To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent (PPM1D) gene and detect its effectiveness of gene silencing in human gliomas, specific siRNA targets with short hairpin frame were designed and synthesized. DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. After the verified plasmids were transfected into 293T cells, the lentivirus was produced and the titer of virus was determined. Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells. PCR and Western blot analyses revealed the optimal interfering target, and the virus with a titer of 6×10(8) TU/mL was successfully packaged. The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection. The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group. The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8 (CCK-8). Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells. We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells. And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.

Objective To investigate the association of red cell distribution width (RDW) with outcome in patients with septic shock.Methods A retrospectively study was performed on a total of 156 cases with septic shock who hospitalized in the Affiliated Hospital of North China University of Science and Technology from January 2012 to December 2014.All cases were divided into two groups according to the outcome:survivor group and non-survivor group.The data of general information,combined disease,RDW,acute physiology and chronic health score(APACHE Ⅱ) were collected and compared between the two groups.All cases were divided into three subgroup in regard of RDW,group A was ≤ 14.0％,group B was 14.1％-15.9％,group C was ＞16.0％.Multiple Logistic regression analysis was performed to analyze the related factor of mortality.ROC analysis was used to test the predictive effect of RDW for the outcome of septic shock.Results Sixty-nine cases died in 156,with a mortality rate of 44.2％(69/156).The RDW in non-survivor group was significantly higher than that of survivor group((15.79±2.64) ％ vs.(12.69±3.09) ％,P＜0.001).Mortality rate in each sub-group were 21.5％ (14/65) in group A,49.0％ (25/51) in group B,75.0％ (30/40) in group C,respectively,and the difference among the three groups was statistically significant (x2 =29.396,P ＜ 0.001).Logistic regression analysis showed RDW was independently associated with mortality of septic shock.In reference with group A,the risk of mortality was 3.504 (95％ CI:1.563-7.858,P =0.002) in group B and 10.924 (95％ CI:4.314 -27.661,P＜0.001) in group C.Conclusion Red cell distribution width is a risk factor of mortality in patients with septic shock.

Objective To observe the expression of CD166 in advanced non-small cell lung cancer (NSCLC),and to explore the relationship between expression of CD166 and clinical characteristics,platinum-based chemotherapy effect of NSCLC patients.Methods A total of 64 cases of NSCLC patients at stage of Ⅲ,Ⅳ were enrolled.Tumor tissue samples were obtained before chemotherapy by bronchoscopy or lung biopsy,and immunohistochemical was used to detect the expression of CD166.All patients received platinum-based chemotherapy(NP/TC/ GP) for 2-4 cycles as the first line treatment.RECIST criterion was used for therapeutic evaluation.Results 46.9% (3 0 cases) of the patients with CD 1 6 6 was positive.The CD 1 6 6 expression was related with smoking (smoking 62.1%,no-smoking 34.3%,x2 =4.916,P =0.027) and degree of differentiation (moderately,low grade 68.2%,high grade 35.7%,x2 =6.112,P =0.013).The effectiveness rate of chemotherapy in CD166 positive patients was 23.3% (7 cases),was significantly lower than 61.8% in CD166 negative patients(21 cases,x2 =9.565,P =0.002).Conclusion NSCLC had CD166 expression,and the expression positive rate was increased in patients with smoking,and low differentiation cancer.The effectiveness rate of platinum-based was decreased in patients with CD166 positive expression,and detection of CD166 can predict the late NSCLC platinum-based chemotherapy effect.

Objective To study the process of removing bacterial endotoxins by ultrafiltration technology in dextran 40 injection. Methods Dextran 40 solution was ultrafiltrated by 100,200,and 300 kDa aperture ultrafiltration membranes with composite, PES and PVDF materials. In order to optimize ultrafiltration process,the content of effective component and endotoxins were detected by HPLC and kinetic-turbidimetry,respectively,and the change of particle size distribution in dextran 40 solution was analyzed before and after ultrafiltration. Results The transmittance of dextran 40 was close to the same MWCO and different membrane material. When MWCO reached 300 kDa,the transmittance was above 91%,which met the requirement of filtration. The endotoxin removal rates by 100-300 kDa composite ultrafiltration membranes were more than 99%. But the endotoxin removal rates of both of PES and PVDF membranes were less than 40%,which were unable to guarantee the removal efficiency of the endotoxin in dextran 40 solution. The particle size declined after ultrafiltration by 300 kDa composite membrane, and level of the insoluble particles decreased. Conclusion The 300 kDa composite ultrafiltration membrane can effectively remove endotoxin in dextran 40 solution with less main components loss. The material can meet requirements for producing dextran 40 injection.

