Objective To study the relationship between genetic polymorphisms of cytochrome P450 2E1(CYP2E1) and susceptibility to gastric cancer. Methods Genotype of CYP2E1 was determined by polymorphism (PCR-RFLP) analysis on DNA in 92 patients with gastric cancer and 92 controls in case-control study. Results The results showed that the frequency of the wild-type genotype (C1/C1) detected by RsaⅠ digestion was 66.3% and 48.9% in gastric cancer group and controls, respectively (P

In INS-1 cells, the insulin secretion was investigated by radioimmunoassay (RIA) after 4 h incubation in medium containing different concentrations of glucose and recombinant human glucagon-like peptide-1 (rhGLP-1) (7-36). Insulin mRNA level in INS-1 cells was assessed by a semi-quantitative RT-PCR method. rhGLP-1 (7-36) is not only a powerful insulin secretagogue, but also can increase insulin gene expression in INS-1 cells.

Objective:To explore the involvement of endoplasmic reticulum molecular chaperone GRP78 in the impairment of inner ear consistented with the mimetic aging model.Method:Twenty-four Wistar rats were randomly divided into two groups. model group was in duced by daily hypodermic injection of 10% D-galactose (800 mg·kg~(-1)·d~(-1)) for 8 weeks and the control group was given saline accordingly. Spatial learning and memory was measured by Morris-Water-Maze. Colorimetry was used to analyze superoxide dismutase (SOD) and malondialdehyde (MDA) extracted from inner ear tissue. Hearing threshold of rats were detected with Auditory brainstem response (ABR).In addition, expression of GRP78 in the inner ear was detected by immunohistochemistry,RT-PCR and Western blot. The control group was studied parallel.Result:The escape latency in the model group injected with D-galactose was markedly longer than that in the control group.accordingly ,the changes of SOD and MDA were more significant in the model group, the difference between two groups was significant(t-test,P<0.01). the variation of ABR in two groups was observed, There was no statistically difference of the hearing in the model group compared with the control group(P>0.05). The expression of GRP78 was significantly different between two groups ,which is increased in the inner ear tissue of model group(P<0.01).Conclusion:The impairment of inner ear tissue partly dued to the oxidative stress in the model, which was induced by D-galactose.and endoplasmic reticulum molecular chaperone was thought to contribute to the impairment mechanism of inner ear in mimetic aging model.

Objective:To investigate the Migration ability toward human pancreatic carcinoma cell line and human colon carcinoma cell line with difference HSP 70 plasma membrane expression .Methods: CD3-CD56+NK cells were obtained from human peripheral blood mononuclear(PBMC)in stem cell growth medium SCGM,2μg/ml TKD was added to the medium on 10th day,the ac-tivating receptor CD94/NKG2C expression levels on NK cells was detected with FAC after 4 days.The human pancreatic carcinoma cell line Colo357 and the human colon carcinoma cell line CW 2 were separated into Colo+and CW2+with high HSP70 expression and Colo-and CW2-with low HSP70 expression;Migration assays of NK to the four difference cell lines were performed in a Transwell cell culture system.The cytolytic activity of TKD-activated NK cells against the four subline with HSP 70 expression on their cell surface was analyzed by MTT assay.Results:Flow cytometry analysis showed that CD 3-CD56+NK cells could expanded after 2 weeks in SCGM medium,and the largest percentage of NK cell was (92.50 ±1.25 )%.CD94 expression levels on NK cells increased obviously after TKD inducement the cell surface HSP 70 expression of Colo+, Colo-were ( 78.2 ±2.2 )% and ( 27.3 ±1.2 )% separately , the cell surface HSP70 expression of CW2+,CW2-were (91.1±2.5)%and (18.2±1.0)%separately after FACS;the Migration of NK cells toward Colo+was (68.6±2.8)%,higher than the migration toward Colo-with (22.8±1.5)%;the Migration of NK cells toward CW2+was(73.5±2.7)%,higher than the migration toward CW2-with (18.2±1.3)%;the cytolytic activity of NK against Colo +was(61.2± 3.0)%compared to (24.5 ±1.5)%against Colo-when the ratio of effector cells and target cell was 20 ∶1,the cytolytic activity of NK against CW2+was (63.8±3.2)%compared to (22.4±1.8)% against CW2-when the ratio of effector cells and target cell was 20∶1.Conclusion:TKD-activated NK cells are highly efficient cytolytic effector cells which have stronger significant migration toward HSP70-positive tumor target cells on their cell surface in vitro .

