Objective To explore the effect of temozolomide on apoptosis and molecular mechanism in small cell lung cancer (SCLC) cell H446. Methods The effect of temozolomide on the viability of H446 cell was measured by MTT assay.The effect of temozolomide on cell cycle was detected by flow cytometry.The activation of phosphatidyl inositol 3-kinase (PI3K)/AKT signaling pathway and expression level of downstream target genes (Cyclin B1), cell division cycle 2 (Cdc2), Bax, Bcl-2 and Survivin were detected by western blot.Results Temozolomide (50, 100, 200 μmol/L) could inhibit H446 cell viability, and the inhibitory rate was highest at 48 h.Moreover, temozolomide made H446 cell cycle arrested in G2 phase.Western blott showed the expression of PI3K, Cyclin B1, Cdc2, Bcl-2, Survivin and the phosphorylation of AKT were reduced, but the expression of Bax were increased by temozolomide.Conclusion Temozolomide could induce SCLC cell H446 apoptosis via blocking PI3K/AKT signal pathway.

We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells,and purified and evaluated for its serological activities.A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide (EAAAK)2 and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The expressed recombinant protein was purified with 6× His purification media,and identified by SDS-PAGE and Western blot,and its antigenicity was analyzed by using an indirect ELISA assay.The recombinant expression vector pET28a-rE was constructed and expressed in BL21 (DE3) successfully,but the recombinant proteins mainly appeared as inclusion bodies.The target protein was obtained with high purity through the purification of affinity chromatography.SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.The established indirect ELISA has high accuracy.This recombinant peptide antigen expressed in E.coli has good potential for serum testing.

Objective To study the cytotoxicity of bi-chimeric antigen receptors modified T lymphocytes (BiCAR-T) on the human acute myeloid leukemia (AML) cell line HL60 in vitro and the anti-tumor effects of BiCAR-T on the NOD SCID mouse model of AML in vivo.Methods The BiCAR-T were prepared and the expression of chimeric antigen receptor (CAR) of prepared BiCAR-T was analyzed by flow cytometry.In vitro study was divided into two groups:the experiment group (BiCAR-T) and the control group (T lymphocyte).The killing rate of BiCAR-T in vitro on HL60 cells was determined by CCK8 assay and the level of interferon-γ (IFN-γ) secreted from BiCAR-T co-culturing with HL60 cells for 48 hours was detected by enzyme linked immunosorbent assay (ELISA) at different effect/target ratios (5∶1,10 ∶ 1,20 ∶ 1).The NOD SCID mice AML model was established by the injection of HL60 cells through tail vein and used to assess the antitumor effects in vivo.The mice were randomly divided into three groups according to the random number table:the blank control group receiving 0.9％ NaCl 0.2 ml through tail vein,the model group and the treatment group receiving 1 × 107 HL60 cells in 0.2 ml phosphate buffer saline (PBS).After 20 days,the treatment group was injected with 2 × 107BiCAR-T in 0.2 ml PBS 3 times a week for 2 weeks,while the other two groups received 0.9％ NaCl 0.2 ml.The pathological changes in the mice livers and spleens were observed after 2 weeks of treatment.Results The CAR expression rates of BiCAR-T were more than 50.00％.In vitro experiments proved that the killing rates of BiCAR-T in the experimental group and T lymphocytes in the control group on HL60 cells were (25.43 ±1.32)％ vs.(16.18 ±0.75)％,(50.33±3.11)％ vs.(25.47±1.27)％,and (85.89 ± 3.96) ％ vs.(49.45 ± 2.77) ％ at different effect/target ratios (5 ∶ 1,10 ∶ 1,20 ∶ 1).The killing efficiency of BiCAR-T and T lymphocytes on HL60 cells was significantly different (F =404.17,P ＜ 0.001);the killing efficiency of BiCAR-T and T lymphocytes on HL60 cells was significantly different at different effect/ target ratios (F =548.09,P ＜ 0.001);and the killing efficiency on HL60 cells in the experimental group (BiCAR-T) was significantly higher than that in the control group (T lymphocytes) at different effect/target ratios (F =45.36,P ＜ 0.001).The IFN-γlevels secreted from BiCAR-T in the experiment group and T lymphocytes in the control group co-culturing with HL60 ceils after 48 h were (435.65 ± 20.44) pg/ml vs.(356.75 ± 19.87) pg/ml,(1 639.98 ± 95.75) pg/ml vs.(1 109.37 ± 80.98) pg/ml,and (3 467.43 ± 187.54)pg/ml vs.(2 245.52 ± 112.66)pg/ml.The IFN-γlevel in the experiment group (BiCAR-T) and the control group (T lymphocytes) was significantly different (F =156.24,P ＜ 0.001);the IFN-γ level was significantly different at different effect/target ratios (F =857.67,P ＜ 0.001);the IFN-γlevel in the experimental group (BiCAR-T) was significantly higher than that in the control group (T lymphocytes) at different effect/ target ratios of 5 ∶ 1,10 ∶ 1,20 ∶ 1,respectively (F =46.31,P ＜ 0.001).The result of hematoxylineosin staining (HE) staining showed that leukocyte infiltration in the treatment group was significantly decreased compared with the model group.Conclusion The experimental results showed that BiCAR-T is a kind of efficient targeted immunocyte modified by gene engineering,and it can significantly inhibit leukocyte infiltration of AML in vivo and in vitro.