BACKGROUND: Previous studies has explored the effects of nerve growth factor (NGF) on cultured rabbit Schwann cells as well as preparation of acellular nerve allografts, in addition, the neural complex was prepared in vitro.OBJECTIVE: To investigate the effect of acellular nerve allografts on the functional recovery and reconstruction of the nerve-muscle structure of the sciatic nerve defect in rabbits.DESIGN, TIME AND SETTING: The randomized controlled animal experiment was performed at the Center Laboratory of Third Hospital of Hebei Medical University from March 2006 to June 2007.MATERIALS: Totally 37 healthy, adult, New Zealand, white rabbits were selected. One rabbit was prepared for extracted nerve bridge, and the others were randomly divided into the experimental and control groups, with 18 animals in each group.METHODS: The double sides of sciatic nerve were removed, divested surrounding tissues, and cut into 3 cm segments, then placed in TdtonX-100 solution for 12 hours, washing with distilled water. The procedure was repeated, and TritonX-100 totally used for 96 hours. Then the extracted nerve bridge was conserved in Hank's solution at temperature of 4 ℃. The concentration of 2~(nd) passage Schwann cell was regulated to 1 ×10~(11)/L, and injected to extracted nerve bridge, followed by DMEM culture, to obtain neural complex. Rabbits in the experimental group were prepared a 20 m defects above knee joints, and the recellularized neural complex were transplanted into the defective sciatic nerve. Rabbits in the control group were prepared by transplanting extracted nerve bridge without Schwann cells.MAIN OUTCOME MEASURES: The elcosis and healing of feet were observed at weeks 4, 8, and 16 after operation, meantime,the recovery of axon of distal end peroneal nerves, myelin sheath, tibialis anterior, as well as motor end plate were detected by electromyogram, light microscope and transmission electron microscope.RESULTS: At weeks 4, 8 and 16 after operation, the rejection was not found in the operating field. The experimental group was better than the control group in healing of ulcers, the number of nerve fibres, the ultramicrostructure of medullary sheaths, the regeneration nerve, the structure of the motor end plate and muscle. At 4 weeks after operation, no nerve conduction was found in 2 groups, but at weeks 8 and 16 after operation, the experimental group was better than the control group in wet weight of tibialis anterior muscle and nerve conduction velocity (P < 0.05).CONCLUSION: The recellularized neural complex can significantly promote the reconstruction of the nerve-muscle structure and functional recovery of injured sciatic nerve.

Background Selective laser trabeculoplasty (SLT) plays lowing-intraocular pressure (IOP)effect by irradiating pigmented trabecular cells selectively using 532 nm Nd:YAG laser.However,its affect pattern is not fully known.Objective This study was to establish a human trabecular cells (HTCs) model of SLT effect,and to observe the morphological changes of HTCs after they were exposed to different energies of SLT.Methods Immortalized HTCs were incubated in DMEM/F12 with pigmental granules for 16 hours (overnight) to prepare pigmented trabecular cells.The cells were irradiated with modified laser lens with the spot of 400 μm and emitting duration of 3 ns.The energy scale of 0.2 mJ was set for standardized energy and 0.1 mJ was used as the low energy.The morphology and ultrastructure of the cells were examined under the light microscope and transmission electron microscope 1 hour,4,8,12,24 hours after irradiation.Trypan blue staining and TUNEL fluorescence staining were used to evaluate the death and apoptosis of the cells after laser irradiation.Results The brown pigment particles were seen in cytoplasm around nuclei of trabecular cells after cultured for 16 hours.After laser irradiation,there was no obvious change in the shape of nonpigmented trabcular cells,but a 400 μm spot was found in the pigmented trabecular cells under the light microscope,and the pigmented particles released as the cell disruption.After 0.2 mJ laser irradiation,cell loss zone was seen at the center of the laser spot,and the positive cells for trypan blue and TUNEL staining were found;while the abnormal manifestations were slight after the 0.1 mJ irradiation.With the lapse of the time,the number of positive staining cells was gradually decreased.Twenty-four hours after laser irradiation,proliferative trabecular cells emerged and migrated to the center area of laser spot.Statistical analysis showed that the numbers of the positive cells for trypan blue and TUNEL staining were significantly reduced in the lower energy group in comparison with the standardized energy group at various time points (all at P＜0.001),and those of both energy groups were gradually reduced with lapse of time with the significant differences between any two time points (all at P＜0.01).Conclusions An in vitro laser effect model of HTCs can be established by exposing pigmented HTCs to SLT laser.After exposed to SLT,the gasification,necrosis-like death,apoptosis-like change appear at the laser center.The laser destroyed cells can be replaced by proliferative cells with the lapse of time.Low-energy laser irradiation cause a similar morphological change of the cells to high-energy irradiation,but the number of abnormal cells is much less.

