幽门螺杆菌CagA+菌株对BGC-823细胞系Cx43表达及细胞增殖的影响

目的:观察体外实验中幽门螺杆菌(Helicobacter pylori, H.pylori)CagA 菌株对BGC-823细胞系Cx43表达和细胞增殖的影响,以及抗H.pylori药物干预后的变化,探讨H.pylori CagA 菌株与胃上皮细胞Cx43表达和细胞增殖的关系. 方法:将H.pylori CagA 菌株(国际标准毒力菌株NCTC J99)和CagA-菌株(NCTC 12908)分别与BGC-823细胞按细菌/细胞比例20:1,100:1,500:1共培养24,48h,以及细菌/细胞比例100:1组共培养16h后予抗H.pylori药物;对照组不加H.pylori或不加药物.用细胞免疫化学链霉亲和素-生物素-过氧化物酶复合物法及计算机图像分析技术检测细胞Cx43表达变化;溴化-3(4,5-二甲基噻唑基-2)-2,5-二苯基四唑比色法检测细胞增殖情况.结果:(1)未加H.pylori的对照组培养48h Cx43表达高于24h(P＜0.05);加CagA 菌株不同比例组48 h时的Cx43表达均低于24h,且低于对照组,24及48h 500:1组均低于100:1组和20:1组,48h 100:1组低于20:1组(P＜0.05);而加CagA-菌株不同比例组48h与24h的Cx43表达比较差异无统计学意义(P＞0.05),24h 100:1组和500:1组均低于对照组,24及48h 500:1组低于20:1组(P＜0.05);抗H.pylori药物干预后48h Cx43表达增强.(2)加CagA 菌株培养24h,100:1组细胞增殖增强;培养48h,20:1组和100:1组细胞增殖增强,500:1组细胞增殖受到抑制(P＜0.05).而加CagA-菌株对细胞增殖无明显影响.抗H.pylori药物干预对细胞增殖有抑制作用.结论:H.pylori CagA 菌株明显下调BGC-823细胞Cx43表达,并与作用时间及H.pylori密度有关,H.pylori密度较低时促进细胞增殖,密度较高时抑制细胞增殖;H.pylori CagA-菌株对细胞增殖无明显影响.抗H.pylori药物干预可上调Cx43表达,抑制细胞增殖.

Effect of CagA+ helicobacter pylori strain on the expressionof connexin 43 and cell proliferation in BGC-823 cells

OBJECTIVE: To determine the effect of CagA(+) Helicobacter pylori(H.pylori)strain and anti-H.pylori drugs on the expression of connexin 43(Cx43) and cell proliferation of BGC-823 cells in vitro,and to investigate the relation between the changes of Cx43 expression, cell proliferation of BGC-823 cells and CagA(+)H.pylori. METHODS: BGC-823 cells were co-cultured with CagA(+) H.pylori strain(NCTC J99) or CagA(-) H.pylori strain(NCTC 12908)at bacteria/cells ratio of 20:1,100:1 and 500:1 for 24 hours and 48 hours respectively. anti-H.pylori drugs was given in the group co-cultured at bacteria/cells ratio of 100:1 after 16 hours. In the control group, BGC-823 cells were cultured for 24 hours and 48 hours respectively,but without H.pylori or antij H.pylori drugs. Immunocytochemical SABC method and the image analysis of the computer were applied to detect the changes of Cx43 expression in BGC-823 cells. The cell proliferation was examined by methyl tetrazolium (MTT) method. RESULTS: (1)The expression of Cx43 in the control group after cultivation for 48 hours was higher than that for 24 hours (P< 0.05). The expression of Cx43 in the groups co-cultured with CagA(+) H.pylori strain after cultivation for 48 hours was lower than that co-cultured for only 24 hours, and that of the groups co-cultured with CagA(+) H.pylori strain was lower than that of the control group for both 24 hours and 48 hours (P< 0.05). The expression of Cx43 in the groups at bacteria/cells ratio of 500:1 was lower than that at bacteria/cells ratio of 20:1 and 100:1 for both 24 and 48 hours (P< 0.05),and that at bacteria/cells ratio of 100:1 was lower than that at bacteria/cells ratio of 20:1 for 48 hours (P< 0.05).However, there was no significant difference in Cx43 expression between 24 and 48 hours in the groups co-cultured with CagA(-) H.pylori strain (P>0.05). Cx43 expression in the groups co-cultured with CagA(-) H.pylori strain at the ratio of 100:1 and 500:1 was lower than that in the control group, and Cx43 expression at the ratio of 500:1 was lower than that at the ratio of 20:1 for 24 hours and 48 hours. Cx43 expression increased after the intervention with anti-H.pylori drugs for 48 hours. (2) In the groups co-cultured with CagA(+)H.pylori strain, the optical density value of MTT indicated that the cell proliferation at the bacteria/cells ratio of 100:1 was higher than that in the control group, but no significant difference was found in other two groups co-cultured for 24 hours. After co-culturing for 48 hours, the cell proliferation at the bacteria/cells ratio of 20:1 and 100:1 was significantly accelerated, while the cell proliferation at 500:1 was inhibited. In the groups co-cultured with CagA(-) H.pylori strain,there was no change in the cell proliferation. Intervention with anti-H.pylori drugs could suppress the cell proliferation. CONCLUSION: CagA(+) H.pylori can down-regulate the expression of Cx43 in BGC-823 cells,which is related to the reaction time and the density of H.pylori.Low density of CagA(+)H.pylori suspensions can accelerate the proliferation of BGC-823 cells, while high density can suppress the cell proliferation. The CagA(-) H.pylori has no effect on the cell proliferation. Intervention with anti-H.pylori drugs can up-regulate the expression of Cx43,and suppress the cell proliferation of BGC-823 cells.