Immune checkpoint inhibitors have become an important alternative for advanced non-small cell lung cancer (NSCLC) patients to surgery, chemotherapy, radiotherapy and targeted therapy. Monoclonal antibodies directed against immune checkpoint have shown better results in the application of first- or second-line treatment of NSCLC and for both squamous and non-squamous cell carcinoma patients, especially for those with positive PD-L1 tumor cells. Some comments will be made in present paper about the efficacy, biomarker, combined therapy and the resistant mechanism of immune checkpoint inhibitors.

AIM: To study the mechanism of butylated hydroxyanisole-induced neural differentiation of fetal liver Sca-1~+ cells in vitro. METHODS: Sca-1~+ cells from 14.5-day-old mouse fetal liver were isolated with a magnetic cell sorting kit. Cultured cells pretreated with or without extracellular signal-regulated kinase (MEK1) inhibitor, PD98059, were induced by 200 ?mol/L butylated hydroxyanisole (BHA) for 24 hours, and then incubated in serum-free medium. Expression of genes in treated or untreated cells were assayed by Western blotting and RT-PCR. RESULTS: There was low level of neuronfilament-L (NF-L) and brain factor-1 (BF-1) in fetal liver Sca-1~+ cells, but no neuronfilament-H (NF-H) and tyrosine hydroxylase (TH) was observed. BHA significantly promoted the expression of neuron-specific NF-L, NF-H, BF-1, and TH in fetal liver Sca-1~+ cells. NF-L, NF-H, BF-1 and TH increased by 6.32 fold, 2.73 fold, 3.37 fold and 2.68 fold, respectively (all P

AIM: To explore the effect of human telomerase reverse transcriptase (hTERT) gene antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) on cisplatin-induced apoptosis in cultured primary acute lymphoblastic leukemia (ALL) cells. METHODS: The expression levels of hTERT protein were detected by immunofluorescence using fluoresce isothiocyanate (FITC) lable. Cell surviving fraction was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis. RESULTS: The expression of hTERT protein was inhibited after treated by hTERT AS PS-ODN. Treatment with cisplatin after 24 h of exposure to AS PS-ODN had significantly reduced the number of viable ALL cells. However,there was no difference on ALL cells survival between sense oligodeoxynucleotide (S PS-ODN) /CDDP combination and CDDP-treated cells alone. In morphological observation of apoptotic cells using Hoechst 33258 and PI double staining techniques,cells displayed classic apoptotic changes treated with CDDP or CDDP combined with hTERT AS PS-ODN or S PS-ODN at 48 h. Apoptotic rates of cells added CDDP and AS PS-ODN were higher than that of cells added CDDP only ( P 0.05). CONCLUSION: hTERT AS PS-ODN inhibited the expression of hTERT protein and increased the CDDP-induced apoptosis in primer acute lymphoblastic leukemic cells.

AIM: To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS: Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS: The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA “ladder”. Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION: The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.

AIM: To explore the effect of hTERT gene antisense phosphorothioate oligodeoxynucleotide (ASODN) on telomerase activity in Raji cells. METHODS: Polymerase chain reaction enzyme-linked immuonassay (PCR-ELISA) was used to determine telomerase activity. The expression levels of hTERT mRNA and protein were assayed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence using fluoresce isothiocyanate (FITC) label,respectively. RESULTS: RT-PCR and immunofluorescence assay showed that the expression levels of hTERT mRNA and protein from Raji cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT mRNA and protein levels between control and SODN-treated cells. Telomerase activity decreased when Raji cells were treated with ASODN for 48 h. Telomerase activity of Raji cells was significantly inhibited when treated with ASODN for 72 h. There was no difference in telomerase activity levels between control and hTERT SODN-treated cells. CONCLUSION: hTERT ASODN could inhibit telomerase activity in Raji cells.

Objective To observe the effects of low frequency electrical stimulation (LFES) on the proliferation of endogenous brain neural stem cells (NSCs) and on the expression of basic fibroblast growth factor (bFGF)and epidermal growth factor (EGF) in rats with acute cerebral infarction; to explore the therapeutic mechanism of LFES in improving neural function. Methods Fifty-four rats were randomly divided into a LFES group, a placebo stimulation group and a sham-operated group. Each group was further divided into 3rd day, 7th day and 14th day subgroups, with 6 rats in each subgroup. An acute cerebral infarction model was induced in the rats of the LFES and placebo stimulation groups by middle cerebral artery occlusion (MCAO). Three days after the operation, rats in the LFES group began LFES treatment (frequency 30 Hz, pulse width 250 μs, current intensity 3 mA, 10 min/d) ,while the placebo stimulation group was treated identically but without electricity. The rats in the sham-operated group had no special treatment. The expression of nestin positive cells in the subgranular zone of the hippocampus and the subventricular zone was detected by immunohistochemical staining. The expression of bFGF, EGF proteins and mRNA in the ischemic hemisphere was detected by Western blotting and RT-PCR analysis. A screen test was applied to evaluate motor function. Results Nestin-positive cells in the subgranular and subventricular zones of rats in the LFES group increased significantly more than in the placebo stimulation group at the 7th and 14th day. The expression of bFGF, EGF proteins and mRNA in the ischemic hemisphere was up-regulated compared to the placebo stimulation group at the 7th and 14th day. At the 14th day a difference in motor function was observed in rats in the LFES group compared with the placebo stimulation group. Conclusion LFES can promote the proliferation of endogenous brain NSCs and the expression of bFGF and EGF in rats with acute cerebral infarction. It can also improve motor function and enhance neural plasticity in the brain.

