小鼠肠道菌群失衡模型的建立及菌群失衡对肠道机械屏障的影响
摘要
目的:
1.以肠道十四种优势菌群变化情况为依据,建立轻度菌群失衡和重度菌群失衡小鼠模型。
2.研究肠道菌群失衡对小鼠肠粘膜机械屏障的影响,探讨优势菌群与肠道粘膜屏障的相互作用机制。
方法:
1.采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型。以头孢曲松钠终浓度5g/kg/d的剂量,连续8天,取盲肠内容物进行肠道菌群分析,建立小鼠轻度菌群失衡模型;以头孢曲松钠终浓度8g/kg/d的剂量,连续8天,分析肠道菌群,建立小鼠重度菌群失衡模型。
2.利用HE染色及电镜技术观察正常组、轻度菌群失衡组、重度菌群失衡组小鼠肠道粘膜绒毛形态变化,用ELISA法检测肠粘膜分泌性免疫球蛋白SIgA分泌量变化,RT-PCR技术及免疫组化法检测肠粘膜粘液素Mucin2、Mucin3,防御素含量的变化。
结果:
1.头孢曲松钠5g/kg/d,连续8天灌胃,肠道优势菌群显著下降,建立轻度菌群失衡小鼠模型;头孢曲松钠8g/kg/d,连续8天灌胃,肠道优势菌群的数量几乎降至零,建立重度菌群失衡小鼠模型。
2.菌群失衡小鼠肠粘膜绒毛出现肿胀、断裂,绒毛顶端肠上皮细胞坏死、脱落,细胞间间隙增宽,胞浆内出现空泡结构,粘膜及粘膜下层有淋巴细胞浸润,重度菌群失衡与轻度菌群失衡小鼠相比较绒毛结构受损加重。
3.菌群失衡小鼠小肠段肠粘膜分泌性免疫球蛋白SIgA分泌水平下降,重度菌群失衡组与正常组比较下降呈显著变化(P<0.05),与轻度菌群失衡组相比下降明显。
4.菌群失衡小鼠粘液层中的粘液素Mucin2、Mucin3、防御素在mRNA水平的表达与正常小鼠相比明显增强,在杯状细胞胞浆内Mucin2蛋白阳性表达增强,且重度菌群失衡组与轻度菌群失衡组小鼠比较,粘液素Mucin2、Mucin3、防御素增强明显。
结论:抗生素干扰肠道优势菌群,建立肠道菌群失衡模型。菌群失衡可导致肠道机械屏障粘膜绒毛结构受损,肠粘膜免疫水平下降,SIgA分泌量随菌群失衡程度呈下降趋势,同时引起粘液层中粘液素和防御素分泌量增加。
Objective:
1. Establish different dysbacteria mice model based on intestinal dominant flora changes.
2. Observe the changes of intestinal physical barrier of dysbacteria mice, to explore the interaction of intestinal dominant flora and intestinal mucosal immunity.
Methods:
1. Mice were dealed by intragastic administration of ceftriaxone sodium, according to the analysis of cecum flora, 5g/kg/d doses, 8 days to establish light-dysbacteria model ; 8g/kg/d doses, 8 days to establish heavy-dysbacteria model.
2. Morphological changes of gastrointestinal mucosa were observed by optical microscope and transmission electron microscope, secreted IgA in intestinal mucosa were detected by ELISA, mucin2、mucin3 and defensin was detected by RT-PCR and immunohistochemical method.
Results:
1. In light-dysbacteria mice, intestinal dominant flora were decreased significantly, and in heavy-dysbacteria mice, intestinal dominant flora were almost dropped to naught.
2. In dysbacteria mice, intestinal mucosal villus were damaged, villus were swell and breakage, the gaps of intestinal epithelial cells were wider, cells showed necrosis and vacuoles, lymph cells infiltrated mucosa layer, heavy-dysbacteria mice were damaged much better than light-dysbacteria mice.
3. Compared with normal mice, mucosal SIgA level was decreased in dysbacteria mice, the heavy-dysbacteria mice SIgA level were lower than light-dysbacteria mice.
4. Compared with normal mice, the levels of mucin2, mucin3 and defensin were higher in dysbacteria mice, the heavy-dysbacteria mice higher than light-dysbacteria mice.
Conclusion: Antibiotics of ceftriaxone sodium can inhibit and kill intestinal dominant flora; In dysbacteria mice, intestinal villus were damaged, the level of intestinal mucosal immunity was decreased, SIgA secretory volume was downtrend followed by dysbacteria degree; intestinal mucin and defensin levels were increased.
