the radiographs at the last follow-up and graded according to the Brooker classification. Risk factors for HO were divided into invariable factors (age, sex, course and with or without ankylosed hip) and variable factors (preoperative C-reactive protein level, preoperative erythrocyte sedimentation rate, intraoperative blood loss, operation time, prosthesis types and anesthesia methods) to determine the pertinent risk factors.RESULTS AND CONCLUSION: (1) Totally 43 hips (32.6%) were found to have developed into HO. (2) Invariable risk factors including male (P=0.029), preoperative ankylosed hip (P < 0.001), and course (P=0.029) increased the prevalence of HO. Among the variable risk factors, prolonged operation time (P=0.031) and general anesthesia (P=0.003)were associated with the increased occurrence of HO. Age, preoperative C-reactive protein level and erythrocyte sedimentation rate, intraoperative blood loss, and prosthesis types had no obvious correlation with HO. (3) These results suggest that to prevent the formation of HO following THA in AS, efforts to reduce the operation time and avoid general anesthesia should be considered.

Objective To explore the effect of Xingnao acupuncture combined with swallowing rehabilitation training on dysphagia after stroke .Methods 66 patients with dysphagia after stroke were selected as the research subjects ,and they were randomly divided into the observation group and the control group ,33 cases in each group .The control group was given rehabilitation training on the basis of conventional treatment ,while the observation group was given Xingnao acupuncture method on the basis of the control group .The therapeutic effect ,swallowing function and adverse reactions were observed before and after treatment in the two groups .Results After treatment,the treatment effect in the observation group was significantly better than that in the control group ,there was statistically significant difference between the two groups (Z=-4.123,P<0.05).The total effective rates of the two groups were 93.94％, 75.76％,respectively,there was no statistically significant difference between the two groups (χ2 =1.906,P>0.05). After treatment,the Watian drinking water test scores,standardized swallowing assessment (SSA) score,dysphagia score in the control group were (1.39 ±0.47) points,(23.02 ±5.24) points,(1.25 ±0.55) points,respectively, which in the observation group were (0.74 ±0.39) points,(18.26 ±3.71) points,(0.74 ±0.28) points,and the differences were statistically significant compared with before treatment ( the control group:t =21.453,10.644, 26.212,all P<0.05;the observation group:t=27.779,14.15,37.469,all P<0.05),which in the observation group were better than those in the control group ,the differences were statistically significant ( t=6.114,4.259,4.747,all P<0.05).There was no statistically significant difference in the incidence of adverse events between the two groups (P>0.05).Conclusion Xingnao acupuncture combined with swallowing rehabilitation training in the treatment of dysphagia after stroke can effectively improve the swallowing function ,improve the quality of life of patients ,and it is safe,reliable and worthy of promotion in clinical practice .