Objective:To artificially design and express a recombinant protein named as ScFv-pLLO by fusing ScFv gene of Rituximab(C2B8)and dominant antigen epitopes from listeriolysin O(LLO),and studying its anti-tumor activity.Methods:VH and VL gene sequences of C2B8 against CD20 were acquired by searching United States Patent database,and ScFv sequence was constructed by linking VL and VH with a short peptide linker.Two CD4+T cell epitopes from LLO were selected and designed to splice ScFv sequence.The recombinant gene of ScFv-pLLO was cloned into prokaryotic expression vector and purified after induction.The capacity of ScFv-pLLO target-binding to B-cell lymphomas was evaluated by flow cytometry ( FCM ) and co-immunoprecipitation ( Co-IP ) .The effects of ScFv-pLLO on B-cell lymphomas proliferation and apoptosis were detected respectively.The immunogenicity of ScFv-pLLO was assessed by lymphocyte proliferation assay.Results: ScFv-pLLO was successfully expressed.It could bind to different B-cell lymphomas cell lines and obviously inhibit the growth of Raji cells as well as inducing apoptosis.Moreover,ScFv-pLLO was able to stimulate proliferation of spleen lymphocytes of immunized mice.Conclusion: The recombinant protein ScFv-pLLO can target-bind to B-cell lymphomas,and perform inhibitory effect and induce apoptosis on Raji cells that indicate ScFv-pLLO retain the capacity of ScFv derived from monoclonal antibody against CD20.Besides, ScFv-pLLO can induce immune response.This study provides a basis for further research about the role of ScFv-pLLO on simulating tumor cell antigens as well as being tumor vaccine adjuvant.

Objective To efficiently builds up and expand breast cancer cells from cancer tissue and to identify their biological properties , provide abundant materials for research and personalized medicine .Methods Feeder cell layer and ROCK inhibitor Y-27632 were employed to faciliate the breast cancer cells;CCK-8 was used to determine the proliferation of the breast cancer cells; Cell cycle distribution was analyzed by flow cytometry; Histochemistry ( FH) assay to show the expression level of CK .The mRNA expression of HER-2, ER, PR and the breast cancer stem cell associated molecules (such as CD44, CD24, etc.) were detected by RT-PCR;STR assay was used for identifying verification of the cells .Results The use of feeder cells and Y-27632 facilitates rapid expand of the original breast cancer cells , and the cells have kept the original features of the tumor .Conclusions To use the method could obtain a large number of cells within a short time , which can promptly be used for the research of per-sonalized medicine .

Objective To identify the species of cultured cells and to detect inter-species cross contamination by polymerase chain reaction (PCR).Methods From references and NCBI database,we outsourced 32 pairs of species-specific primers targeted to genomic DNA of ten common species of cultured cells.Then we screened for optional primers with high specificity and high sensitivity.PCR amplification with these primers,the genomic DNAs isolated from the tested cell line and agarose gel electrophoresis of the PCR products were done.Mixed DNA of 10 species was used as positive-control template,and water as negative-control template.Results Ten pairs of species-specific and highly sensitive primers were selected.By PCR amplification with these primers and agarose gel electrophoresis,we may easily identify the origin of cell lines and find whether the tested cell lines are contaminated by cells of other species.Conclusion This PCR assay provides a simple,rapid,sensitive,and cost-effective method to identify cell species and detect interspecies cross-contamination.

Objective To investigate the effects and possible mechanisms of nicorandil on lung injury of the collapsed lung in one-lung ventilation.Methods Twenty-four clean Japanese big-ear rabbits were randomly divided into sham group (group S) (two-lung ventilation + thoracotomy),negative control group (group C) (one-lung ventilation + thoracotomy + saline),nicorandil group (group N) (one-lung ventilation+thoracotomy+nicorandil) and antagonist group (group J) (onelung ventilation+ thoracotomy+ nicorandil+ glyburide) equally.Mechanical ventilation was implemented through self-made double-lumen endotracheal tube after intravenous induction through ear marginal vein.Intravenous maintenance medicine was infused by trace injection pump after anesthesia induction.Thoracic surgery was simulated through one-lung or two-lung ventilation determined by auscultation,bubble test and direct observation.Then wet and dry weight ratio (W/D) and content of MDA were measured after non-ventilatory lung was processed and preserved.The expression of Akt,p-Akt and NF-κB protein in non-ventilatory lung tissue were detected by Western-blot in all groups.Results In respects of W/D and content of MDA,the other three groups had significant differences compared with group S (P ＜ 0.05).It was significantly lower in group N than in group C (P ＜0.05),and it was significantly higher in group J than in group N (P＜0.05).The expressions of pAkt protein and p-Akt/Akt in group N were significantly higher than those in group S and group C (P＜0.05).Those of group J were significantly lower than group N (P＜0.05).Compared with group S,the expression of NF-κB protein in group C was significantly higher (P＜0.05).That of group N was significantly lower than that of group C (P＜0.05).But that in group J was higher than that in group N (P ＜ 0.05).Conclusion Nicorandil has a protective effect on the collapse and inflation of non-ventilatory lung in rabbits under one-lung ventilation,acting on mitoKATP through PI3K/Akt,and down-regulating NF-κB to reduce IR-induced lung injury.