1.以肠道十四种优势菌群变化情况为依据,建立轻度菌群失衡和重度菌群失衡小鼠模型。
2.研究肠道菌群失衡对小鼠肠粘膜机械屏障的影响,探讨优势菌群与肠道粘膜屏障的相互作用机制。
方法:
1.采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型。以头孢曲松钠终浓度5g/kg/d的剂量,连续8天,取盲肠内容物进行肠道菌群分析,建立小鼠轻度菌群失衡模型;以头孢曲松钠终浓度8g/kg/d的剂量,连续8天,分析肠道菌群,建立小鼠重度菌群失衡模型。
2.利用HE染色及电镜技术观察正常组、轻度菌群失衡组、重度菌群失衡组小鼠肠道粘膜绒毛形态变化,用ELISA法检测肠粘膜分泌性免疫球蛋白SIgA分泌量变化,RT-PCR技术及免疫组化法检测肠粘膜粘液素Mucin2、Mucin3,防御素含量的变化。
结果:
1.头孢曲松钠5g/kg/d,连续8天灌胃,肠道优势菌群显著下降,建立轻度菌群失衡小鼠模型;头孢曲松钠8g/kg/d,连续8天灌胃,肠道优势菌群的数量几乎降至零,建立重度菌群失衡小鼠模型。
2.菌群失衡小鼠肠粘膜绒毛出现肿胀、断裂,绒毛顶端肠上皮细胞坏死、脱落,细胞间间隙增宽,胞浆内出现空泡结构,粘膜及粘膜下层有淋巴细胞浸润,重度菌群失衡与轻度菌群失衡小鼠相比较绒毛结构受损加重。
3.菌群失衡小鼠小肠段肠粘膜分泌性免疫球蛋白SIgA分泌水平下降,重度菌群失衡组与正常组比较下降呈显著变化(P<0.05),与轻度菌群失衡组相比下降明显。
4.菌群失衡小鼠粘液层中的粘液素Mucin2、Mucin3、防御素在mRNA水平的表达与正常小鼠相比明显增强,在杯状细胞胞浆内Mucin2蛋白阳性表达增强,且重度菌群失衡组与轻度菌群失衡组小鼠比较,粘液素Mucin2、Mucin3、防御素增强明显。
结论:抗生素干扰肠道优势菌群,建立肠道菌群失衡模型。菌群失衡可导致肠道机械屏障粘膜绒毛结构受损,肠粘膜免疫水平下降,SIgA分泌量随菌群失衡程度呈下降趋势,同时引起粘液层中粘液素和防御素分泌量增加。
Objective:
1. Establish different dysbacteria mice model based on intestinal dominant flora changes.
2. Observe the changes of intestinal physical barrier of dysbacteria mice, to explore the interaction of intestinal dominant flora and intestinal mucosal immunity.
Methods:
1. Mice were dealed by intragastic administration of ceftriaxone sodium, according to the analysis of cecum flora, 5g/kg/d doses, 8 days to establish light-dysbacteria model ; 8g/kg/d doses, 8 days to establish heavy-dysbacteria model.
2. Morphological changes of gastrointestinal mucosa were observed by optical microscope and transmission electron microscope, secreted IgA in intestinal mucosa were detected by ELISA, mucin2、mucin3 and defensin was detected by RT-PCR and immunohistochemical method.
Results:
1. In light-dysbacteria mice, intestinal dominant flora were decreased significantly, and in heavy-dysbacteria mice, intestinal dominant flora were almost dropped to naught.
2. In dysbacteria mice, intestinal mucosal villus were damaged, villus were swell and breakage, the gaps of intestinal epithelial cells were wider, cells showed necrosis and vacuoles, lymph cells infiltrated mucosa layer, heavy-dysbacteria mice were damaged much better than light-dysbacteria mice.
3. Compared with normal mice, mucosal SIgA level was decreased in dysbacteria mice, the heavy-dysbacteria mice SIgA level were lower than light-dysbacteria mice.
4. Compared with normal mice, the levels of mucin2, mucin3 and defensin were higher in dysbacteria mice, the heavy-dysbacteria mice higher than light-dysbacteria mice.
Conclusion: Antibiotics of ceftriaxone sodium can inhibit and kill intestinal dominant flora; In dysbacteria mice, intestinal villus were damaged, the level of intestinal mucosal immunity was decreased, SIgA secretory volume was downtrend followed by dysbacteria degree; intestinal mucin and defensin levels were increased.
