Asthma ; diagnosis ; Exhalation ; Humans ; Nitric Oxide ; metabolism ; Respiratory Function Tests

BACKGROUND: Osteosarcoma cell OS-9607 is a cell line cultured from human osteosarcoma tissue. To construct cDNA library of this cell line so as to clone target gene from it.OBJECTIVE: To construct cDNA library of osteosarcoma cell OS-9607 by switching mechanism at 5' end of RNA transcript (SMART) technique and analyze its quality.DESIGN: Verified experimental study SETTING: Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA MATERIALS: This experiment was carried out at the Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA in May 2005. Osteosarcoma cell OS-9607 was cultured in CO2 incubator. Cells were pre-treated with DM for 30 minutes, recovered for 24 hours in standard culture medium. The whole RNA of osteosarcoma cell OS-9607 was isolated by RNAease reagent kit. The quantity and integrity of total RNA was detected by ultraviolet spectrometer and electrophoresis on a denaturing formaldehyde agarose gel stained by ethidium bromide (EtBr). METHODS: Total RNA was extracted from human osteosarcoma cell OS9607 and mRNA was isolated, cDNA of OS-9607 cells was acquired by in situ polymerase chain reaction (PCR) and long-distance PCR (LD-PCR),then the cDNA was ligated with dephosphorylated λ phage vector. The recombinants were constructed with SMART cDNA library construction kit.The size of cDNA inserts and the diversity of library were detected by PCR.MAIN OUTCOME MEASURES: The quality and integrity of total RNA, the titer of un-amplified and amplified library and recombination fraction; analysis of cDNA length of library RESULTS: ①The concentration of the isolated total RNA was determined and the measuring absorbance was between 260 nm and 280 nm. Its absorptance was about 1.9 A. 0.8％ denaturing formaldehyde agarose gel electrophorosis showed clear 28S rRNA and 18S rRNA,and the brightness rate of 28S rRNA and 18S rRNA (28S/18S) was 2:1. ②After linked to λ phage in vitro, packed and transfected host bacteria, the titer of unamplified library was 2.2×107 pfu/mL, and the titer of amplified 1 ibrary arrived to 4.5×1010 pfu/mL. The recombination fraction analysis showed that there were 8 blue plaques and 1 360 colorless plaques in one plate, so the recombination fraction was 99.5％. ③ The obtained cDNA library consisted of 1.6×106 recombinant bacteriophages and the average exogenous inserts of the recombinants was 1.5 kb.CONCLUSION: These results suggest that the obtained osteosarcoma cell OS-9607 cDNA library has high quality, which sets up a firm foundation for the further research on osteosarcoma cell OS-9607 and its elated genes screen.

Objective To express the major allergen of Blattella germanica (Bla g 2) in Pichia pastoris and obtain the soluble protein. Methods The known Bla g 2 gene was used to design the primers which had the restriction enzyme sites. PCR method was applied to obtain the Bla g 2 gene. The gene fragment was then cut and ligated with the Pichia expression vector pGAPZaA, resulting in a recombinant plasmid pGAPZaA-Bla g 2. The linearized pGAPZaA-Bla g 2 was transformed into Pichia pastoris GS115 through electroporation, then screened to positive transformants, and the protein was expressed in YPD medium. Purification of the recombinant protein was achieved by metal (Ni2+) chelating affinity chromatography and Western-blotting assay indicated its IgE binding capacity. Results With the expressed reeombinanl protein, SDS-PAGE showed the presence of the product in the supernatant of the culture with Mr 45 000. After 3 days culture, the recombinant protein occupied 50% of the total proteins in the supernatant. The recombinant protein was purified and Western-blot demonstrated an adequate IgE binding capacity of the product. Conclusion A recombinant protein of Bla g 2 has been obtained, which is soluble in the supernatant and therefore can avoid a process of denaturalization and renaturation of the recombinant.