Background Selective laser trabeculoplasty (SLT) can increase the outflow of aqueous humor and reduce the intraocular pressure of patients with open angle glaucoma,but its mechanism is unknown.To investigate the mechanism of SLT would improve the therapeutic effect of SLT.The aqueous outflow resistance in trabecular meshwork was affected by the extracellular matrix (ECM).Matrix metalloproteinase-3 (MMP-3) and MMP-9 were closely related to ECM degradation in trabecular meshwork.Objective This study was to observe the effects of SLT on the expressions MMP-3 and-9 in human trabecular ceils in vitro.Methods Immortallized human trabecular cells were cultured with pigment particles mixed suspension for 16 hours and incubated overnight.Then the cells were irradiated with Q switch frequency doubling 532 nm Nd:YAG laser to establish SLT-effective cells with the energy of 0.2 mJ,spot diameter of 400 μm and pulse duration of 3 ns.The expressions of MMP-3 mRNA and MMP-9 mRNA in the cells were detected by fluorescence quantitative real time PCR before and 1 hour and 4,8,12,28 hours after exposure of laser.The concentrations of MMP-3 and MMP-9 in the medium were assayed using ELISA 1 hour and 4,8,12,28 hours after exposure of laser and compared between the non-irradiation group and the irradiation group.Results The relative expressing levels of MMP-3 mRNA were 1.00,1.32±0.12,2.08±0.05,2.34±0.04,2.77± 0.05 and 2.49±0.27 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of laser SLT,showing a significant difference among the groups (F =15.966,P=0.007),and relative expressing levels of MMP-3 mRNA were significantly higher in various time points after laser irradiation than those of the non-irradiation group (all at P＜0.05).The relative expressing levels of MMP-9 mRNA were 1.00,0.91 ±0.10,1.27 ± 0.07,1.46±0.07,1.69±0.09 and 0.87±0.09 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of SLT,which was considerably different among the groups (F =30.526,P =0.005),and significant increased values were seen in the 4,8 and 12 hours after irradiation compared with the non-irradiation group (all at P＜0.05),with highest expression in the irradiation for 12-hour group.The concentrations of MMP-3 and MMP-9 proteins in medium were significantly increased in various time points after laser exposure in comparison with the control group (all at P＜0.05).Conclusions The expressions of MMP-3 and MMP-9 in human trabercuolar cells upregulate and the secretion ability of human trabecular cells to MMP-3 and MMP-9 proteins improves in early stage of SLT in vitro.However,these procedures gradually diminish with the lapse of time.