引文
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15. Ramsay , A . J . , Husband , A . J . , Ramshaw , I . A . , Bao , S . , Matthaei , K . I . ,Koehler , G . et al. The role of interleukin-6 in mucosal IgA antibody responses in vivo . Science, 1994, 264(5158) , 561-563.
16. Harriman GR, Bogue M, Rogers P, Finegold M, Pacheco S, Bradley A, Zhang Y.Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes. J Immunol, 1999 ,162(5):2521-2529.
17. Lycke N, Erlandsson L, Ekman L, Schon K.Lack of J chain inhibits the transport of gut IgA and abrogates the development of intestinal antitoxic protection. J Immunol, 1999, 163(2):913-919.
18. Uren TK, Johansen FE, Wijburg OL, Koentgen F. Role of the polymeric Ig receptor in mucosal B cell homeostasis. J Immunol, 2003,170(5):2531-2539.
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2. Bowman EP, Kuklin NA, Youngman KR, Lazarus NH, Kunkel EJ, Pan J, et al. The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. J Exp Med ,2002,195(2): 269–75.
3. Kunkel EJ, Campbell JJ, Haraldsen G, Pan J, Boisvert J, Roberts AI, et al. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immun compartment: Epithelial expression of tissue-specific chemokines as an orga-nizing principle in regional immunity. J Exp Med, 2000,192(5):761–8.
4. Agace WW, Amara A, Roberts AI, Pablos JL, Thelen S, Uguccioni M, et al. Constitutive expression of stromal derived factor-1 by mucosal epithelia and its role in HIV transmission and propagation. Curr Biol, 2000,10(6):325–8.
5. Ziegler and liu.Thymic stromal lymphopoietin in normal and pathogenic T cell development and function. Nat. Immunol,2006, 7(7), 709-714.
6. Rescigno, M et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunology,2001,2(4), 361-367.
7. Rmoldi M, Chieppa M, et al. Monocyte-derived dendritic cells activated by bacteria or by bacteria-stimulated epithelial cells are functionally different. Blood,2005 ,15;106(8):2818-26 .
8. Fagarasan, Honjo. Intestinal IgA synthesis: regulation of front-line body defences . Nat Rev Immunol, 2003, 3(2): 63-72.
9. Bergqvist P, Gardby E, Stensson A, et al. Gut IgA class switch recombination in the absence of CD40 does not occur in the lamina propria and is independent of germinal centers. J Immunol ,2006,177(1):7772–7783.
10. Litinskiy MB, Nardelli B, Hilbert DM, et al. Antigen presenting cells induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat Immunol ,2002, 3(9):822–829.
11. He , B . , Xu , W . , Santini , P . A . , Polydorides , A . D . , Chiu , A . , Estrella , J . et al. Intestinal bacteria trigger T cell-independent Immunoglobulin A(2) classswitching by inducing epithelial-cell secretion of the cytokine APRIL . Immunity, 2007, 26(6) , 812– 826.
12. McCarthy DD, Chiu S, Gao Y, et al. BAFF induces a hyper-IgA syndrome in the intestinal lamina propria concomitant with IgA deposition in the kidney independent of LIGHT. Cell Immunol, 2006, 241(2):85–94.
13. Castigli E, Scott S, Dedeoglu F, et al. Impaired IgA class switching in APRILdeficient mice. Proc Natl Acad Sci U S A ,2004,101(11):3903–3908.
14. Castigli E, Wilson SA, Scott S, et al. TACI and BAFF-R mediate isotype switching in B cells. J Exp Med ,2005,201(1):35–39.
15. Mora JR, Iwata M, Eksteen B, et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science ,2006, 314(5802):1157–1160.
16. Singh M , Vohra H , Kumar L , et al . Induction of Systemic and Mucosal Immune Response in Mice Immunised with Porins of Salmonella Typha [ J ] . J Med Microbiol ,1999 ,48(1) :79.
17. Lanning DK, Rhee KJ, Knight KL. Intestinal bacteria and development of the B-lymphocyte repertoire. Trends Immunol, 2005,26(8):419–25.
18. Crottet , P . & Corthesy , B . Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab )2: a possible implication for mucosal defense . J. Immunol,1998,161(10) , 5445– 5453 .
19. Phalipon , A . & Corthesy , B . Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins . Trends Immunol, 2003,24(2), 55– 58.
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