Objective To study the expression of SGK1 in T lymphocytes from pediatric asthma,and the effect of SGK1 on the differentiation of T cells,also to explore the function of SGK1 regulating the differen-tiation of T subset in pediatric asthma. Methods Twenty-eight children with asthma were recruited in Xi′an children′s hospital and divided into moderate group and severe group according to diagnostic guideline of asth-ma. The serum levels of IL-4,IL-13 and IL-17A were analyzed by ELISA. The CD4 +T cells from PBMC and na?ve T cells were selected using magnetic beads. Na?ve T cells were differentiated in vitro under cytokines. SGK1 expression were analyzed with Real-time PCR. The ability of Th2 and Th17 on secreting IL-4 and IL-17A were detected after SGK1 was inhibited by siRNA. In vivo,shRNA-SGK1 Na?ve T cells were transferred into the mice asthma models by intravenous injection. The airway inflammation were observed in shRNA-SGK1 Na?ve T models. Results Compared with healthy children,the serum levels of IL-4、IL-13 and IL-17A increased signifi-cantly in the children with asthma. Importantly,the levels of these three cytokines were much higher with the de-velopment of asthma. SGK1 were up-regulated remarkably in CD4 +T cells from the children with asthma and were positively correlated with IL-13 and IL-17A. Besides,SGK1 expression increased in the differentiated Th2 and Th17 in vitro,but had no change in the differentiated Th1. The levels of IL-4 and IL-17A associated with Th2 and Th17 decreased after SGK1 was inhibited by siRNA. Similarly,In vivo,the serum levels of IL-13 and IL-17A and airway inflammation were reduced in shRNA-SGK1 Na?ve T models. Conclusion The over-expres-sion of SGK1 in pediatric asthma enhances the asthma progress by promoting the differentiation of T subsets.

Objective To evaluate neurofunctional and radiographic results of transpedicular screw fixation reduction and anterior column fixation with use of screw-red system in treatment of multiple segment lumbar pedicle and vertebral body fractures combined with spondylolisthesis. Methods A consecutive series of 12 patients with unstable multiple segment lumbar pedicle fracture, vertebral body fracture, spondylolisthesis and neurologic deficit were managed with posterior transpedicular screw fixation including fractured pedicle and anterior screw-rod fixation instrumentation from January 2002 to December 2007. Results Patients were followed up for 24-30 months (mean 26 months). All the patients with incomplete neurologic deficits got improvement by at least one Frankel grade. Transpedicular screw brought satisfactory reduction. At the time of the latest follow-up, no screw breakage occurred. Con-clusions Excellent reduction of unstable multiple segment lumbar pedicle fractures combined with spon-dylolisthesis can attain better reduction and maintenance by means of selective pedicle screw fixation via fractured pedicle and anterior screw-rod instrument.

BACKGROUND: Along with the establishment and development of in vitro culture technique for human keratinocyte, skin would no longer be considered only as the physiological barrier, it's of important significance in immunity and endocrinology and so on.OBJECTIVE: To explore the experimental cultivating technique for human keratinocytes to provide reliable cell resource forthe appliance of keratinocytes in many ways.DESIGN: An opening study with keratinocytes as the subjects of the experiment.SETTING: Department of Immunology and Department of Dermatology,Xijing Hospital, Fourth Military Medical University of Chinese PLA MATERIALS: This experiment was carried out in the clinical laboratory of Department of Immunology of Xijing Hospital of the Fourth Military Medical University of Chinese PLA between March 2003 and March 2005.Prepuce sample was postoperatively obtained from a 6-year-old boy who was admitted to the department of urology surgery of Xijing hospital in April 2003, prepuce was used as the source for keratinocytes.METHODS: ①Two step digestion technique was used for the isolation of epidermal cells: Firstly the skin flap was digested with dispase of mass concentration of 2.5 g/L at 4 ℃ for overnight, followed by separating epidermis the next day; Secondly it was dipped in trypsin and EDTA mixture for subsequent digestion. ② Improved serum-free cultivating technology was applied to the culture of human keratinocytes. The culture medium is composed of human keratinocyte serum free culture medium +2.5 mg/L bovine pituitary lixivium +5 μg/L epidermal growth factor+438 mg/L glutamine, glutamine can promote the growth of the keratinocyte. ③Human keratinocyte in exponential phase were cryopreserved and rewarmed, then made into cell suspension with additional serum free medium, cells were incubated in culture bottle of 75 cm2 by density of 5×105/mL for subculture. Cell morphological changes were observed under microscope and transmission electron microscope.MAIN OUTCOME MEASURES: ① The growth of primary cells and passage cells. ② The cryopreservation and resuscitation of human keratinocyte from different passages. ③ Morphological observation of Keratinocytes RESULTS: ①The growing state of the primary cells and passage cells:cells suspended primarily and gradually adhered to bottom of the culture dish at about 6-24 hours, most of cells were round and gradually stretched to ellipse shape, at about day 3, some keratinocyte clones and clusters can be observed with satellite-like proliferating epidermal cells scattered around, most of them were polygon and cell homogeneity and transparency became strengthened. Cell fusion amount to 70% at around 5 days, reaching to 90% at day 9 and even completely fused into cell diaphragm at day 1 1. Keratinocyte cultured in vitro can survive 2-3 months without obvious changes in cell morphology and growth speed. ② The cryopreservation and resuscitation of human keratinocyte in different passages: Different passage keratinocytes were separately cryopreserved in liquid nitrogen pot and resuscitated 3 months later, the morphology and growth speed of keratinocytes did not change obviously. ③Morphological observation on Keratinocytes: Cells possess typical epidermal cell characteristics under microscope, displaying higher nucleus/plasma ratio, cells arranged closely with clear boundary and good refraction. Transmission electron microscope revealed that cultured epidermal keratinocyte possessed multiple keratinocyte characteristics, such as massive bundles of tonofibrilla in the plasma, clearly observed mitochondria and rough endoplasmic reticule, cytoplasm sticking out short prominence and cells were found connected by desmosome.CONCLUSION: Keratinocyte can keep its normal morphological characeristics during in vitro culture process by using improved cultivating technique, which can be taken as reliable and abundant cell resource for the experimental and clinical study of human keratinocytes.

