Objective To study the effect of substance P ( SP) on int racellular f ree calcium concent ration in human poorly-differentiated gast ric cancer cell in vitro. Methods Human gast ric cancer cell line MKN45 was cultured in RPMI 1640. Then the cells were loaded with specific calcium fluorescent probe Furu23/ AM. ASN21377642 (NK21 receptor antagonist) , Nicardipine (calcium channel blocker) and different concent rations of SP were used to treat gast ric cancer cells. The concent ration changes of int racellular free calcium were detected by laser scanning confocal microscope. Results It was found that 10 , 50 and 100 nmol/ L SP could significantly increase the int racellular free calcium concent ration of gast ric cancer cells in Hanks solutions , which contain ext racellular calcium ( P lt;0. 05) , and the change was in a dose-dependent manner ( P lt; 0. 05) . When there was ext racellular calcium existed ,the increasing amplitude of intracellular f ree calcium concent ration was significantly higher than that when there was no extracellular calcium ( Plt; 0. 05) . And when Hanks solutions were pretreated with ASN21377642 and Nicardipine , the effects of 100 nmol/ L SP were partly inhibited , and the concent rations of int racellular f ree calcium were significantly lower than those in group s without pret reatment s ( P lt; 0. 05) . Conclusion SP can significantly increase free calcium concent ration in the gastric cancer cells. Releasing of stored calcium in the cells and influx of extracelluar calcium may contribute to the elevation of int racellular free calcium concentration.
Objective To investigate the effect of CO2 pneumoperitoneum on the tumor cell port site implantation in laparoscopic surgery. Methods Male SpraqueDawley rats were intraperitoneally injected with gastric cancer cells (cell line SGC-7901). Continuous CO2 pneumo of 15 mm Hg or 30 mm Hg were established for 5 mins, 60 mins, 120 mins and 180 mins with the injection of different concentrations of tumor cells (104/ml, 106/ml respectively). Several samples of peritoneal washing served as positive control. All collecting dishes were incubated at 37℃ with 5% CO2 concentration for one week and then examined for the presence of tumor cell under microscope. Results After one week of incubation, some of the dishes with continuous flow of CO2 gas (5 L/min) at pneumo 30 mm Hg for 60 mins or longer demonstrated tumor growth, and all peritoneal washing samples showed tumor growth, while other dishes showed negative. Conclusion The research suggests that gastric cancer cells can cause port site implantation and the concentration of tumor cells, pneumoperitoneum pressure and duration may affect the occurrence of port site implantation. It may help to find a suitable way to prevent the port site implantation in operations.
目的 研究CD14过表达对胃癌细胞核转录因子-κB（NF-κB）活性以及人β防御素-2（hBD-2）表达的影响，探讨CD14在胃癌发生发展中的作用。 方法 体外培养CD14稳定转染的胃癌SGC-7901细胞系及空质粒转染的对照细胞，CD14蛋白受体胞壁酰二肽刺激细胞，凝胶迁移实验检测NF-κB的活性，蛋白质印迹法检测NF-κB蛋白的表达，同时分别用逆转录-聚合酶链反应以及蛋白质印迹法检测hBD-2 mRNA及蛋白的表达。 结果 与对照组相比，CD14过表达的细胞中NF-κB的活性明显增强，蛋白表达量也大幅度增加，同时hBD-2的mRNA及蛋白的表达都有所提高。 结论 胃癌细胞中CD14在介导NF-κB的激活以及hBD-2的表达中发挥重要作用。
目的 研究胃癌细胞SGC-7901培养上清液及转化生长因子-β1（TGF-β1）是否可促进人类腹膜间皮细胞表达βig-h3蛋白。方法 培养胃癌细胞SGC-7901，取第3天培养液上清与DMEM培养液的混合液 （1∶4）以及0、1.0、10.0和50.0ng/ml的 TGF-β1分别刺激人类腹膜间皮细胞HMrSV50、3、6、12及24h，ELISA方法检测上清液中βig-h3蛋白浓度，Western blot法检测细胞内βig-h3蛋白浓度。结果 对照组有基础量的βig-h3蛋白表达； 胃癌细胞SGC-7901培养上清液及TGF-β1均可明显增加HMrSV5细胞上清液及细胞内的βig-h3蛋白浓度（P＜0.05），且TGF-β1的刺激作用呈时间及浓度依赖性。结论 胃癌细胞SGC-7901培养上清液及TGF-β1可明显刺激HMrSV5细胞表达和分泌βig-h3蛋白。
Objective To construct the eukaryotic expressive vector of human tissue factor （TF），and to abserve the effect of TF on invasion and metastasis of gastric cancer cells line. Methods The human TF cDNA was obtained from human placenta by nest PCR, and the constructed eukaryotic expressive vector TF-pcDNA3 was transfected into SGC7901 cells by lipofectamine. Stable-transfected cells were screened by G418. The expressions of TF mRNA and protein on the cells were detected by RT-PCR and Western blot. Cell motility was assessed by using Transwell experiments and wound-healing assays. Results The eukaryotic expressive vector TF-pcDNA3 was successfully constructed and transfected into SGC7901. Compared with blank control group and negative control group, the expressions of TF mRNA and TF protein in transfection group were increased, the cell motility in vitro was enhanced. Conclusion TF can enhance the ability of invasion and metastasis of gastric cancer cells in vitro.
