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 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 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 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.

OBJECTIVETo construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro.

METHODSTotal RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA.

RESULTSHuman γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, P<0.05) and elevated the adherence of SW1116 cells to HUVECs(3.08±0.36 vs. 1.22±0.21, P<0.05).

CONCLUSIONExpression of γ-synuclein can strengthen colon cancer cell SW1116 potentiality of invasion and metastasis in vitro.

Cell Adhesion ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Genetic Vectors ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; gamma-Synuclein ; genetics

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.
Animals ; Extracellular Vesicles ; metabolism ; Female ; Follicular Fluid ; chemistry ; Granulosa Cells ; drug effects ; MicroRNAs ; pharmacology ; Oogenesis ; drug effects