壳寡糖减轻幽门螺杆菌对胃癌AGS细胞的诱导凋亡作用

发布时间：2018-01-08 08:03

本文关键词：壳寡糖减轻幽门螺杆菌对胃癌AGS细胞的诱导凋亡作用　出处：《青岛大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究目的:观察壳寡糖(Chitooligosaccharide,COS)体外抑制幽门螺杆菌(Helicobacter pylori,HP)增殖的活性,观察壳寡糖对AGS细胞和HP感染的AGS细胞的诱导凋亡的作用,探讨壳寡糖在治疗HP感染及治疗胃癌中的潜在应用价值。实验方法:1.培养HP菌株并传代。37℃微需氧环境中培养48h后,向BHI培养基表面滴加100u L COS溶液,对照组不做处理,继续培养48h,肉眼及显微镜下观察两组HP菌落形成情况。制备含壳寡糖培养基,HP传代培养48h,观察细菌菌落状态。2.观察壳寡糖减轻HP对AGS细胞的诱导凋亡作用:细胞共分2组进行:AGS细胞组和HP感染的AGS细胞。按照感染复数MOI=200建立HP感染AGS细胞的模型。分别向两组细胞中分别加入按比例0.5:100、1:100、2:100、4:100稀释的COS原液,37℃,5%CO2饱和湿度培养箱中培养24h后固定细胞,10ug/ml Hoechst33258工作液染色,荧光显微镜下观察AGS细胞的凋亡情况和细胞核的形态学改变。3.向AGS细胞中加入按比例稀释的壳寡糖与100mol/L,200mol/L,300mol/L和400mol/LBi Cl3混合溶液,37℃,5%CO2饱和湿度培养箱中培养24h后固定细胞,Hoechst33258染色,荧光显微镜下观察AGS细胞的凋亡情况和细胞核的形态学改变。实验结果:1.肉眼观,对照组细菌形成良好的无色透明的菌落形成,布满培养皿表面的接种区域,加入壳寡糖的区域细菌菌落明显稀少,散在分布;显微镜下观察,与未加壳寡糖的区域相比,加入壳寡糖区域内的菌落明显稀少。2.我们通过Hoechst33258染色荧光染色观察AGS细胞凋亡情况。结果显示:(1)AGS细胞组:与对照组相比,随着壳寡糖浓度的增加,细胞核呈均匀蓝色的细胞比例明显降低,而呈亮蓝色颗粒块状荧光及核形态异常的细胞比例明显上升,表明壳寡糖具有诱导AGS细胞凋亡的作用,其强度具有浓度依赖性。(2)感染HP的AGS组:HP感染的AGS细胞存在一定的凋亡现象,与其相比,加入低浓度的壳寡糖亮蓝色颗粒块状荧光及核形态异常的细胞比例下降,继续增加壳寡糖浓度,亮蓝色荧光的比例上升,这表明在较低的浓度壳寡糖减弱HP对AGS细胞的诱导凋亡作用。(3)观察Bi3+与壳寡糖联合对AGS细胞凋亡的影响,我们发现Bi3+与壳寡糖联合,其高亮染色的比例比单独使用壳寡糖或的比例更高,即二者联合比壳寡糖或Bi3+单独具有更强的诱导AGS细胞凋亡作用。结论:1.壳寡糖具有一定的体外抑制HP增殖活性。2.壳寡糖具有诱导AGS细胞凋亡的作用,强度呈浓度依赖性,Bi3+可增强壳寡糖的促凋亡作用。3.壳寡糖在一定浓度可以减轻HP对AGS细胞促凋亡作用,即对HP感染的AGS细胞具有保护作用。
[Abstract]:Objective: To observe the chitosan oligosaccharide (Chitooligosaccharide, COS) in vitro inhibition of Helicobacter pylori (Helicobacter pylori HP) to observe the activity of proliferation, apoptosis induced by oligochitosan on AGS cells and HP AGS cells infected with the role of the potential application of chitosan oligosaccharide in the treatment of patients with HP infection and gastric carcinoma. Methods: 1. HP and 48h were cultured strains cultured.37 C micro aerobic environment, the surface of the medium adding 100u L COS solution to BHI, the control group without treatment, cultured 48h, two groups were observed under microscope and naked eye HP colony formation. The preparation of chitosan oligosaccharide containing culture medium, HP culture 48h, observe the bacteria.2. observation of chitosan reduced colony apoptosis inducing effect of HP on AGS cells: the cells were divided into 2 groups: AGS cell group and HP infected AGS cells. In accordance with the establishment of HP infected AGS cells model m.o.i.of MOI=200 respectively to two groups of cells. Were added by COS solution, the proportion of 0.5:100,1:100,2:100,4:100 diluted 37 DEG 5%CO2 saturated humidity in cultured 24h cells after fixed box, 10ug/ml Hoechst33258 staining and morphological observation of AGS cells apoptosis and cell under fluorescent microscope.3. adding chitosan oligosaccharide and 100mol/L, according to the proportion to AGS cells in 200mol/L, 300mol/L 400mol/LBi and Cl3 mixed solution, 37 DEG C, 5%CO2 saturated humidity in cultured 24h fixed cells, Hoechst33258 staining in the box, change the fluorescence microscope and the apoptosis of AGS cell and cell nuclear morphology. Results: 1. eye view group of bacteria to form a good transparent colony forming control, with cultured regional dishes the surface area of bacterial colonies adding chitosan oligosaccharide was scarce and scattered; under the microscope, compared with not added oligosaccharide, plus In the area of the colony of oligochitosan was rare.2. we stained by Hoechst33258 fluorescent staining observation the apoptosis of AGS cells. The results showed: (1) AGS group: compared with the control group, with the increase of chitosan concentration, the nuclei were uniform blue cell ratio was significantly lower, and have bright blue fluorescence and massive nuclear particles abnormal cell morphology ratio significantly increased, showed that the chitosan oligosaccharide has induced AGS cell apoptosis, its strength is concentration dependent. (2) AGS group HP infection: HP infected AGS cells apoptosis phenomenon, some contrast, adding chitosan oligosaccharide with low concentration of bright blue particulate fluorescence and nuclear morphological abnormalities the proportion of cells decreased, continue to increase the concentration of chitosan, the bright blue fluorescence ratio rise, suggesting that weakened the effect of apoptosis induced by HP on AGS cells in the lower concentration of chitosan oligosaccharide. (3) observation of Bi3+ and chitooligosaccharides it The effects on the apoptosis of AGS cells, we found that the combination of Bi3+ and chitosan, the bright staining proportion is higher than the single use of chitosan oligosaccharide and the proportion of the two combined ratio of chitosan or Bi3+ alone has a stronger effect induced apoptosis in AGS cells. Conclusion: 1. chitosan oligosaccharide has certain inhibition on proliferation of HP.2. chitosan oligosaccharide has induced AGS cell apoptosis, the intensity in a concentration dependent manner, Bi3+ can enhance the apoptosis inducing effect of.3. chitosan chitosan oligosaccharide in a certain concentration can reduce the HP induced apoptosis of AGS cells, which has protective effect on HP infected AGS cells.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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