OBJECTIVE:To investigate the effects of etomidate combined with propofol on painless gastrointestinal endoscopy and cognitive function of elderly patients. METHODS:Totally 90 elderly patients with painless gastrointestinal endoscopy were ran-domly divided into propofol group(30 cases),etomidate group(30 cases)and combination group(30 cases). Propofol group was given Fentanyl injection 1 μg/kg+Propofol injection 1.5 mg/kg;etomidate group was given Fentanyl injection 1 μg/kg+Etomidate injection 0.3 mg/kg;combination group was given Fentanyl injection 1 μg/kg+Propofol injection 1 mg/kg+Etomidate injection 0.15 mg/kg. The levels of SBP,DBP,HR,MAP and SpO2 at different time points,examination time,anesthesia induction time,recov-ery time and recovery time of orientation were observed in 3 groups. NCSE and MMSE score at different time points,the occur-rence of ADR were also observed in 3 groups. RESULTS:During examination,SBP,DBP,MAP and SpO2 of propofol group were significantly lower than before anesthesia,after examination and those of combination group,with statistical significance(P<0.05);but there was no statistical significance in above indexes of propofol group between during examination and before anesthesia (P>0.05). The recovery time and recovery time of orientation in combination group were significantly shorter than propofol group and etomidate group,with statistical significance(P<0.05),but there was no statistical significance in above indexes between etomidate group and propofol group(P>0.05). The proportion of patients'memory ability,computing ability,orientation ability passing examination in combination group were significantly higher than propofol group,with statistical significance(P<0.05). There was no statistical significance in above indexes between combination group and etomidate group,between etomidate group and propofol group(P>0.05). There was no statistical significance in MMSE score of combination group at different time points (P>0.05);MMSE score of propofol group 15,30 min after anesthesia and that of etomidate group 15 min after anesthesia were significantly lower before anesthesia and combination group,with statistical significance(P<0.05). There was no statistical signifi-cance in MMSE score of propofol group 1 h after anesthesia,etomidate group 30 min and 1 h after anesthesia,compared to before anesthesia(P>0.05). The incidence of apnea,hypotension and injection pain in combination group and etomidate group were low-er than propofol group,the motor response,myoclonus,nausea and vomiting in combination group and propofol group were lower than etomidate group,the tachycardia ease in combination group was lower than propofol group,with statistical significance(P<0.05). CONCLUSIONS:Etomidate combined with propofol anesthesia shows good therapeutic efficacy for painless gastrointestinal endoscopy of elderly patient,can reduce cognitive dysfunction and shortens recovery time,but doesn't increase the occurrence of ADR.

Objective To discuss the surgical skills and clinical value of laparoscopic splenectomy with a safe approach to the splenic hilum through first mobilizing the lower pole of the spleen.Methods A total of 88 patients with hypersplenism secondary to liver cirrhosis who underwent laparoscopic splenectomy in our department from September 2015 to September 2016 were selected into this study.And these patients were divided into two groups based on whether to take the safe approach to the splenic hilum through first mobilizing the lower pole of the spleen in laparoscopic splenectomy.The control group included 40 cases who underwent the traditonal laparoscopic splenectomy,while the observation group included 48 cases who took the safe approach to the splenic hilum through first mobilizing the lower pole of the spleen in laparoscopic splenectomy.The clinical data were collected retrospectively by medical clinical records review.Results There was no mortality occurred in this study.The blood loss of the observation group was (247.50±135.89)mL,which was obviously lower than (361.75±144.43)mL of the control group,and the difference was statistically significant (P<0.05).The operation time of the two groups were (194.69±47.99)min and (232.75±45.26)min respectively,and the difference was statistically significant (P<0.05).No significant difference was found in terms of rate of conversion to laparotomy,rate of intraoperative blood transfusion,postoperative hospital stay and complications (P>0.05).Conclusion It is very crucial for a safe approach to the splenic hilum through first mobilizing the lower pole of the spleen to establish a tunnel behind the splenic hilum in laparoscopic splenectomy.It is safe and feasible to perform a safe approach to the splenic hilum through first mobilizing the lower pole of the spleen in laparoscopic splenectomy.The technique is generalized in clinics,especially for freshmen.

Objective To discuss the clinical distribution and antibiotic resistance of 1909 strains of Pseudomonas aeruginosa(PA)to 19 antibiotics.Methods To collect and isolate clinical specimens from Jan to Dec in 2007,and analysis the drug resistance of 1909 stains of PA.Results The top three departments of infection rnte are Senior Officials inpatient Ward(38.9%),Neurology Department(28.1%)and Pneumology Department(19.58%).The drug with the highest level of antibiotic resistance is Trimethoprin/Sulfa(96.3%),the second one is Tetracycline(95.3%),and drugs with low level antibiotic resistance are imipenem(39.8%),Ceftazidime(29%),Piperacillin/Tazobactam(28%),amikacin(14%),respectively.Conclusion PA is a main pathogenic bacteria for bedridden patients and critically ill patients in ICU.It's important to choose antibiotics reasonably,and it's necessary to acontrol the path of hospital-acquired infection.

Objective A method was developed for the determination diazepam and its main metabolites,nordiazepam, and oxazepam and then in urine by GC-ECD. Method The urine samples were hydrolyzed with ?-glucuronidase were extracted in organic solvent. The extractives were derived with BSTFA and the analytes as trimethylsilyl derivatives were determined by GC. Results Sensitive of the method is as low as snglme, the recovery is high then 70%. Conclusion This method is sensitive enough for the analysis of urine from the subjects over a 48 hour period after receiving a 10mg dose of diazepam orally.

On the basis of the bioluminescent detection technology of the lux gene being a kind of new and progressing environmental detection technology,in this paper,the constitution,function,expression and regulation of the lux gene were introduced,moreover,the recent advance of its application in the study of environmental detection was reviewed