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

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 investigate the NAD+ - dependent protein deacetylase SIRT1 expression in the cochlea of C57BL/6 mice ,a mouse model of age - related hearing loss(AHL) .Methods A total of 46 C57BL/6 mice were used ,and were divided into 2 groups ;according the age ,there were young group (1 ~ 2 months old ,23 mice) and old group (12 ~ 16 months old ,23 mice) .ABR measurements were performed on young and old C57BL/6 mice . The expression of SIRT1 in the cochlea was detected by qRT - PCR and immunofluorescence .Results The ABR thresholds in the old mice(4 kHz :82 .7 ± 7 .32 dB SPL ,8 kHz :80 .9 ± 7 .8 dB SPL ,16 kHz :89 .3 ± 5 .5 dB SPL ,32 kHz :89 .9 ± 4 dB SPL) were significantly higher than those in the young C57BL/6 mice(4 kHz :52 .1 ± 8 .3 dB SPL ,8 kHz :40 .5 ± 6 .1 dB SPL ,16 kHz :50 .7 ± 9 .4 dB SPL ,32 kHz :57 .6 ± 11 .9 dB SPL)(P < 0 .001) .SIRT1 mRNA expression was significantly decreased in the cochlea of old C57BL/6 mice in comparison with young mice ( P <0 .01) .SIRT1 protein was abundantly expressed in the inner hair cells ,outer hair cells ,supporting cells ,strial marginal cells ,strial intermediated cells ,and spiral ganglion neurons .The positive area and the average flourescence intensity of SIRT1 protein were reduced in old C57BL/6 mice(P< 0 .001) .Conclusion The down - regulation of SIRT1 mRNA and protein expression in the older C57BL/6 mouse cochlea may be correlated with the pathogenesis of AHL .

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 establish an HPLC method for determination of gastrodigenin concentration in brain tissue of mice and investigate its pharmacokinetics after intragastric administration of gastrodin.Methods The brain homogenate was extracted with acetoacetate and analyzed by HPLC method.The separation was performed on a Diamonsil C18 column(250 mm ? 4.6 mm,5 ?m) under the following chromatographic conditions: mobile phase,acetonitrile-water(10.5∶89.5);column temperature,25 ℃;flow rate,1.0 mL/min;detection wavelength,221 nm;and sampling amount,20 ?L.Results The calibration curve showed good linearity within the concentration range of 50-1 616 ng/mL(r= 0.999 6).The relative recoveries were 93.8%-95.1%,and the RSDs of the intra-and inter-day precision were less than 10%.The concentration-time profile of gastrodigenin in brain tissue of mice showed double peaks(tmax1=15 min,tmax2=90 min).The AUC was 52 822.5 ng?min/g,and t1/2(ke) was 54.8 min.Conclusion The analytical method established for assay of gastrodigenin in brain tissue of mice is sensitive and accurate.The result indicates that gastrodin could rapidly distribute to the brain,be metabolized into gastrodigenin,and be eliminated after oral administration.

Objective To explore the possible mechanism of miR 34a/Bcl-2 apoptotic pathway in the mouse model of age-related hearing loss.Methods A C57BL/6 mouse model of age-related hearing loss was conducted,and 4-week-old and 12-month-old mice were considered as the objects.The auditory brainstem response (ABR) was used to test the hearing function.The TdT-mediated dUTP nick end labeling (TUNEL) was used to observe the apoptosis of neuron in auditory cortex.The mRNA and protein levels of miR-34a,Bcl-2 and caspase 3 were detected by real-time PCR and Western bloting,respectively.Results The ABR showed that the hearing threshold level at 4,8,16,32 kHz were higher in 12-month-old mice than in 4-week-old mice [(80.0±2.5) dBHL vs.(32.0 ±4.5) dBHL,(74.0±3.5) dBHL vs.(51.0±1.2) dBHL,(86.0±4.6) dBHL vs.(51.0±3.5) dBHL,(87.0±6.6) dBHL vs.(56.0±1.5) dBHL,all P＜0.05].Compared with 4 week-old mice,the total number of neurons in auditory cortex was decreased,the number of apoptosis neurons was increased,the expressions of miR-34a (t=6.02,P=0.001),Bax (t=6.51,P=0.012) and Caspase 3 (P=0.023) rised,and the expression of Bcl-2 (t=7.12,P=0.032) declined in 12 month-old mice.Conclusions The activation of miR-34a/Bcl-2 apoptotic pathway may be one of the mechanisms of age-related hearing loss.

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 explore the expression of Bcl-2, on mRNA and protein levels, in the different age of C57BL/6 mice cochleae and the expression localization in the cochleae.METHODS Using ABR to test the hearing level in C57BL/6 mice. Surface preparation of cochlear basil membrane is used to observe the morphology and amout of the outer and inner hair cells in different age of C57BL/6 mice. Fluorescent quantitative real time PCR, immunofluorescence histochemical method and western blot are used to detect the expression of Bcl-2 on the mRNA and protein levels in the C57BL/6 mice cochlea of different age groups ('young group', 'elderly group').RESULTS ABR results showed that the hearing threshold of 'older' C57BL/6 mice is much higher than that in the 'young' mice, and surface preparation of cochlear basil membrane showed the hair cell localized in the cochlear basil turn of 'old' mice arranged in a disorder station and part of hair cells were lost. Also, the spiral ganglion cells arranged sparsely and messily. Fluorescence quantitative real-time PCR results suggest the expression of Bcl-2 on/at the mRNA level of the 'old' mice cochleae decreases significantly, compared to that in the 'young' mice. The results of Immunofluorescence and Western blot suggest the expression of Bcl-2 on/at the protein levels of the 'old' mice cochlea decreased, compared to that in the 'young' mice. Also, the Bcl-2 is located in the cytoplasm, and the expression of Bcl-2 in the inner hair cells seems higher than that in the outer hair cells. CONCLUSION The expression of Bcl-2 significantly deceased in the 'old' C57BL/6 mice cochleae, both on mRNA and protein level, which may be related to the hearing loss and loss of hair cells.

OBJECTIVE: To investigate the risk factor,type and characteristic nystagmus of the otolith abnormal migration during diagnosis and treatment for posterior semicircular canal benign paroxysmal positional vertigo (PSC-BPPV). The therapy and prevention is also discussed. METHOD: Four hundred and seventy-nine patients with PSC-BPPV were treated by Epley's canalith repositioning procedures(CRP) from March 2009 to March 2012. We observed otolith abnormal migration complicating during diagnosis and treatment. According the type of otolith abnormal migration, the additional repositioning maneuver was performed. RESULT: The rate of complication was 8. 1%(39/479), with canal conversion in 5.4%(26/479) and primarily canal reentry in 2.7%(13/479). The rate of incidence of conversion to horizontal canal conversion and anterior canal were 4. 8%(23/479)and 0. 6%(3/479) respectively. All the patient was cured in follow up. The risk factors were unappropriated head movement during or after CRP, including another Dix-Hallpike were performed immediately. CONCLUSION To prevent the complications,the pathognostic positioning sequence and angle of head rotation are commenced during CRP. Appropriate short time postural restrictions post-treatment is necessary. Careful observation of nystagrnus variation is crucial to determine the otolith abnormal migration.
Benign Paroxysmal Positional Vertigo ; therapy ; Head ; Humans ; Incidence ; Nystagmus, Pathologic ; etiology ; Otolithic Membrane ; Patient Positioning ; adverse effects ; Semicircular Canals ; Vertigo