Objective To investigate the influence of different pressures and duration of CO2 pneumoperitoneum on the adhesive and invasive ability of gastric cancer cells based on the expressions of adhesive and invasive molecules. Methods With an artificial CO2 pneumoperitoneum model in vitro, human gastric cancer cell lines including MKN-45, SGC-7901, and MKN-28 were exposed to CO2 in different environments: 0 mm Hg （1 mm Hg=0.133 kPa）, 9 mm Hg （2 h, 4 h）, and 15 mm Hg （2 h, 4 h）. The expressions of mRNA of E-cadherin, intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor-A (VEGF-A) in the different environments were measured by RT-PCR. The expressions of protein of E-cadherin and ICAM-1 in the environments of 0 mm Hg and 15 mm Hg （4 h） were measured by FCM. Results With the increase of duration or pressure, RT-PCR showed that there was a downward trend in the expression of E-cadherin mRNA as well as there were upward trends in the expressions of ICAM-1, MMP-2, and VEGF-A mRNA; FCM showed that there was a downward trend in the expression of E-cadherin protein while the expression of ICAM-1 protein showed the opposite change. But there were no obvious differences under different environment (P＞0.05). Conclusions Under low pressure (≤15 mm Hg) and short time (≤4 h) of CO2 pneumoperitoneum, the adhesive and invasive ability of gastric cancer cells could not be affected, which means that under this environment, CO2 pneumoperitoneum will not increase the possibility of neoplasm metastasis.
Objective To observe the effects of vascular endothelial growth factor antisense oligonucleotide (VEGF-ASODN) on expression of vascular endothelial growth factor (VEGF) and growth in gastric cancer cells. Methods The VEGF-ASODN was synthesized artificially with phosphorothioic acid. After transfecting with VEGF-ASODN in gastric cancer cells SGC-7901, the initial copy number of mRNA was detected by real-time RT-PCR, and the quantity of VEGF protein in both cell and supernatant were detected by ELISA. The levels of expression of survivin protein in cells were measured by Western blot. FCM and MTT method were used to detect cellular apoptosis and the activity of cells, respectively. The effect of transfection on the growth of cells was evaluated by growth curve. Results The copy number of VEGR mRNA, protein levels of VEGF in the cells and in culture fluid all decreased when the concentration of transfected VEGF-ASODN increased, as well as the levels of survivin protein （P＜0.05）. The ratio of apoptosis increased, the activity of cells also decreased as the concentration of transfected VEGF-ASODN increased （P＜0.05）. Conclusion Transfection with VEGF-ASODN in gastric cancer cells SGC-7901 can inhibit the expressions of VEGF and survivin remarkably. It can enhance cellular apoptosis and suppress growth of cells.
ObjectiveTo study the expression levels of miR-339-3p and miR-339-5p in the gastric carcinoma cell lines(SGC-7901, BGC-823, and MKN-45) and gastric surface epithelium(GES-1);detect the relationship between miR-339-3p and miR-339-5p and the gastric carcinoma cell lines in vetrio experiment through the gain of function, and further significance is suggested. MethodsSYBR greenⅠreal time PCR was performed to access the expression of miR-339-3p and miR-339-5p in different cell lines(SGC-7901, BGC-823, MKN-45, and GES-1). The expression levels of miR-339-3p and miR-339-5p were verified by real time PCR experiment again after transfecting miR-339-3p mimics and miR-339-5p mimics. After that, the changes of MKN-45 cells apoptosis and proliferation at 72 h after transfection were detected by flow cytometry and CCK-8 method. ResultsThe expression levels of miR-339-3p and miR-339-5p in gastric carcinoma cell lines(SGC-7901, BGC-823, and MKN-45) were down regulated. Compared with the control group, the apoptosis of MKN-45 cell line was significantly higher(P < 0.05), the ability of proliferation of MKN-45 cell line decreased after transfecting miR-339-3p mimics and miR-339-5p mimics within 72 hours(P < 0.01). ConclusionThe expression levels of miR-339-3p and miR-339-5p significantly decreased in the gastric carcinoma cell lines(SGC-7901, BGC-823, and MKN-45) in contrast with gastric surface epithelium. MiR-339-3p and miR-339-5p may be involved in the apoptosis and proferation of the gastric carcinoma.