Objective To investigate the clinical characteristics of laminar shelling decompression for the treatment of thoracic spinal stenosis.Methods One hundred and twenty-one patients with thoracic spinal stenosis were reviewed.Ages of these 51 male and 70 female patients ranged from 45 to 71 years (mean 54.8 years).There were 72 patients with thoracic ossification of ligamentum flavum(OLF),21 patients with thoracic ossification of posterior longimental ligament(OPLL)and 28 patients with thoracic OLF and OPLL.The lesion segmentum,kyphosis angle of thoracic vertebra and residual area of vertebral canal(RAVC)were measured.All these patients were treated with laminar shelling decompression.Preoperative and postoperative functional statuses were evaluated using a Japanese Orthopaedic Association(JOA)score.Results Thoracic OLF were found between T7 to T12 in 77.0% of the lesions;thoracic OPLL were found between T1 to T6 in 81.1% of the lesions.Of the 121 patients,the mean kyphosis angle was 31.5°±6.8° in upper thoracic spine and,9.4°±3.5° in lower thoracic spine.In patients whose RAVC were more than 80%,the pre- and postoperative mean JOA score was 7.7±1.4 and 9.5±1.6 respectively;RAVC more than 50%,5.2±1.8 and 8.6±2.1 respectively;RAVC less than 5%,4.8±1.4,and 5.6±1.3 respectively.Conclusion Thoracic OLF mostly occurred in lower thoracic spine,while thoracic OPLL mostly occurred in upper thoracic spine.The RAVC is a significant factor to the prognosis of thoracic spinal stenosis.As long as the clinical symptoms correspond with imaging findings,it is better to resect the whole ossification part as much as possible.Thoracic spinal stenosis often recurs after surgery.More attention to decompression ranges and decompression skills shoud be paied during revision surgery.

Objective To immunologically characterize the binding activity of a genetic engineering human antibody against keratin. Methods The specific anti-keratin Fab was screened from a phage antibody library. After Fab monoclonal antibody was induced by isopropylthio-?-D-galactoside (IPTG), the binding activity with antigens and antigenic specificity of the antibody were verified by ELISA,Western blot, and competitive inhibition ELISA. Results The antibody possessed good antigenic specificity as well as excellent binding activities with antigens, and could recognize 46 000 keratin specifically. It was identified as anti-keratin 17 antibody. Conclusions The immunologic characteristics of the genetic engineering antibody are very good. It is necessary to further study the biological activities and clinical application of it.

This study was aimed to design the distal radial anatomic splint which was more in line with the human anatomy and treatment characteristics using the computer design software 3D-MAX. According to ergonomic design principles, the forearm and wrist plaster splint were selected from volunteer models. Then, data of the plaster was measured and input into the computer for the design of the three-dimensional model of distal radial anatomic splint with 3D-MAX software. Finally, the blueprint was drawn for the factory to make the distal radial anatomic splint. The results showed that the distal radial anatomic splint was more in line with human anatomy, which did not require shaping in clinical using. It did not affect the biomechanical properties. And the patients never complain the squeez-ing discomfort of the splint. It was concluded that the three-dimensional model of the distal radial anatomic splint, which designed with 3D-MAX software, provided key parameters of the important part of the distal radial anatomic splint. Therefore, the produced splint was more in accordance with the human anatomy and the clinic treatment re-quirements. It avoided the loss of biomechanical properties after shaping, which was more convenient and effective in the clinical using. Its clinical promotion has a broad prospect.

