Adoptive cell transfusion based on chimeric antigen receptor (CAR) is a new approach for treating malignant diseases and even advanced malignancies. More patients with advanced malignancies are expected to benefit from CAR T-cells once the suitable target molecules are selected, the safe method of gene transduction is applied, and the side effects of CAR T-cells are prevented.

Background and purpose:Small cell lung cancer(SCLC) is a highly aggressive malignancy with a 5-year survival rate of less than 10%.These features suggest the enrichment in cancer stem cells.Our study was aimed to establish a small cell lung cancer cell line from primary cultured from lung cancer tissue,and to identify and isolate cancer stem cells from the early generations of the established cell line.Methods:By using a serum-free medium,lung cancer primary cells were cultured from a small fresh lung cancer cell sample,and a small cell lung cancer cell line was established after passaging the cultured cells.Lung cancer stem cell markers were searched by using flow cytometry analysis to sort through the third or forth generation cells of the established cell line.Biological characteristics of lung cancer stem cells were studied by using the single cell clone formation test,plat colony formation test and cell sphere formation test.Results:A small cell lung cancer cell line was established by primarily culturing a fresh lung cancer sample.The cell line could easily passaged more than 25 generations.When the third or fourth generation cells of the established cell line were checked by flow cytometry,there was a small population of cells that were obviously with CD44 stronger positive(CD44++ cells,5.1%) than the main population cells,which were CD44 weak positive(CD44+ cells).CD44++ cells showed stronger colony formation ability than the CD44+ cells.Furthermore,only the CD44++ cells could form stem cells when a single cell was seeded in a well of 96 well plates.Most importantly,only the CD44++ cells could form cell spheres in ultra low attachment 96 well plates.These results indicated that the CD44++ cells enriched more cancer stem cells in the small cell lung cancer cell line.When CD44 and CD90 antibodies were co-stained,the cells of the established cell line could be separated into 4 populations,i.e.CD44+CD90-,CD44+ CD90+,CD44++ CD90+ and CD44++ CD90-cells.CD44+ CD90+ cells were the smallest population cells in the cell line,with a ratio of about 1.9%.When cultured in ultra low attachment 96 well plates,CD44++ CD90+ cells had the strongest cell sphere formation ability when compared with other population cells,indicating that CD90 might also be a small cell lung cancer stem cell marker.Conclusion:There were cancer stem-like cells in primary cultured small cell lung cancer cell lines,CD44 and CD90,which might mean that they could be lung cancer stem cell markers.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells at different stages of maturation.MDSCs mediate the suppression of the anti-tumor immunity,and play a crucial role in cancer tolerance through suppressing the activity of T cells and natural killer cells,inducing T-regs and involving the angiogenesis,and then contribute to the tumor development and metastasis.Promoting the differentiation of MDSCs,reducing its quantity and inhibiting its function by using various methods may contribute to the recovery of patients normal immune status,the tumor progression control,and improvement of the efficacy of other anti-neoplastic therapies.Therefore,possible novel therapeutic approaches targeted at MDSCs could be considered and developed rapidly now.

Objective: Construction mouse GM-CSF gene effective eukatyotic expressive vector, selection high GM-CSF expressing mouse leukemia cell line RMA after transfected with the constructed vector, and study the method of treatment leukemia with tumor cells transfected with GM-CSF gene.Methods:770 bp of GM-CSF 3' end cDNA was amplified by PCR and inserted into pcDNAS vector.The constructed vector was transfected into RMA cells by electroporation. After screening by G418 and cloning by limiting dilution, a relative high GM-CSF expressing cell clone was selected by RT-PCR, hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. The cells of this clone were inactivated by mitomycin-C and vaccinated mice to induce antitumor immune reaction. Results: The orientation and sequence of the insert was found to be correct, and a GM-CSF high expressing cell line RMA-GM was selected , which can induce mice obtain anti-tumor protective immune ability after inactivated by mitomycin-C.Conclusion:Tumor cells transfected with GM-CSF gene may be used an effective anti-T lymphycoma tumor vaccine.

