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Objective@#To investigate the role of adenovirus-mediated short hairpin RNA (shRNA) in down-regulating the expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) on p130Crk-related substrates(p130Cas) and paxillin signal transduction to activate hepatic stellate cell (HSC) in vitro.@*Methods@#The rat hepatic stellate cell line, HSC-T6 was cultured and activated in vitro. The adenovirus was used as a vector to transiently transfect shRNA targeting PTEN to activate HSC in vitro, and then PTEN low expression model of activated HSC in vitro was established. Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of PTEN, p130cas and paxillin in activated HSC. The experiment was divided into control group (HSC were transfected with DMEM medium instead of adenovirus), Ad-GFP group (HSC were infected with empty the adenovirus expressing green fluorescent protein (GFP) alone), and Ad-shRNA/PTEN group (HSC were infected with the recombinant adenovirus containing both shRNA targeting PTEN and GFP gene). One-way analysis of variance was used for comparison of multiple groups, and LSD test was used for inter-group comparison.@*Results@#shRNA targeting PTEN was successfully transfected and significantly down-regulated the PTEN protein and mRNA expression of HSC in vitro (P < 0.05), and the PTEN low expression model of HSC in vitro was successfully constructed. Compared with the expression of p130cas mRNA in the three groups of HSC, the expression fold of p130cas mRNA in the Ad-GFP group and the Ad-shRNA / PTEN group was 1.01 times and 1.52 times, respectively. The expression of p130cas mRNA in HSC of the Ad-shRNA / PTEN group was significantly higher than control group and Ad-GFP group (P < 0.05), but there was no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). The expression of p130cas protein in the three groups was higher than that in the control group (0.74 ± 0.07) and the Ad-GFP group (0.72 ± 0.02); P < 0.05, but there was no statistically significant difference between the Ad-GFP group and the control group (P > 0.05). The expression of paxillin mRNA in the three groups of HSCs was compared with the expression of paxillin mRNA in the control group of HSC being 1, the expression folds of paxillin mRNA in the Ad-GFP group and Ad-shRNA / PTEN group were 0.97 times and 1.58 times, respectively. The expression of paxillin mRNA in the Ad-shRNA / PTEN group was higher than that in the control group and the Ad-GFP group (P < 0.05), and there was no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). The expression of paxillin protein in the three groups of HSCs was higher in the Ad-shRNA / PTEN group (0.91 ± 0.05) than control group (0.46 ± 0.03) and Ad-GFP group (0.50 ± 0.04), P < 0.05, and there was no statistically significant difference between the Ad-GFP group and the control group (P > 0.05).@*Conclusion@#Down-regulation of PTEN expression can significantly boost p130cas and signal transduction activity of paxillin protein in activated HSC in vitro.
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Objective@#To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro.@*Methods@#The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca2+ concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups.@*Results@#Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca2+ compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05).@*Conclusion@#The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.
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Objective To investigate the down regulation of phosphatase and tensin homolog deleted on chromosome 10 ( PTEN) gene by adenovirus mediated short hairpin RNA ( shRNA) on the adhesion in activated hepatic stellate cells( HSC ) in vitro and the related signal transduction mechanism .Methods The recombinant adenovirus ( Ad-shRNA/PTEN) with shRNA targeting PTEN and expressing green fluorescent protein ( GFP) were transient trans-fected into the cultural activated HSC in vitro.The experimental group as follows:1)Control group, viral medium was replaced by DMEM at virus transfection step .2 ) Ad-GFP group , HSC were infected with adenovirus expressing GFP alone.3)Ad-shRNA/PTEN group, HSC were infected with adenovirus both taking shRNA targeting PTEN and expressing GFP.PTEN mRNA expression was detected by RT-qPCR, and western blot was used for detecting pro-tein expressions of PTEN , focal adhesion kinase ( FAK) and phosphorylated FAK ( Thr397 ) [ p-FAK( Tyr397 ) ] in HSC.The toluidine blue stain method and MTT colorimetric method were used to determine the adhesion ability of HSC.Results When HSC were infected by adenovirus for 48 hours, PTEN protein and mRNA expressions in Ad-shRNA/PTEN group significantly decreased ( P<0.05 ) , compared to control group and Ad-GFP group, and the expressions of p-FAK ( Tyr397 ) in Ad-shRNA/PTEN group were significantly higher than those in control group and Ad-GFP group ( P<0.05 ) .The adhesion cell counting and the adhesion rate of HSC in Ad-shRNA/PTEN group significantly increased as compared with control group and Ad-GFP group ( P<0.05 ) .Conclusions The down-regulation of PTEN expression can promote the adhesion by increasing the activation of FAK signaling trans -duction in activated HSC in vitro.