In this study we implanted magnetically labeled neural stem cells (NSCs) in PD rats and then monitored their survival and migration in the host brain by magnetic resonance imaging (MRI). The mesencephalic NSCs were obtained from the brain of SD rats. Superparamagnetic iron oxide (SPIO) was transferred to NSCs by Lipofectamine transfection. Eighteen PD lesioned rats were selected for transplantation by evaluation of their rotational behavior in response to amphetamine and randomly assigned to 3 groups, i.e., sham group, PBS group and NSCs transplanted group, with 6 rats in each group. MR scanning was performed at 1, 2, 4, 6, 8 and 10 week(s) following transplantation. At the meantime, rotational behavior was assessed in each group. Our results showed that SPIO particles were clearly visible with Prissian blue staining in neurospheres and cells derived from NSCs. The rotational behavior of the NSCs transplanted group was remarkably improved compared with that of sham group and PBS group (P < 0.05). In vivo MR tracking of NSCs showed that SPIO labeling led to a strong susceptibility change of signal 1 week after transplantation on T2 weighted images. And a large circular hypointense signal appeared in the transplanted area on T2* gradient echo images. Ten weeks following transplantation, the hypointense signal on T2 weighted and T2* gradient echo images was still displayed. It is concluded that SPIO particles could label NSCs effectively, and MRI detection of SPIO labeled cells is a promising method and novel approach to analyzing the NSCs following transplantation in the treatment of PD.

Objective To observe the effects of acute normovolemic hemodilution(ANH) plus tranexamic acid on blood loss and blood transfusion in patients with total knee replacement.Methods 98 cases of patients with total knee replacement were divided into ANH group and combination group by computer randomization,49 cases in each group.ANH was performed in ANH group,and ANH plus tranexamic acid was given in combination group.The changes of hemoglobin(Hb),red blood cells deposited(Hct),platelet count(PLT) were observed before and after autologous blood transfusion in two group.The changes of prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT),fibrinogen(FIB),D-dimer were observed at preoperative and postoperative in two group.The amount of blood loss and allogeneic blood infusion,the urine volume and the postoperative flow volume were recorded in two groups.Results The average levels of Hb,Hct after autologous blood transfusion in combination group were higher than those in ANH group,the differences were statistically significant(P<0.05).The average level of D-dimer at postoperative in combination group were higher than that in ANH group,the difference was statistically significant(P<0.05).The amounts of blood loss and allogeneic blood infusion,the postoperative flow volume were lower than those in ANH group,the differences were statistically significant(P<0.05).Conclusion The joint application of ANH and tranexamic acid can effectively reduce the amounts of blood loss and allogeneic blood infusion in patients with total knee replacement,and has a certain clinical value.

Objective To test whether the changes of partial end-tidal carbon dioxide pressure (PETCO2) during passive leg raising (PLR) predict fluid responsiveness in mechanically ventilated patients with septic shock.Methods Forty-two mechanically ventilated patients with septic shock admitted from January 2012 to November 2012 were prospectively recruited.Hemodynamic parameters monitored by a pulse indicator continuous cardiac output(PiCCO) device and PETCO2 monitored by an expiratory-CO2 device were studied at baseline,after PLR,and after volume expansion.Fluid responsiveness was defined as an increase in cardiac index (CI) of 15％ or greater after volume expansion.The correlation between PLR-induced CI change (△CIPLR) and PETCO2 (△PETCO2-PLR) was analyzed.The value of △PETCO2-PLR to predict fluid responsiveness was evaluated by receiver operating characteristic (ROC) curves.Results A total of 42 patients were enrolled in this study,of whom,24 had a CI increase of ≥ 15％ after volume expansion (responders).After PLR,CI and PETCO2 were both significantly increased in the response group compared with baseline [(21.4 ± 12.9) ％ of CI and (9.6 ± 4.7) ％ of PETCO2,P ＜ 0.05],while no significant changes were observed (P ＞ 0.05) in the non-response group.Both △CIPLR and △PETCO2-PLR were significantly higher in responder group than in the non-responder group (both P ＜ 0.05).△CI and △PETCO2 after PLR were strongly correlated (r =0.64,P ＜ 0.05).In responders after PLR,the area under ROC curve of △PETCO2-PLR was 0.900 ± 0.056 (95％ CI 0.775-1.000,P ＜ 0.05).An increase of ≥ 5％ in △PETCO2-PLR predicted fluid responsiveness with a sensitivity of 88.0％ and specificity of 88.2％.Conclusions The change of PETCO2 induced by passive leg raising is a non-invasive and easy way to predict fluid responsiveness in mechanically ventilated patients with septic shock.