Adoptive cellular immunotherapy (ACI) achieves the elimination and control of tumor by mobilizing the body's immune function.It also has targeted efficacy and mild untoward effects.Cytokineinduced killer cells and tumor-infiltrating lymphocytes have been widely used in clinic and have obtained preliminary efficacy.With the development and clinical application of the specific gene transfer of T cell,it will further increase the efficacy of immunotherapy.At present,improving cell culture technology and cell function and using with other treatment are the key links to improve the efficacy of ACI.

Objective:To construct pIRES-I-Ad?? bicistronic eukaryotic expression vector. Methods: Total RNA was acquired by TRIZOL method, the genes of I-Ad ?? chains were amplified through RT-PCR, respectively. The target genes were connected to pGEM-T vector and sequenced. Then the target genes were subcloned into pIRES bicistronic eukaiyotic expression vector and NIH3T3 cell line was transfected. Transfectant was screened by G418 antibiotics. Total RNA of transfectant was obtained by TRIZOL method, mRNA of foreign gene was examined by RT-PCR. Flow cytometry( FCM) was used to detennine foreign gene expression in protein level and surface expression in NIH3T3 cell line. Results: Bicistronic eukaryotic expression vector pIRES-I-AdaB was established. Foreign gene in mRNA level in transfectants was examined. Verified Ⅰ-Ad ?? chain wa3 expressed in high level expressed on transfected NIH3T3 cell line surface by FCM. Conclusion:Bicistronic eukaryotic expression vector pIRES-I-Ad ?? was constructed successfully. It is useful for studying antigen presentation and interaction between epitopes and MHC- Ⅱ molecule of BALB/c mouse.

Objective To investigate the effect of Wushen decoction on levels of serum cardiac troponin Ⅰ (cTnI),cardiac muscle enzyme and clinical parameters in patients with sepsis heart failure,and to analyze the correlations between cTnI and myocardial enzyme level and clinical parameters.Methods Forty-two patients diagnosed as sepsis admitted to Wuhan Hospital of Traditional Chinese Medicine from March 2014 to March 2016 were enrolled,and they were divided into a Wushen decoction treatment group and a control group by principle of single blind complete randomized method,21 cases in each group.The patients in control gToup were treated by conventional western medicine,while the patients in Wushen decoction treatment group,on the basis of conventional western medicine,they were treated additionally by Wushen decoction (composed of ginseng,radix sophorae flavescentis,radix glehniae,radix adenophorae,salvia,astragalus,notoginseng radix,rosewood,etc.),one dose a day,the therapeutic course in both groups being 7 days.The changes of biochemical indicators [cTnI,creatine kinase (CK),CK isoenzyme (CK-MB)],haemodynamics parameters [cardiac index (CI),central venous pressure (CVP),extravascular lung water index (ELWI),global ejection fraction (GEF),mean arterial pressure (MAP),heart rate (HR)],treatment condition and prognostic parameters [vasoactive drug dosage index,acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score,duration of mechanical ventilation,the length of stay in intensive care unit (ICU) and total hospitalization time] were compared before and after treatment for 7 days in the two groups.The correlations between the level of cTnI on admission before treatment and CK,CK-MB,APACHE Ⅱ,vasoactive drug dosage index,duration of mechanical ventilation,the length of stay in ICU and total hospitalization time were analyzed.Results The levels of cTnI,CK,CK-MB,CVP,ELWI,HR,vasoactive drug dosage index,APACHE Ⅱ score in two groups after treatment were obviously lower than those before treatment,the levels of CI,GEF,MAP were markedly higher than those before treatment,the duration of mechanical ventilation,the length of stay in ICU and total hospitalization time were significantly shorter than those before treatment,and the changes of above indexes were more remarkable in Wushen decoction group than those in control group [cTnI (mg/L):0.94-± 0.29 vs.1.30 ± 0.67,CK (U/L):96.00 ± 24.30 vs.101.38 ± 24.55,CK-MB (U/L):31.14 ± 6.78 vs.36.48 ± 8.17,CI (mL· s-1 · m-2):64.51 ± 5.83 vs.53.34 ± 4.67,CVP (cmH2O,1 cmH2O =0.098 kPa):10.56 ± 1.84 vs.11.94--2.16,ELWI (mL/kg):8.81±1.61 vs.11.66±2.30,GEF:(33.62±3.88)％ vs.(27.14±4.55)％,MAP (mmHg,1 mmHg =0.133 kPa):84.67 ± 5.58 vs.79.52 ± 5.74,HR (bpm):87.86 ± 9.02 vs.82.95 ± 5.26,vasoactive drug dosage index:2.44 ± 0.53 vs.2.89 ± 0.68,APACHE Ⅱ score:10.66 ± 1.66 vs.14.43 ± 1.82,duration of mechanical ventilation (days):1.67 ± 2.11 vs.2.10 ± 2.26,the length of stay in ICU (days):8.86 ± 2.59 vs.10.67 ± 2.96,total hospitalization time (days):13.24 ± 4.53vs.16.76 ± 5.04,all P ＜ 0.05].On admission before treatment,the correlations between the level of cTnI and CK,APACHE Ⅱ score,vasoactive drug dosage index,duration of mechanical ventilation and the length of stay in ICU were all positive (r =0.322,0.335,0.327,0.328,0.338,P =0.038,0.030,0.030,0.034,0.029).Conclusions The elevation of cTnI level may reflect the degree of myocardial damage in patients with sepsis cardiac failure,and it can be used as an indicator to predict the prognosis of the disease;the changes of many biochemical and clinical indexes suggest that the addition of Wushen decoction might elevate the clinical efficacy for treatment of patients with sepsis heart failure.