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Objective To compare the effects of Dexmedetomidine and Midazolam for sedation in elderly patient with upper cervical spine fracture in awake tracheal intubation.Methods A total of 68 patients with upper cervical spine fracture undergoing awake tracheal intubation who treated in our hospital from Jan.2010 to Jan.2015 were considered as the objects,who was randomly divided into group A and group B.34 cases in group A were treated with Dexmedetomidine for sedation,and the other 34 cases in group B were treated with Midazolam for sedation.The Heart rate (HR),mean arterial pressure (MAP) and BIS value on the before anesthesia (T1),immediately before intubation (T2),immediately after intubation (T3),PaCO2 in before and after intubation,and the adverse reactions were compared between the two groups.Results There was no difference in HR,MAP and BIS at time of T1 between the two groups (P＞0.05).The HR,MAP and BIS were lower in group A than in group B at time of T2 and T3 (P＜0.01).The PaCO2 had no difference between the two groups at before and after intubation (P＞ 0.05).The rate of adverse effects had no difference between the two groups (x2 =1.308,P =0.253).Conclusions Compared with Midazolam,Dexmedetomidine can stable HR,MAP and BIS effectively and has a good safety in the treatment of elderly upper cervical spine fracture in awake tracheal intubation,which is worthy of clinical application.
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Objective To observe the effects of general anesthesia combined with epidural anesthesia on the short-term cognitive function in the elderly patients after orthopedics surgery.Methods 185 elderly patients undergoing orthopedics surgery were treated in our hospital from Jan.2011 to Jan.2014.The patients were divided into observation group (with general anesthesia combined with epidural anesthesia,n=94) and control group (with general anesthesia,n=91).The short term cognitive function,mean arterial pressure and heart rate were compared at 30 min before,during and at the end of the operation between the two groups.Results There were no statistically significant differences in awakening time,extubation time and response time between the two groups [(28.7±7.8) min vs.(30.9±8.1) min,(29.2±8.1) min vs.(32.2±8.4) min,(30.4±7.9) min vs.(33.1±8.6) min,t=1.881,2.472,1.943,respectively,P=0.031,0.007,0.027].The minimental state examination (MMSE) scores were higher in observation group than in control group at 6,12 and 24 hours after surgery [(26.1±1.4) vs.(24.9±1.5),(25.0±1.5) vs.(24.1±1.4),(27.9 ±1.4) vs.(26.3±1.3),t=5.627,3.279,8.049,all P＜0.001].The incidence of cognitive dysfunction was less in observation group than in control group at 6 and 12 hours after surgery (7.5％ vs.17.6％,8.5％ vs.19.8％,x2=4.363,4.862,respectively,P=0.037,0.027).Conclusions Compared with general anesthesia,the general anesthesia combined with epidural anesthesia can reduce the effects of anesthesia on cognitive dysfunction,and has a good effect of anesthesia.It is more suitable for the elderly patients with anesthesia for surgery.
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Objective To investigate the effects of cerebral hemodynamics and oxygen metabolism of dexmedetomidine on patients with intracranial aneurysm surgery.Methods Sixty-four patients with intracranial aneurysm surgery were collected and randomly divided into study group and control group (32 cases for each group).Patients in the study group before induction of anesthesia were given dexmedetomidine and patients in the control group were given saline but anesthesia.Mean arterial pressure (MAP),heart rate (HR),cerebral metabolic rate of oxygen(CMRO2) in different time points were observed and time in intubation and intracranial aneurysm clamp before anesthesia were rescored.Cerebral blood flow (CBF),intracranial pressure (ICP) were observed and the recovery situation.Results At the intubation,MAP and HR in the study group after 15 min of intubation,time in intracranial aneurysm clamp and extubation were significantly lower than those of the control group(P ＜ 0.05).CMRO2 in study group at the intubation and intracranial aneurysm clamp were (34.2 ± 5.0) ％ and (27.1 ± 4.2),significantly higher than that of the control group ((33.9 ± 4.3) ％,(26.5 ±3.6) ％; P ＜ 0.05).CBF in study group at the intubation and intracranial aneurysm clamp were (53.5 ±8.8) ml/(100 g · min) and (56.8 ±9.2) ml/(100 g · min),significantly lower than that of control group ((67.3±11.2) ml/(100 g· min),(67.3 ±11.2) ml) (100 g· min); P＜0.05) ; The same trend was seen in terms of ICP.Spontaneous breathing recovery time and extubation time in study group were (7.35 ± 1.12) h and(12.98 ± 3.76),significantly earlier than those of the control group((9.27 ± 1.45) h and (14.89 ±4.88) h; t =10.92,9.23,P ＜0.01).Steward scores in study group was (5.12 ±0.33),significantly higher than control group ((3.98 ± 0.28) ; t =5.55,P ＜ 0.05).Conclusion Dexmedetomidine can certainly keep hemodynamic stability in patients with intracranial aneurysm surgery,improve rate of cerebral oxygen uptake and recovery performance,which is worthy of clinical application and promotion.