Objective To investigate the expression of neural-nitric oxide synthase (nNOS) in renal clear cell carcinoma and its clinical significance.Methods The expression of nNOS mRNA in 533 samples of TCGA database was analyzed with Student t test,and statistical analysis was performed to assess the relationship between nNOS expression and clinical prognosis with Kapla-Meier test.Western blot analysis of nNOS protein expression in 10 cases of clear cell renal cell carcinoma(ccRCC) from department of urology of Wuhan union hospital with student t test.Results The mRNA levels of nNOS in 72 cases of ccRCC in tumor tissues and adjacent tissues and were 2.99 ± 0.28 and-1.57 ± 0.17,it is significantly lower than those in adjacent tissues (P ＜ 0.01).The mRNA levels of nNOS in 533 cases of ccRCC,in tumor tissues and adjacent tissues and were 2.99 ± 0.28 and-1.76 ± 0.05,it is significantly lower than those in adjacent tissues (P ＜ 0.01).A total of 533 sample studies showed a low correlation between nNOS expression and clinical T stage,T1-1.59 ±0.08,T2-1.96 ±0.13,T3-1.90 ±0.09,T4-2.38 ±0.28 (P =0.0029) and -1.63 ±0.06 and-2.16 ± 0.13 between non-metastasis and no-metastasis (P =0.0009),and-1.57 ± 0.08 and-2.03 ± 0.11 between non-recurrence and recurrence (P =0.008).Survival analysis showed that the overall survival time were (40.3 ± 5.6) months and (48.3 ± 5.7) months in lower and higher nNOS expression,and disease free survival time were (37.1 ± 2.1) months and (40.3 ± 5.6) months in lower and higher nNOS expression,both with shorter time in low expression of nNOS (P ＜ 0.01).nNOS proteins were 1.02 ± 0.16 and 0.61 ± 0.1 1 in tumor tissues and adjacent tissues with significantly lower expression(P＜0.05).Conclusions The mRNA and protein of nNOS are lower in ccRCC with a poor prognosis of ccRCC.

OBJECTIVETo observe the relationship between the amount of HBV DNA in serum/liver tissue and HGV infection in patients with chronic hepatitis B (CH-B) for exploring the effect of HGV infection on hepatitis B virus (HBV) replication of CH-B.

METHODSHGV RNA in serum, HGV nonstructural region 5 (NS5) antigen (HGV Ag) in liver tissue and the amount of HBV DNA in serum, liver tissue were detected for 56 patients with CH-B by reverse transcription-polymerase chain reaction (RT-PCR) assay, peroxidase antiperoxidase (PAP) immunohistochemical method and fluorescence quantitative PCR assay, respectively. Then the relationship between HGV Ag expression in liver tissue and HGV RNA expression in serum was analysed and the amount of HBV DNA in serum and liver tissues from the serum HGV RNA or liver tissue HGV Ag positive patients were compared with those of the serum HGV-RNA or liver tissue HGV Ag negative patients, respectively.

RESULTSTen (17.9%) and eight (14.3%) patients were positive for serum and liver tissues,respectively.HGV RNA expression in serum was closely related to HGV Ag expression in liver tissues, but there was HGV RNA in serum from some of the liver tissues HGV Ag negative patients ?cases of HGV RNA and HGV Ag positive or negative,HGV RNA positive but HGV Ag negative, HGV RNA negative but HGV Ag positive, respectively: 5,43,5,3,(P<0.01). There was no significant difference in the amount of HBV DNA in serum and liver tissues between HGV RNA or HGV Ag positive and negative patients (P>0.05).

CONCLUSIONSHGV infection may not affect HBV replication. Liver is the site of HGV replication, but HGV probably also replicates in extrahepatic tissues. HGV hepatic pathogenicity is probably mild and further studies are still needed.

Adult ; DNA, Viral ; analysis ; blood ; Female ; Flaviviridae Infections ; complications ; virology ; GB virus C ; genetics ; immunology ; pathogenicity ; Hepatitis Antigens ; analysis ; Hepatitis B virus ; genetics ; physiology ; Hepatitis B, Chronic ; complications ; virology ; Hepatitis, Viral, Human ; virology ; Humans ; Liver ; virology ; Male ; RNA, Viral ; blood ; Virus Replication