With the development of tumor precise immunotherapy,it is a hot topic to find biomarkers to predict the response ability of programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1).So far,many predictors,such as the PD-L1 expression in tumor tissue,tumor-infiltrating lymphocyte,tumor mutational burden,serum markers and radiographic markers,have shown predictive value in the process of anti-PD-1/PD-L1 immunotherapy.But each predictor has its limitations.

Objective To explore the influence of AKT inhibitors on tumor infiltrating T lymphocytes (TIL)in patients with liver metastasis of colorectal cancer.Methods The tumor tissues from the patients with liv-er metastasis of colorectal cancer in Department of General Surgery,The Affiliated Cancer Hospital of Zhengzhou University from January 2016 to December 2016 were collected.TIL and tumor cells were isolated by percoll densi-ty gradient centrifugation. The profiling of AKT inhibitors on TIL were analyzed by flow cytometry. Results AKT inhibition enhances the expansion of TIL with memory cell without affects its proliferation,also the cells obtained under AKT inhibitor with IL-2 showed higher frequency of IFN-γproducing cells than IL-2. Conclusion Add AKT inhibitors in TIL cultivation system can strengthen the proliferation of central memory T cells,and does not affect the number of CD8+T cells.This might be developed for cell-based immunotherapy of cancer.

Objective:To investigate the ability of the anti-PD-1(scFv)/hIL-15 fusion protein(PD-15) to specifically bind to PD-1 in vitro and the effect of the combination of PD-15 with GF-hIL-15Ra-K562(G15Ra-K562) feeder cells to expand NK/T cells. Methods:Overlap PCR was used to construct G15Ra expression vector. pMXs-G15Ra-IP was transfected into K562 by electroporation. G15Ra-K562 feeder cell lines were obtained by limiting dilution method. pUC57-PD-15 was constructed by digestion and ligation. Lipofectamine? 2000 was used to transiently transfect pUC57-PD-15 into HEK293T cells and the conditioned medium containing PD-15 fusion protein was obtained. Density gradient centrifugation was used to obtain human peripheral blood mononuclear lymphocytes(PBMC), and CFSE staining was used to mark active proliferating cells. Flow cytometry was used to detect the ability of PD-15 to specifically bind to PD-1 and its effect on the proliferation of human PBMC and the proportion of different subpopulations of lymphocytes.Results:The feeder cells G15Ra-K562 with high expression of fusion protein G15Ra was successfully constructed. The addition of hIL-15 can increase the ability of G15Ra-K562 to expand human PBMC by more than 5 times( P<0.05). PD-15 fusion protein has PD-1 specific binding ability( P<0.001), combined with G15Ra-K562 can efficiently expand human peripheral blood-derived NK/T cells in vitro( P<0.05). The cells expanded by PD-15 and G15Ra-K562 are mainly natural killing cell, CD8 + T and CD4 + T cells. Conclusions:The PD-15 fusion protein can specifically target the PD-1 molecule and has a strong human peripheral blood-derived NK/T cell expansion ability when combined with G15Ra-K562 feeder cells. These results shed light on selective expansion of PD-1 + lymphocytes in vitro.