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Objective To compare the effects of remifentanil infused at different rates on median effective target plasma concentration (EC50) of propofol inhibiting responses to laryngeal mask airway (LMA) insertion and determine the optimum infusion rate of remifentanil when used for fiberoptic bronchoscopy in pediatric patients.Methods Eighty-four ASA Ⅰ or Ⅱ pediatric patients,aged 7 months-3 years,scheduled for elective fiberoptic bronchoscopy,were randomly assigned into 3 groups (n =28 each):normal saline group (group C),remifentanil infused at 3 ng· kg-1 ·min-1 group (group R1) and remifentanil infused at 5 ng· kg-1 · min-1 group (group R2).Responses to LMA insertion were defined as body movement and/or bucking during insertion.The initial target plasma concentrations of propofol were 5.2,4.8 and 4.4 μg/ml in groups C,R1 and R2,respectively.The target plasma concentration of propofol was determined by up-and-down sequential allocation.Each time the target plasma concentration increased/decreased by 0.2μg/ml.EC50 and 95 ％ confidence interval of propofol blunting responses to LMA insertion were determined by probit method.Results EC50 (95 ％ confidence interval) of propofol was 5.03 (4.92-5.12) μg/ml,4.71 (4.58-4.84) μg/rnl and 4.46 (4.20-4.94) μg/ml in groups C,R1 and R2,respectively.There was no significant difference in EC50 of propofol between groups R1 and C (P ＞ 0.05).EC50 of propofol was significantly lower in group R2 than in groups C and R1 (P ＜ 0.05).Conclusion The infusion rate of remifentanil should not be lower than 5 ng· kg-1· min-1 when combined with propofol in pediatric patients undergoing fiberoptic bronchoscopy.
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Objective To investigate the prevalence of influenza virus infections in infants and young children during the pandemic period of 2009 influenza A(H1N1)in Beijing.Methods Throat swabs were collected from children visited the affiliated Children's Hospital to Capital Institute of Pediatrics for influenza-like illness from June 1,2009 to February 28,2010.The specific gene segments of 2009 pandemic influenza H1N1 and seasonal influenza viruses were amplified from samples by real-time RT-PCR recommended by WHO and National Influenza Reference Center of China.Results Out of 4363 clinical samples tested by real-time RT-PCR,the total positive rate of influenza A viruses was 29.3%,including 623(14.3%)identified as 2009 pandemic influenza A(H1N1)and 657(15.1%)influenza A viruses without subtype identity.Among those pandemic influenza H1N1 positive,23 were severe cases with 5 deaths.The ages for 618 pandemic influenza H1N1 infected children with completed information were from 14 days to 16 years.The ratio of male to female wag 1.3:1.Among them,25.2% were patients in age group of 1 to 3 years old and distribution of children in age groups of 3 to 6 years old and 6 to 12 years old were similar(about 30.0%).During the survey period,it appeared only one prevalence wave of pandemic influenza H1N1.The positive rate of pandemic H1N1 increased in September and the peak(36.5%of positive rate)was in November and then declined to 2.7%in February 2010.The data from routine influenza virus surveillance from 20-30 clinical samples collected each week indicated an alternative prevalence of seasonal H3N2,pandemic H1N1 and influenza B during this study period.Respiratory syncytial virus(RSV)became predominant in children after the circulating of pandemic H1N1.Conclusion There was an epidemic of pandemic influenza H1N1 in children in Beijing from June 2009 to February 2010,especially in those of preschool and school aged children.Seasonal influenza viruses and pandemic influenza H1N1 were contributed alternatively.