Objective To investigate the effects of nicorandil on hypoxia-inducible factor (HIF)-1α mRNA and protein in lung tissue of one-lung ventilation.Methods Twenty-four clean New Zealand white rabbits were randomly divided into sham group (group S) (two-lung ventilation+thoracotomy),negative control group (group C) (one-lung ventilation + thoracotomy + saline),nicorandil group (group N) (one-lung ventilation+ thoracotomy+ nicorandil) and antagonist group (group J) (one-lung ventilation + thoracotomy + nicorandil + glibenclanide) equally.The implementation of mechanical ventilation depended on self-made double-lumen endotracheal tube after intravenous induction through ear marginal vein.Intravenous maintenance medicine was infused by trace injection pump after anesthesia induction.The implementation of thoracic surgery was simulated through one-lung and two-lung ventilation by auscultation,bubble test and direct observation.Group S was given anaesthesia only,no one-lung ventilation group S,the other three groups had single lung ventilation,and the drug was injected before the operation.Group N was infused nicorandil 100 ptg· kg-1 · h-1 before the implementation of single lung ventilation for 1 h.Group C was injected with the same amount of normal saline.Group J was intravenous infusion of glibenclamide 75 μg· kg-1 · h-1 and nieorandil 100μg · kg-1 · h-1 the implementation of single lung ventilation for 1 h.Then wet and dry weight ratio(W/D) and superoxide dismutase (SOD) activity were measured after non-ventilatory lung was processed and preserved.The expression of HIF-1α protein of non ventilatory lung tissue was detected by Western-blot in the four groups.The transcription of HIF-1α mRNA was detected by real-time quantitative real-time PCR (qRT-PCR) in all groups.Results W/D in groups C and J were significantly higher compared with that of groups S and N (P＜0.05).The activity of SOD in groups C and J was significantly lower compared with groups S and N (P＜0.05).The expression of HIF1α protein and transcription of HIF-1α mRNA in groups C,N and J were significantly higher than those in group S,and that of group N was significantly higher than those of groups C and J (P＜0.05).Conclsion Nicorandil has a protective effect on the collapse and inflation of non-ventilatory lung in rabbit with one-lung ventilation,reducing oxidative stress by SOD,acting on mito KATP and coming into play by up-regulation of HIF-1α.

Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.

Objective: To clarify the virus hosting status of human cell lines. Methods: The DNA of Epstein-Barr virus (EBV), Hepatitis B virus (HBV), Human pappilomavirus (HPV), Human immunodeficiency virus (HIV), Human T-cell leukemia virus (HTLV) in 135 human cell lines were checked using PCR, and HCV RNA sequences were checked by RT-PCR. The transmission electron microscopy (TEM) was used to examine the virus particles in cells. The high-risk genotypes of HPV were tested by PCR. Results: By PCR assaying, among the 135 human cell lines, four cell lines(Daudi, Raji, EB-3, NCI-BL2009) harbored EBV DNA sequences; two(Hep3B, PLC/PRF/5) harbored HBV DNA sequences; seven (HeLa, HeLa 229, H1HeLa, HeLaS3, SiHa, Caski, CNE-2Z) harbored HPV DNA sequences, including two cell lines (SiHa, Caski) with HPV16, five cell lines(HeLa, HeLa 229, H1HeLa, HeLaS3, CNE-2Z) with HPV18; one cell line(HUT 102) harbored HTLV DNA sequences. No cell line harbored HCV RNA sequences and HIV DNA sequences. No viral particles were observed in the positive cell lines by TEM, but some viral inclusion bodies in certain cell lines. Conclusions The virus hosting status of human cell lines can be checked by PCR or RT-PCR. The viral DNA sequences were integrated in the cellular genome.

