益生菌增加细胞表面半乳糖在抑制RV感染中的作用

发布时间：2018-03-22 10:04

  本文选题：益生菌　切入点：半乳糖　出处：《东北农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：真核细胞表面的糖蛋白或者糖脂中都含有半乳糖基化的糖链,研究表明半乳糖不仅广泛地参与各种糖链的合成,作为终末残基,半乳糖残基影响细胞的识别、黏附、迁移和生长。轮状病毒(RV)在附着到肠上皮细胞的过程中需要识别细胞表面糖链,所以半乳糖与轮状病毒感染细胞的过程紧密相关。为阐明益生菌上清成分是否可通过诱导肠黏膜细胞表面半乳糖的增加而提高抗RV感染的效果。本研究通过3种不同的益生菌上清孵育猪小肠上皮细胞(IPEC-J2),诱导出3种不同分化型IPEC-J2。以不同分化型IPEC-J2检验益生菌在抗RV感染过程中的作用。同时,用D-半乳糖取代高糖DMEM培养基中的葡萄糖培养IPEC-J2细胞得到新的分化型,与益生菌诱导的分化型比较细胞表面半乳糖基化程度以及抗RV能力。实验设干酪乳杆菌诱导组(g组)、枯草芽孢杆菌诱导组(k组)、食淀粉乳杆菌诱导组(s组)、半乳糖诱导组(gal组)和未诱导细胞组。以上处理组又分为猪轮状病毒(PRV)感染和未感染处理,采用凝集素荧光技术观测各处理组IPEC-J2细胞表面半乳糖的(半定)量的变化;利用Reed-Muench法检测各感染细胞组轮状病毒的滴度变化;用双抗体夹心ELISA方法检测感染12h、24h、48h、72h后各感染组细胞中轮状病毒、β-半乳糖苷酶、β-半乳糖基转移酶含量,以各组未感染细胞作为对照。由凝集素荧光图可知,以三株益生菌的上清或半乳糖孵育细胞,都能够改变细胞表面半乳糖的含量。半数感染量检测结果为当轮状病毒感72h时,未诱导细胞组pRV感染后TCID50/0.1m L为10-7.15±0.14,g组pRV TCID50/0.1m L为10-3.875±0.125,食淀粉乳杆菌p RV TCID50/0.1mL为10-4.125±0.138,枯草芽孢杆菌p RV TCID50/0.1m L为10-4.16±0.144。病毒定量检测结果显示未诱导细胞组在感染的第12h到24 h RV含量变化不明显,24h到48h病毒量持续上升,并且达到最大值,48h-72h病毒量有所下降。而gal组在RV感染后24h内,RV量低于其他组。提示半乳糖的抗病毒作用主要在病毒进入复制期前最为明显。三个益生菌上清组在RV感染的48h内,RV量显著低于未处理组,12h内三个益生菌上清组组之间RV含量差异不显著(P≥0.05)。各组细胞的半乳糖苷酶含量检测结果显示:在未感染的各组细胞中,正常细胞组半乳糖苷酶的浓度高于g组、k组和s组(P0.05)。在RV感染的各组细胞中,在感染后第12h,s组和k组显著低于正常细胞和g组。在RV感染的第24h,正常细胞组和g组细胞酶浓度无差异(P≥0.05),s组和k组显著低于正常细胞组和g组。在RV感染的第48h,正常细胞组和k组间酶浓度无差异(P≥0.05),s组和g组的酶浓度显著低于正常细胞和k组(P0.05),s组和g组间无差异。在RV感染的第72h,s组和k组显著低于正常细胞组和g组(P0.05)。各组细胞的半乳糖基转移酶含量检测结果显示:各组细胞在感染RV前,g组、k组和s组三个组的β-半乳糖苷酶浓度低于正常细胞(P0.05);在RV感染后的第12h,g组细胞β-半乳糖苷酶浓度和正常细胞相比无差异(P0.05),s组和k组则显著低于正常细胞和g组(P0.05),s组和k组间无差异(P0.05)。在RV感染的第24h,正常细胞和g组细胞β-半乳糖苷酶浓度无差异(P0.05),s组和k组显著低于正常细胞和g组(P0.05),s组和k组间无差异(P0.05)。在RV感染的第48h,正常细胞和k组间β-半乳糖苷酶浓度无差异(P0.05),s组和g组的β-半乳糖苷酶浓度显著低于正常细胞和k组(P0.05),s组和g组间无差异(P0.05)。在RV感染的第72h,正常细胞和g组间β-半乳糖苷酶浓度无差异(P0.05),s组和k组显著低于正常细胞和g组(P0.05),s组和k组间无差异(P0.05)。综上所述,干酪乳杆菌上清,食淀粉乳杆菌上清,枯草芽孢杆菌上清能够提高IPEC-J2细胞表面的半乳糖基转移酶含量,增加细胞表面的半乳糖含量,从而减少RV对细胞的黏附,增强细胞抗RV感染的能力。
[Abstract]:Sugar chain galactosyl containing eukaryotic cell surface glycoprotein or glycolipid in the study showed that the synthesis of galactose is not only widely involved in various sugar chain, as the terminal residues, galactose residues affect cell recognition, adhesion, migration and growth. Rotavirus (RV) in attachment to recognition cell surface carbohydrate chain process of intestinal epithelial cells, closely related to the process so galactose and rotavirus infected cells. To clarify whether the ingredients can be obtained by adding probiotics supernatant induced intestinal mucosal cell surface galactose and improve the effect of anti RV infection. This study through 3 different incubation supernatant of porcine intestinal probiotics epithelial cells (IPEC-J2), induced by 3 different differentiated IPEC-J2. in different differentiated IPEC-J2 test of probiotics in anti RV infection process. At the same time, with D- galactose substituted high glucose DMEM medium glucose culture I PEC-J2 cells have been differentiated new, compared with differentiated probiotic induced cell surface galactose level and anti RV ability. The Lactobacillus casei induced group (group G), Bacillus subtilis induced group (K Group), food starch Lactobacillus induction group (s group), galactose induced group (Group Gal) and non induced cells. Treatment group was divided into porcine rotavirus (PRV) infection and non infection treatment, using lectin fluorescence technique observation of each treatment group IPEC-J2 cell surface galactose (semidefinite) quantity change; change the titer of rotavirus infected cells by Reed-Muench method; using double antibody sandwich ELISA method for detection of 12h infection, 24h, 48h, 72h after the infection of cells of rotavirus, beta galactosidase, beta galactosyltransferase content were detected by uninfected cells as control. The fluorescent lectin shows to three strains of probiotics. Clear or galactose incubating the cells were able to change the cell surface galactose. MID50 detection results when rotavirus 72h, did not induce cell group after pRV infection TCID50/0.1m L 10-7.15 + 0.14, G group pRV TCID50/0.1m L 10-3.875 + 0.125, food starch Lactobacillus P TCID50/0.1mL as RV 10-4.125 + 0.138, Bacillus subtilis P RV TCID50/0.1m L for the detection of 10-4.16 + 0.144. virus quantitative results showed that cells induced by 12h infection in the group to change 24 h RV content is not obvious, 24h to 48h virus continues to rise, and reached the maximum value, the 48h-72h virus decreased. In the group gal after RV infection 24h, RV was lower than that of other groups. That the antiviral effect of galactose in the main virus copy into the period before the most obvious. Three probiotics supernatant group in RV infected 48h, RV was significantly lower than the untreated group, 12h three probiotics supernatant 缁勭粍涔嬮棿RV鍚�閲忓樊寮備笉鏄捐懀�

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