Objective To investigate the effects of sevoflurane pretreatment on the myocardial injury in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Twenty NYHA class Ⅱ or Ⅲ patients,aged ＜ 60 yr,undergoing cardiac valve replacement with CPB,were randomly divided into 2 groups (n =10 each):sevoflurane group (group S) and control group (group C).The patients were premeditated with intramuscular morphine and scopolamine.Anesthesia was induced with iv injection of midazolam 0.05-0.08 mg/kg,fentanyl 10-15 μg/kg and pipecuronium 0.08-0.10 mg/kg.Anesthesia was maintained with intermittent iv boluses of midazolam,fentanyl and pipecuronium and in addition sevoflurane was inhaled before aortic clamping and the end-tidal concentration was rapidly increased to 1.0％ and maintained at the level for 5 min in group S.Blood samples were taken from the central vein before skin incision (T1),immediately after aortic clamping (T2 ),at 0 and 30 min after aortic unclamping (T3-4),and at 2,6,12 and 24 h after operation (T5-8) for determination of the concentration of serum cardiac troponin Ⅰ (cTnI) and activities of creatine kinase (CK) and creatine kinase isoenzyme-MB (CK-MB).Myocardial specimens were taken from right auricle before aortic clamping and at 10 min after aortic unclamping for electron microscopic examination.Results The concentration of serum cTnI and activities of CK and CK-MB were significantly increased at T4-8 in both groups ( P ＜ 0.05).The serum cTnI concentration at T4-8 and the activities of CK and CK-MB at T8 were significantly lower in group S than in group C ( P ＜0.05).Different degrees of mitochondrial swelling were observed after aortic unclamping in both groups,but the changes were milder in group S than in group C.Conclusion Sevoflurane pretreatment can attenuate the myocardial injury in patients undergoing cardiac valve replacement with CPB.

OBJECTIVETo investigate the effect and explore its possible mechanisms of epidermal growth factor receptor(EGFR) expression in macrophages on the anti-cancer effect of berberine (BER) on the growth of colorectal cancer.

METHODSMice with EGFR gene defects in macrophages (Egfr(fl/fl) LysM-Cre) and with EGFR gene expression in macrophages (LysM-Cre) (control group) were treated with azoxymethane (AOM) to establish colorectal tumor models. These models were treated with or without berberine (BER) intervention. The number of colorectal tumors and the gut length in the two groups were measured. The proliferation of tumor cells was detected by Ki-67 immunohistochemistry and apoptosis was detected by annexin V-FITC fluorescence labeling. Western blot was used to detect the expression of cleaved-caspase-3 protein.

RESULTSAfter treated with AOM, the colorectal tumor number was 10.26 ± 1.43 in the LysM-Cre group and 7.62 ± 1.05 in the Egfr(fl/fl) LysM-Cre group, showing a significant difference (P = 0.021). The gut length was (6.04 ± 1.06) cm in the LysM-Cre group and (6.39 ± 0.92) cm in the gfrfl/flLysM-Cre group, with a non-significant difference between the two groups (P = 0.075). After treated with AOM plus BER intervention, the colorectal tumor number of the LysM-Cre group was 8.35 ± 1.22 and that in the Egfr(fl/fl) LysM-Cre group was 2.66 ± 0.38, showing a very significant difference between the two groups (P = 0.006). The gut length of the LysM-Cre group was (7.34 ± 1.16) cm and that of the Egfr(fl/fl) LysM-Cre group was (10.01 ± 1.72) cm (P = 0.028). After treated with AOM, the ratio of Ki-67-positive tumor cells in the LysM-Cre group was (78.31 ± 3.43)% and that in the Egfr(fl/fl) LysM-Cre group was (75.85 ± 2.92)% (P = 0.282). After AOM plus BER treatment, the ratio of Ki-67-positive tumor cells in the LysM-Cre group was (42.43 ± 3.09)% and that in the Egfr(fl/fl) LysM-Cre group was significantly lower (29.65 ± 2.47)% (P = 0.018). The ratio of annexin V-positive tumor cells was (0.95 ± 0.13)% in the LysM-Cre group, not significantly different from (1.13 ± 0.16)% in the Egfr(fl/fl) LysM-Cre group (P = 0.175). After AOM plus BER treatment, the ratio of annexin V-positive tumor cells in the LysM-Cre group was (32.10 ± 1.97)%, significantly lower than the (47.08 ± 2.83)% in the Egfr(fl/fl) LysM-Cre group (P = 0.010). The level of cleaved-caspase-3 protein expression was 235.92 ± 19.73 in the Egfr(fl/fl) LysM-Cre group, significantly higher than the 119.71 ± 12.87 in the LysM-Cre group (P = 0.012).

CONCLUSIONSThe growth of colorectal cancer cells in mice can be inhibited by BER treatment, and this anti-cancer effect of BER can be further enhanced by EGFR gene knockout in macrophages. The mechanisms may be related to the inhibition of proliferation and promotion of apoptosis in colorectal cancer cells.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; Berberine ; pharmacology ; therapeutic use ; Caspase 3 ; Colorectal Neoplasms ; drug therapy ; Genes, erbB-1 ; physiology ; Immunohistochemistry ; Macrophages ; metabolism ; Mice