Objective: To construct the third generation chimeric antigen receptor based on a novel humanized anti-HER2 H1-2 scFv, and to investigate the specific cytotoxicity of H1-2 CAR modified T lymphocytes(CAR-T) against HER2⁺ tumor cells. Method: The expression cassette of the third generation CAR gene and anti-HER2 H1-2 scFv were constructed and cloned into lentivirus transfer plasmid, and then the third generation H1-2 CAR was transduced into human T lymphocytes using lentivirus.Enzyme linked immunosorbent assay was used to detect the expression of cytokines IL2, and LDH release assay was used to detect the cytotoxic effect of the H1-2 CAR-T.Finally, NOD/SCID mice and HER2⁺ breast cancer cell line SKBR3 were used to detect the anti-tumor effect of H1-2 CAR-T in vivo. Results: The third generation H1-2 CAR was successfully constructed.H1-2 CAR-T secreted high dose of IL2 after confrontation with HER2⁺ breast cancer cells.In vitro, the cytolytic rate of H1-2 CAR-T on high expression HER2⁺ tumor cells was significantly higher than that in low expression HER2 or non-expression HER2 tumor cells. At the efficacy to target ratio of 20, the cytolytic rate of H1-2 CAR-T against breast cancer cell SK-BR-3 could reach (90.1±2.8)%, while the cytolytic rate of H1-2 CAR-T against HER2^- breast cancer cell MDA-MB-231 was only (13.5±4.7)%. In the mouse xenograft tumor model, H1-2 CAR-T cells inhibited breast cancer growth in vivo.At the end of the experiments, the average tumor weight in the H1-2 CAR-T cell treatment group was (0.7±0.1) g, the non-transfected T cell therapeutic group was (1.2±0.2) g, and the PBS group was (1.2±0.2) g. There was significant difference between the H1-2 CAR-T therapeutic group and the non-transfected T cell therapeutic group (P<0.05). However, there was no significant difference between the non-transfected T cell therapeutic group and the PBS treatment group (P>0.05). Conclusion The HER2-sepcific H1-2 CAR-T cells specifically kill HER2 positive cells, and further studies on CAR-T cells for the treatment of HER2⁺ cancers are useful.

Objective To establish human and mouse pancreatic cancer cell strains stably expressing Cas9 protein,green fluorescent protein,red fluorescent proteins,luciferase-tdTomato,and to validate the activity of luciferase and gene editing of Cas9 function for pancreatic cancer research using luciferase and CRISPR/Cas9 system.Methods In human pancreatic cancer cells(AsPC-1,CFPAC-1,HPAC,BxPC-3,HS 766T,MIA PaCa-2,PANC-1,and SW 1990),and mouse pancreatic cancer cell(Pan02),the cells were infected with Cas9-expressing plas-mid pLv-EF1α-Cas9m1.1-Puro,and single-cell clones were selected for culture and expansion.After extracting the total protein,Western blot verified the expression level of Cas9;Infected with fluorescent protein expression plasmids pLv-EF1α-EGFP,pLv-EF1α-mCherry,pLv-EF1α-tdTomato,pLv-EF1α-Luc2-tdT,and selected single cell clones stably expressing fluorescent proteins were cultured and amplified under fluorescence microscope.Cas9 stable expression cell line was selected to be infected with pLv-EF1α-Luc2-tdT,and the mono-clonal culture of stable expression of fluorescent proteins was selected for expansion under fluorescence micro-scope.One of the cell lines were selected to be infected with Lv-EF1a-mCherry,and the mCherry-positive cells were sorted out by flow cytometry,and then the guide RNA of mCherry gene was then infected by lentivirus to tar-get the mCherry gene,and after cell expansion,mCherry knockdown was detected by fluorescence microscope observation and flow cytometry;5 BALB/c Nude mice were subcutaneously inoculated with MIA PaCa-2-Luc2-tdT cells(1.0×107/cells each),and imaged in vivo after 36 days.Results 48 human pancreatic cancer cell strains with stable Cas9 expression were screened(including 23 cells expressing Cas9m1.1,25 cells expressing Cas9m1.1-Luc2-tdT),33 pancreatic cancer cell strains with stable expression of fluorescent proteins were screened(8 cells expressing EGFP,7 expressing mCherry,and 9 each expressing Luc2-tdT and tdTomato).Cells expressing mCherry and Cas9 were infected with mCherry gRNA and mCherry was knocked down.In vivo imaging showed that both bioluminescence and fluorescence luminescence were present in MIA PaCa-2 cells ex-pressing Luc2-tdT.Conclusions 33 pancreatic cancer cell strains with stable expression of fluorescent proteins are successfully established,in which the Luc2-tdT-expressing cell strains have luciferase activity;48 pancreatic cancer cell strains with stable expression of Cas9 are successfully established,and the Cas9 protein has gene edi-ting activity,gene editing activity varies depending on the original cell strains.

OBJECTIVETo evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms.

METHODSA549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot.

RESULTSBoth GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax.

CONCLUSIONSFor single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.

Apoptosis ; Benzimidazoles ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin B1 ; Drug Synergism ; Enzyme Inhibitors ; Humans ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins p21(ras) ; Signal Transduction ; TOR Serine-Threonine Kinases ; ras Proteins ; metabolism