The paper is to establish a method for simultaneous determination of 5 kinds of alkaloids in ephedra and poppy which are in Kechuanning tablets. Solid-phase extraction (SPE) was adopted in pretreatment, and a UPLC method with 2 different wavelengths had been developed: 210 nm for the detection of morphine, codeine phosphate, ephedrine hydrochloride and pseudoephedrine hydrochloride, and 251 nm for papaverine hydrochloride. The column used was Acquity UPLC BEH C18 (100 mm x 2.1 mm ID, 1.7 microm) with linear gradient elution using acetonitrile and 0.1% phosphoric acid. The flow rate was 0.4 mL.min-1, and the column temperature was 30 degrees C. The linear response range was 0.375 0 - 12.50 microg.mL-1 for morphine, 0.064 32 - 2.144 microg.mL-1 for codeine phosphate, 0.030 06 - 1.002 microg.mL-1 for papaverine hydrochloride, 1.126 - 37.52 microg.mL-1 for ephedrine hydrochloride, 0.287 8 - 9.592 microg.mL-1 for pseudoephedrine hydrochloride (r = 0.999 7). The average recoveries of these compounds were 99.26%, 100.6%, 95.29%, 100.1% and 97.48%, respectively. This is a more reasonable and credible method of quality control for Kechuanning tablets.

Objective To obtain the specific antigens of the expressed major capsid proteins from Noroviruses with different sub-genotypes in Beijing area. Methods The full-length genes of the major capsid proteins (VP1)were obtained through the amplification of the VP1 encoding gene in the recombinant plasmids pBST-CR2987(G Ⅱ-3)and pBST-CR2932(GⅡ-4),which represented different Norovirus geno-types. The full-length genes were sub-cloned into the expression vector pET-30a(+),resulting in a recombinant plasmid, with which the BL21 competent cells were transformed, and the expression of the gene was induced by adding IPTG to the growth culture. The expression of the major capsid proteins were analyzed with Coomassie blue staining after SDS-PAGE, and assayed by Western blot with serum from human. Results (1)The major capsid protein genes of CR2987 and CR2932 were sub-cloned into expression vector pET-30a(+). The VP1 encoding genes were 1647 bp in length for CR2987 and 1623 bp for CR2932. The open reading frames(ORF)coded for 549 and 541 amino acids for these two proteins, respectively. (2)The expressed VP1s were present primarily as inclusion bodies,and the maximal amount of the expressed proteins occurred at 4-6 h after IPTG induction.(3)These VP1s could be recognized by specific immune serum against VP1 of Norovirus as well as His-tag antibody. Conclusion The VP1s of CR2987 and CR2932 are expressed in BL21 E.coli cells.The expressed VP1s could react with specific immune serum against VP1 of Norovirus, indicating that the expressed VP1s are of antigenicity.

Objective To understand the undergraduate nursing students’ awareness of the basic situation of occupational exposure in Kunming Medical University, and to understand their knowing status of occupational exposure knowledge. Methods We used self- designed questionnaire to investigate 449 undergraduate nursing students in Kunming Medical University to obtain the status of occupational exposure and protection knowledge level of students.Results (1) 449 students were investigated, and 258 of them had been in clinical practice, and occupational exposure occurred in 48.8% of them. (2) Occupational exposure factors included grade, washing hands and wearing gloves;The results showed that:Grade was a risk factor and wearing glove and washing hands were protective factors. (3) There was relevance between accepting the occupational protection education and the occupational exposure. Conclusions A useful measure to reduce the occurrence of occupational exposure is Pre-service training for undergraduates nursing students. Operation should be in strict accordance with operating specifications. Schools and departments should implement occupational protection system, so as to strengthen the related knowledge vocational education,enhance self-protection awarenes,and reduce or minimize the occurrence of occupational exposure.

Objective To study the effect of gastrin on the proliferation and angiogenesis of human umbilical vascular endothelial (HUVE) cell in vitro.Methods The immunocytochemistry assay,realtime-PCR,and Western blot were used to detect the gastrin receptor (CCK-BR) expression in HUVE cells.After HUVE cells were treated with 10 and 100 nmol/L gastrin for 72 h,MTT and soft agar colony formation assay were used to test the cell proliferation rate and colony formation rate,and the vascular-like structures were observed by three-dimensional culture of HUVE cells.The half-ring and ring vascular numbers were counted with five random visions.The vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1a (HIF-1a) mRNA and protein levels in HUVE cells were detected by realtime PCR and ELISA assay respectively.Results HUVE cells expressed CCK-BR.After the treatment with 10 and 100 nmol/L gastrin,the cell proliferation rate was increased by 48.48％ and 82.82 ％ compared to the control group,and colony formation rate was increased to 31.33％ and 45.67 ％ as compared with 15.33％ in the control group(P＜0.01).Relative expression quantities of VEGF and HIF-1a genes were 2.3 and 4.6 folds (VEGF) and 20.76 and 26.77 folds (HIF-1 a) than those in the control group.The concentration of VEGF protein in culture medium was 221 and 392 μg/mg protein higher than that in control group.The numbers of half-ring and ring vascular structures were (14.00 ± 3.00,39.33 ± 7.57 and 34.33 ± 4.50)/vision and (8.33 ± 2.51,41.33 ± 5.85 and 37.67 ± 3.51)/vision in control,10 and 100 nmol/L gastrin-treated groups,respectively (P＜0.01).Conclusion Gastrin up-regulates the expression of VEGF and HIF-1a genes in HUVE cells and promotes cell proliferation and vascular-like structure formation of HUVE cells in vitro by being combined to CCK-BR,which may be involved in the development and metastasis of gastric cancer.