Objective:To investigate the antitumor effect of TKD-activated NK cells on tumor growth inhibition of human pancreatic carcinoma in nude mice .Methods:CD3-CD56+NK cells were obtained from human peripheral blood mononuclear ( PBMC) in stem cell growth medium SCGM , 2 μg/ml TKD was added to the medium on day 10.The activating receptor CD 94/NKG2C expression levels on NK cells was detected with FAC after 4 days.The cytolytic activity of TKD-activated NK cells against human pancreatic carcinoma subline with HSP 70 expression on their cell surface was analyzed by MTT assay .Established a new model of orthotopic-transplantation tumor of human pancreas .NK cells were injected i.v.into the tail vein of tumor-bearing mice on day 15,the antitumor activity of the NK were evaluated .The capacity to infiltrate Colo 357 tumors in SCID/beige mice was detected with Immunohis-tochemistry.Results:Flow cytometry analysis showed that CD3-CD56+NK cells could expanded in SCGM medium ,and the average percentage of NK cell was (87.50 ±1.35 )%.CD94 expression levels on NK cells increased obviously ,the mean fluorescence intensity of CD94 was(220.56±1.82),compared to (68.72±1.85)of control group cell.The cytolytic activity against HSP70 membrane-positive pancreatic carcinoma sublines Colo 357 cells was high and there was significantly statistical difference between TKD-activated NK cells and unactivated NK cells.The cytolytic activity was(68.72±2.55)%when ratio of effector cells and target cell was 40:1.TKD-activated NK cells had a stronger suppressive effect on tumor growth in BALB /c nude mice bearing Colo 357 cells in vivo ,Median inhibitory rates was ( 61.3 ±1.5 )% .There was significant statistical difference compare to control group ( P <0.01 ) .The result of Immunohistochemistry indicated that predominantly NK cells induced with TKD had the capacity to infiltrate Colo 357 tumors in SCID/beige mice.Conclusion: TKD-activated NK cells are highly efficient cytolytic effector cells which have a stronger significant suppression against pancreatic carcinoma growth in vivo .

Objective: To provide an effective hGM CSF gene transferring vector mediated by gene gun and a basis for study of hGM CSF gene modified tumor cell vaccines. Method: The gastric tumor cell line (SGC) was transfected with eukarytic expression plasmid deoxyribonucleic acid containing the human granulocyte macrophage colony stimulating (hGM CSF) gene using the gene gun. The SGC cell clones (SGC GM CSF 1～5)secreting high hGM CSF level were obtained after G418 resistance selection. The hGM CSF gene had been integrated into chromatosome of SGC by the assay of RT PCR.Results: There was hGM CSF production whose lane was about 30 kD in the culture medium of SGC GM CSF by the assay of SDS PAGE and Western blot. SGC GM CSF had the ability of the high level of GM CSF for a long time(mean 247ng/(10 6 cell?24 h).Conclusion: The hGM CSF gene transferring vector mediated by gene gun was effective and safe. These results provide a basis for study of GM CSF gene therapy for cancer.

Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.

Objective:To investigate the mechanism of intense noise-induced cochlea cells death in guinea pig,and the effect of JNK signal transduction pathway in the procedure of cochlea cells apoptosis by intense noise-induced.Method:Thirty-two guinea pigs were randomly divided into 4 groups.The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h.After the noise expose for 1,4,14 days of the experiment guinea pigs,ABR of the guinea pigs on experiment and control groups were tested before put them to death.Four guinea pig's cochleas of every group were taken to paraffin section,and the rest was extracted the total cochlear's protein.Apoptosis was tested by terminal deoxynucleotidyl Transferase(TdT)-mediated deoxyuridine triphosphate(d-UTP)nick and labeling method(TUNEL).The phosphorylation of JNK and c-Jun were tested by immunohistochemistry and western blot methods.Result:Tunel-Positve cells in the Corti's,SGC and SV of experiment groups,and there have significant differences compared with the control group(P＜0.01)and Tunel-Positve cells are most in 1 d experiment group.The positive cells of P-JNK and p-e-Jun could be dectected in guinea pig's cochleas after noise exposed,but no positive cells were found in the control.Protein levels of p-JNK,and P-c-Jun were risen up and activated quickly after noise exposed,and achieved peak in 1 d,4 d,and then fallen-offs,but still maintained higher levels within 14 d.Conclusion:Intense noise causes cochlea cell lesion by inducing apoptosis to result in and JNK signal transduction pathway plays an important role in the procedure of apoptosis.