ObjectiveTo explore the effect of Poria cocos on xenograft tumors of gastric cancer SGC-7901 cell line in mude mice. Method①After establishment of xenograft tumor of gastric cancer SGC-7901 cell line, 10 nude mice were equally divided into normal control group and Poria cocos group. The nude mice of each group were gavaged with normal saline (NS) and Poria cocos (0.5 mL) for 32 days, respectively. Tumor volume were measured to draw tumor growth curves and the tumor weight inhibitory rate was calculated with tumor weight (on the 32-day, nude mice were sacrificed to get the xenograft tumors). The expressions of B cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), Caspase-3, Caspase-9, and vascular endothelial growth factor (VEGF) were detected by immunohistochemical staining. ②Preparation of drug serum containing Poria cocos. Gastric cancer SGC-7901 cell line were be divided into 2 groups: normal control group and Poria cocos group. Cells of normal control group were treated with serum containing NS, and cells of Poria cocos group were treated with drug serum containing 10% Poria cocos. After 24 hours and 48 hours, Western-blot was used to detect the expressions of Bcl-2 and Bax. ResultsOn 32-day, the volume and weight of xenograft tumors in normal control group〔(2 652.17±225.01) mm3 and (2.48±0.21) g〕were both higher than those of Poria cocos group〔(1 247.56±277.23) mm3 and (1.28±0.28) g〕, P<0.050. The tumor inhibitory rate in Poria cocos group was 48.39%. The results of immunohistochemical staining showed that, compared with normal control group, Poria cocos could down-regulate the expressions of Bcl-2〔(4.20±1.10）score vs. (8.00±1.20) score〕and VEGF〔(3.80±0.45) score vs. (7.80±1.10) score〕, while up-regulate the expressions of Bax〔(7.40±1.34) score vs. (3.00±0.71) score〕, Caspase-3〔(6.60±1.34) score vs. (2.60±0.55) score〕, and Caspase-9〔(7.20±1.79) score vs. (4.00±1.22) score〕, P<0.050. Compared with normal control group (1.72±0.03), the expression value of Bcl-2 was all higher in 24 h-Poria cocos group (0.96±0.04) and 48 h-Poria cocos group (0.77±0.04), P<0.050, and the expression value was higher in 48 h-Poria cocos group than that of 24 h-Poria cocos group (P<0.050). Compared with normal control group (0.15±0.01), the expression value of Bax was higher in 48 h-Poria cocos group (0.55±0.01), P<0.050, but there was no significant difference between the normal control group and 24 h-Poria cocos group（0.19±0）, P>0.050. ConclusionsPoria cocos can restrain the growth of xenograft tumors for gastric cancer SGC-7901 cell line in mude mice, and the mechanism may be related to mitochondrial apoptosis pathway and the inhibition of expression of VEGF.
Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552 (miR-552) on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods ① The MKN-45 cells were divided into MKN-45 blank control group (no transfection), MKN-45 miR-552 inhibitor group [transfection of plasmid inhibiting mir-552 expression (mir-552 inhibitor plasmid)], and MKN-45 negative control group [transfection of negative control plasmid (empty plasmid)], the exosomes were extracted, purified, and identified. Western blotting was used to detect the protein expression of exosomal markers [CD63, CD9, and tumor susceptibility gene 101 (TSG101)]. ② The HUVEC cells were divided into HUVEC control group (added PBS), HUVEC-exosome group (co-cultured with exosomes of MKN-45 cell), HUVEC-negative control exosome group (co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid), and HUVEC-miR-552 inhibitor exosome group (co-cultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid), exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells. Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552, the MTT method was used to detect the proliferation of HUVEC cells, the Transwell chamber method was used to detect the migration of HUVEC cells, the angiogenesis test was used to detect the angiogenesis ability. Results This study successfully extracted exosomes from MKN-45 gastric cancer cells. Observed by transmission electron microscope, the exosomes were all round or elliptical, with a diameter of 100–150 nm, and the exosomal vesicle structure could be seen. Western blotting detection showed that the surface markers of exosomes (CD63, CD9, and TSG101 protein) were expressed in exosomes. The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells. After MKN-45 cells were transfected with miR-552 inhibitor plasmid, compared with the MKN-45 blank control group and MKN-45 negative control group, the relative expression level of miR-552 in the exosomes decreased (P<0.05). Compared with the HUVEC control group, the cell proliferation rate at 24, 48 and 74 h increased, as well as number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-exosomes group increased (P<0.05). Compared with the HUVEC-negative control exosome group, the cell proliferation rate at 24, 48 and 74 h decreased, as well as the number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased (P<0.05). Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation, migration, and angiogenesis of HUVEC cells.