Objective To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional(3D)culture environments of the gels on the morphology,free migration ability,and cell killing function of natural killer(NK)cells.Methods Type Ⅰ collagen was isolated from the tails of Sprague Dawley(SD)rats and collagen gels with different levels of stiffnesses were prepared accordingly.The microstructure of the collagen gels was observed by laser confocal microscopy.The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer.NK-92MI cells were cultured in collagen gels with different levels of stiffness.The morphology of NK-92MI cells was observed by inverted microscope.High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance.NK-92MI cells were cultured with type Ⅰcollagen gels with different levels of stiffness for 24 h and 48 h and,then,co-cultured with human colorectal DLD-1,a human adenocarcinoma epithelial cell line.CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells.Results Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰcollagen gel with the respective stiffness of(10.970±2.10)Pa and(114.50±3.40)Pa were successfully prepared.Compared with those cultured with the low-stiffness type Ⅰ collagen gel,the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape(P<0.05),the mean area of the cells was reduced([69.88±26.97]μm2 vs.[46.59±21.62]μm2,P<0.05),the roundness of the cells decreased(0.82±0.12 vs.0.78±0.18,P<0.05),cell migration speed decreased([2.50±0.91]μm/min vs.[1.70±0.72]μm/min,P<0.001)and the migration distance was shortened([147.10±53.74]μm vs.[98.03±40.95]μm,P<0.0001),with all the differences being statistically significant.Compared with those cultured with the low-stiffness type Ⅰ collagen gel,NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation,with the proliferation rate being(46.39±12.79)%vs.(65.87±4.45)%(P<0.05)and reduce the cell killing ability.Comparison of the cells cultured for 48 h led to similar results,with the proliferation rates being(31.36±2.88)%vs.(74.57±2.16)%(P<0.05),and the differences were all statistically significant.Conclusion The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology,migration ability,and killing function of NK-92MI cells.This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells,as well as laying the theoretical foundation for optimizing immunotherapy protocols.

Objective: To evaluate whether human NK cells expanded in vitro can be used as carrier cells of reovirus and to investigate its clinical application value. Methods: Expansion of human NK cells in vitro, and flow cytometry was used to analyse the purity of CD3-CD56+ cells. Expanded NK cells were loaded with reovirus and observed by confocal microscopy, to determining the location of reovirus on NK cells. CCK-8 assay was used to detect reovirus-induced oncolysis of expanded NK cells carrying reovirus (Reo-NK) to tumor cells in the presence of neutralizing antibodies; Real-time fluorescence quantitative PCR was used to assess the relative expression of viral RNA in tumor cells. Cytotoxicity assay were performed to detect Reo-NK cells against KRAS mutant (DLD-1) and KRAS wild type (CaCo-2, HT29) colorectal cancer cell lines, ELISA matched paired antibodies assay was performed to measure the perforin level released by NK cells. Results: Confocal microscopy demonstrated that NK cells retained reovirus on the surface. Expanded NK cells could delivery reovirus to tumor cells in the presence of neutralizing antibodies, and the reovirus after delivery still had significant oncolytic activity (P<0.01); Corresponding qPCR result displayed that the expression of viral RNA in tumor cells significantly increased over time (P<0.01). Compared with NK group, Reo-NK group evidently enhanced the cytotoxicity on colorectal cancer cell lines with both KRAS gene mutant and wild (all P<0.05), and significantly increased the release of perforin (all P<0.05). Conclusion: In vitro expanded NK cells provide a convincing cell carrier for reovirus, while reovirus enhances the cytotoxicity of NK cells, and the combination of the two show a stronger killing effect on colorectal cancer cells，that has important clinical application value.

Objective:To observe the effect of different concentration HMGB1 on the secretion of TNF-αand NO from Ana-1 infected with DEN2 and virus copy.Methods:DEN2 were proliferated and identified by conventional methods.The adherence of DEN2 to Ana-1 was observed by direct immunofluorescence and RT-PCR.The level of virus mRNA were detected by qRT-PCR.The concentration of TNF-αwas detected by ELISA.The concentration of NO was detected with Griess reagent.Results:Ana-1 was able to adhered for DEN2.Compared with DEN group,the inhibition ratio(%) of the level of virus mRNA in D-HMGB1-1 group,D-HMGB1-10 group,D-HMGB1-100 group,D-HMGB1-1000 group was 41.53 ±2.12,55.30 ±1.59,74.75 ±1.12,86.35 ±1.42.Compared with DEN group,the level of TNF-αand NO decreased in D-HMGB1 groups(P<0.05).Conclusion:HMGB1 can be effectively regulated of Ana-1 secreted inflammation factor of infected with DEN2,and inhibited DEN2 replication.

Objective To investigate the predictive value of serum uric acid for patients with traumatic brain injury.Methods A total of 330 patients with traumatic brain injury (Glasgow Coma Scale score,GCS:3-14) admitted to the First Affiliated Hospital of Soochow University between November 2010 and October 2012 were enrolled.They were divided into a survival group (GOS:2-5) and a non-survival group (GOS:1).The levels of serum uric acid were measured from venous blood in the morning of the second day after admission.Clinical data were analyzed by logistic regression model,spearman correlation,and ROC curve analysis.Results Spearman correlation analysis showed that serum uric acid was significantly correlated with GCS (r =-0.270 1,P =0.000) and GOS (r =-0.251 2,P =0.000).Age,GCS,pupil reaction and serum uric acid were determined as independent predictors for death by logistic model.The adjusted OR of uric acid was 1.0048,(95％ CI:1.001 9-1.007 6,P =0.001).The area under the ROC curve was 0.718,(95％ CI:0.666-0.766),the optimal cut-off value determined by the Youden index was 304 μmol/L (sensitivity:60.24％,specificity:78.14％,correctly classified:73.64％).In the core model (Age + GCS + Pupil reaction),theR2 value was 0.476 4.With uric acid added into,the predictive power of the model increased to R2 =0.510 5 (7.2％ increased).Conclusions The level of serum uric acid is significantly correlated with the severity of TBI and could be used as an independent predictor for death.