Objective To investigate the difference of apparent diffusion coefficients (ADCs) changes in three major salivary glands after gustatory stimulation using two different stimuli. Methods Thirty healthy volunteers were examined with a 1.5 T MR unit. A diffusion-weighted MR imaging (MR DWI) sequence was performed once at rest and continuously repeated 13 times after gustatory stimulation using a commercially available lemon juice and vitamin C tablets in the same volunteer by using self-controlled method. The subsequence of two stimuli was random. In addition, the salivary flow rates at rest and after stimulation were measured. Characteristics and differences in ADCs curves of three salivary glands before and after stimulation between two stimuli were analyzed. Comparison of maximum ADCs, maximum ADCs increase rates (IRs) and times to maximum ADCs(Tmax) between two stimuli was performed by using independent-samples t test. Correlation analysis between rest salivary flow rates and rest ADCs, the maximum salivary flow rates and ADCs after stimulation, the maximum salivary flow IRs and ADC IRs after stimulation were performed by using Pearson correlation test. Results In lemon juice stimulation group, the mean ADCs mostly showed a steady increase to peak values during the first DW MRI scan after stimulation in all glands, followed by a gradually decrease fluctuating slightly around the baseline values. In vitamin C stimulation group, the mean ADCs were significantly increased in all glands during the first DW MRI scan after stimulation, followed by a gradual upward trend till peak values. In lemon juice stimulation group, the mean Tmax of submandibular and sublingual glands[(184±122)s, (345±232)s, respectively] were significantly earlier than those[(454 ± 301)s, (528 ± 297)s, respectively] in vitamin C stimulation group (t=-3.517 and-2.548 respectively, P<0.01 for all). The mean maximum ADCs of three glands in lemon juice stimulation group[(1.05 ± 0.12) × 10-3 mm2/s, (1.22 ± 0.10) × 10-3 mm2/s and (1.26 ± 0.21) × 10-3 mm2/s, respectively] were all lower than those in vitamin C stimulation group[(1.13±0.13) ×10-3 mm2/s, (1.32±0.25) × 10-3 mm2/s and (1.57 ± 0.36) × 10-3 mm2/s, respectively], and the differences in parotid and sublingual glands between two groups were significant(t=-2.894 and-3.681 respectively, P<0.01 for all). The mean maximum ADC IRs of three glands in lemon juice stimulation group[(11.35±4.07)%, (8.81±5.40)%, (34.08±21.66)%, respectively] were significantly lower than those[(17.80 ± 12.72)%, (18.16 ± 18.93)%, (67.49 ± 46.04)% , respectively] in vitamin C stimulation group (t=-2.252,-2.330 and-3.432 respectively, P<0.05 for all) . In two groups, the mean maximum ADC IRs of parotid and submandibular gland were all significantly lower than sublingual gland (t=-5.994 and-6.443 respectively, P<0.01 for all). No correlation was observed between ADCs and salivary flow rates, ADC IRs and salivary flow rate IRs in two groups (P>0.05). Conclusion MR DWI with transient stimulation using lemon juice is more stable for evaluating the physiologic changes of salivary glands in vivo.

In order to study the effect of 5, 6-Dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose-and time-dependent manner. After being treated with 0, 10, 20, 40, 80 microm mol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68+/-0.19)%, (1.95+/-0.12)%, (8.51+/-0.26)%, (11.26+/-0.17)% and (14.99+/-0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 microm mol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.