本文選題：益生菌　切入點(diǎn)：半乳糖　出處：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：真核細(xì)胞表面的糖蛋白或者糖脂中都含有半乳糖基化的糖鏈,研究表明半乳糖不僅廣泛地參與各種糖鏈的合成,作為終末殘基,半乳糖殘基影響細(xì)胞的識別、黏附、遷移和生長。輪狀病毒(RV)在附著到腸上皮細(xì)胞的過程中需要識別細(xì)胞表面糖鏈,所以半乳糖與輪狀病毒感染細(xì)胞的過程緊密相關(guān)。為闡明益生菌上清成分是否可通過誘導(dǎo)腸黏膜細(xì)胞表面半乳糖的增加而提高抗RV感染的效果。本研究通過3種不同的益生菌上清孵育豬小腸上皮細(xì)胞(IPEC-J2),誘導(dǎo)出3種不同分化型IPEC-J2。以不同分化型IPEC-J2檢驗(yàn)益生菌在抗RV感染過程中的作用。同時(shí),用D-半乳糖取代高糖DMEM培養(yǎng)基中的葡萄糖培養(yǎng)IPEC-J2細(xì)胞得到新的分化型,與益生菌誘導(dǎo)的分化型比較細(xì)胞表面半乳糖基化程度以及抗RV能力。實(shí)驗(yàn)設(shè)干酪乳桿菌誘導(dǎo)組(g組)、枯草芽孢桿菌誘導(dǎo)組(k組)、食淀粉乳桿菌誘導(dǎo)組(s組)、半乳糖誘導(dǎo)組(gal組)和未誘導(dǎo)細(xì)胞組。以上處理組又分為豬輪狀病毒(PRV)感染和未感染處理,采用凝集素?zé)晒饧夹g(shù)觀測各處理組IPEC-J2細(xì)胞表面半乳糖的(半定)量的變化;利用Reed-Muench法檢測各感染細(xì)胞組輪狀病毒的滴度變化;用雙抗體夾心ELISA方法檢測感染12h、24h、48h、72h后各感染組細(xì)胞中輪狀病毒、β-半乳糖苷酶、β-半乳糖基轉(zhuǎn)移酶含量,以各組未感染細(xì)胞作為對照。由凝集素?zé)晒鈭D可知,以三株益生菌的上清或半乳糖孵育細(xì)胞,都能夠改變細(xì)胞表面半乳糖的含量。半數(shù)感染量檢測結(jié)果為當(dāng)輪狀病毒感72h時(shí),未誘導(dǎo)細(xì)胞組pRV感染后TCID50/0.1m L為10-7.15±0.14,g組pRV TCID50/0.1m L為10-3.875±0.125,食淀粉乳桿菌p RV TCID50/0.1mL為10-4.125±0.138,枯草芽孢桿菌p RV TCID50/0.1m L為10-4.16±0.144。病毒定量檢測結(jié)果顯示未誘導(dǎo)細(xì)胞組在感染的第12h到24 h RV含量變化不明顯,24h到48h病毒量持續(xù)上升,并且達(dá)到最大值,48h-72h病毒量有所下降。而gal組在RV感染后24h內(nèi),RV量低于其他組。提示半乳糖的抗病毒作用主要在病毒進(jìn)入復(fù)制期前最為明顯。三個(gè)益生菌上清組在RV感染的48h內(nèi),RV量顯著低于未處理組,12h內(nèi)三個(gè)益生菌上清組組之間RV含量差異不顯著(P≥0.05)。各組細(xì)胞的半乳糖苷酶含量檢測結(jié)果顯示:在未感染的各組細(xì)胞中,正常細(xì)胞組半乳糖苷酶的濃度高于g組、k組和s組(P0.05)。在RV感染的各組細(xì)胞中,在感染后第12h,s組和k組顯著低于正常細(xì)胞和g組。在RV感染的第24h,正常細(xì)胞組和g組細(xì)胞酶濃度無差異(P≥0.05),s組和k組顯著低于正常細(xì)胞組和g組。在RV感染的第48h,正常細(xì)胞組和k組間酶濃度無差異(P≥0.05),s組和g組的酶濃度顯著低于正常細(xì)胞和k組(P0.05),s組和g組間無差異。在RV感染的第72h,s組和k組顯著低于正常細(xì)胞組和g組(P0.05)。各組細(xì)胞的半乳糖基轉(zhuǎn)移酶含量檢測結(jié)果顯示:各組細(xì)胞在感染RV前,g組、k組和s組三個(gè)組的β-半乳糖苷酶濃度低于正常細(xì)胞(P0.05);在RV感染后的第12h,g組細(xì)胞β-半乳糖苷酶濃度和正常細(xì)胞相比無差異(P0.05),s組和k組則顯著低于正常細(xì)胞和g組(P0.05),s組和k組間無差異(P0.05)。在RV感染的第24h,正常細(xì)胞和g組細(xì)胞β-半乳糖苷酶濃度無差異(P0.05),s組和k組顯著低于正常細(xì)胞和g組(P0.05),s組和k組間無差異(P0.05)。在RV感染的第48h,正常細(xì)胞和k組間β-半乳糖苷酶濃度無差異(P0.05),s組和g組的β-半乳糖苷酶濃度顯著低于正常細(xì)胞和k組(P0.05),s組和g組間無差異(P0.05)。在RV感染的第72h,正常細(xì)胞和g組間β-半乳糖苷酶濃度無差異(P0.05),s組和k組顯著低于正常細(xì)胞和g組(P0.05),s組和k組間無差異(P0.05)。綜上所述,干酪乳桿菌上清,食淀粉乳桿菌上清,枯草芽孢桿菌上清能夠提高IPEC-J2細(xì)胞表面的半乳糖基轉(zhuǎn)移酶含量,增加細(xì)胞表面的半乳糖含量,從而減少RV對細(xì)胞的黏附,增強(qiáng)細(xì)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 緇勭粍涔嬮棿RV鍚噺宸紓涓嶆樉钁，

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