【作者】 刘磊；

【导师】 倪学勤；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2020， 硕士

【摘要】 随着益生菌益生作用的不断发现并被报道,加之其应用优势,越来越多的益生菌被用于研究它们对肠道疾病的改善作用。本实验室早期从大熊猫的粪样中分离到一株具有良好生物学特性的乳酸菌,融合魏斯氏菌BSP201703（Weissella confusa BSP201703,W.confusa BSP201703）。动物试验初步表明W.confusa BSP201703对肠道屏障功能具有保护作用,而其机制尚未阐明。故此,本研究利用细胞模型开展体外试验,旨在验证W.confusa BSP201703对肠上皮屏障的保护作用并探究其调控肠上皮细胞闭锁蛋白（Occludin,Ocld）表达的可能机制。研究选择Caco-2细胞为试验对象,采用脂多糖（Lipopolysaccharide,LPS）刺激处理建立肠上皮屏障受损模型,通过测算有无W.confusa BSP201703预处理的肠上皮屏障受损模型单层细胞跨膜电阻值（Trans-epithelial electrical resistance,TEER）和异硫氰酸荧光素标记的葡聚糖4（Fluorescein isothiocyanate dextran 4,FD-4）的表观渗透系数(Apparent permeability coefficient,P_app),并与完全空白组比较,体外分析W.confusa BSP201703对肠上皮屏障功能的影响;随后,参考CCK-8试验结果,结合实时荧光定量PCR（Real-time fluorescent quantitative polymerase chain reaction,RT-qPCR）和蛋白质免疫印迹（Western blot,WB）技术,在转录水平和翻译水平分析不同因子对Ocld表达的影响,为后续开展探究W.confusa BSP201703的作用机制试验优化LPS的使用浓度、益生菌的作用浓度和作用时间等参数;最后,使用特定激酶的抑制剂或受体的拮抗剂处理细胞单层膜,运用WB方法分析目标蛋白表达变化,通过与W.confusa BSP201703处理组和LPS处理组比较,以此说明特定激酶或受体是否参与了W.confusa BSP201703调控Ocld蛋白表达的过程。结果:在Caco-2细胞模型中,W.confusa BSP201703预处理可有效缓解LPS诱导的TEER值降低和FD-4 P_app升高,保护肠上皮屏障功能;以紧密连接蛋白Ocld的表达为依据,同时参考细胞增殖-毒性试验数据,试验参数优化结果表明:用100μg/m L的LPS刺激Caco-2细胞单层膜2 h可建立合适的肠上皮屏障损伤模型,使用浓度为1×10⁶CFU/m L的W.confusa BSP201703预处理肠上皮屏障6 h,对LPS刺激细胞Ocld的表达改善作用最佳;机制探究试验结果显示,W.confusa BSP201703预处理通过抑制LPS诱导的ROCK1（P<0.01）和磷酸化p38 MAPK（P<0.01）水平升高,增加细胞MLCK表达量（P<0.01）,进而发挥对Ocld蛋白表达的影响。使用TLR2的拮抗剂Cu-CPT22预处理Caco-2细胞单层膜消除了W.confusa BSP201703对Ocld蛋白表达的促进作用,表明TLR2是W.confusa BSP201703在肠上皮细胞上的作用受体。综上所述,本研究在体外证实了W.confusa BSP201703对紧密连接蛋白Ocld表达和肠上皮屏障功能维持的积极作用,并发现ROCK/p38 MAPK/MLCK信号通路是W.confusa BSP201703调控肠上皮细胞紧密连接蛋白Ocld表达的作用机制之一,TLR2为W.confusa BSP201703在Caco-2细胞上的作用受体。本试验的研究结果为W.confusa BSP201703的进一步应用奠定了研究基础。然而,W.confusa BSP201703发挥作用的有效结构元件尚未被发现,还有待进一步研究。更多还原

【Abstract】 With the continuous discovery and reporting of probiotics’beneficial effects in our life,coupled with its advantages in application,more and more probiotics have been used to explore their effects of improvement in preventing enteric disease.Previously work in this laboratory have isolated a lactic acid bacteria with good biological characteristics from giant panda feces,named Weissella confusa BSP201703（W.confusa BSP201703）.Animal experiments preliminarily showed that W.confusa BSP201703 had a protective effect on gut barrier function,but the mechanism had not been expounded.Therefore,this study will carry out trials in vitro,using a typical cellular model to verify the protective effects of W.confusa BSP201703 on epithelial barrier and further investigate its mechanism by which W.confusa BSP201703regulates occludin（Ocld）expression in intestinal epithelial cells.In the study,Caco-2 cells were selected as experimental subject,and lipopolysaccharide（LPS）stimulation was used to establish a model of impaired intestinal epithelial barrier,the trans-epithelial electrical resistance（TEER）of monolayer and the apparent permeability coefficient(P_app)of fluorescein isothiocyanate dextran 4（FD-4）of the impaired intestinal epithelial barrier with or without W.confusa BSP201703 pretreatment were measured and calculated,the results were compared with those of total control group so that to analyze the effects of W.confusa BSP201703 on intestinal epithelial barrier function in vitro.Subsequently,according to the results of CCK-8 assay,real-time fluorescent quantitative polymerase chain reaction（RT-qPCR）and western blot（WB）were used for analyzing the effects of different factors on Ocld expression at the level of transcription and translation,in order to optimize parameters such as LPS concentration,W.confusa BSP201703 concentration and treatment time for the follow-up mechanism exploration experiment of W.confusa BSP201703.At last,Caco-2 monolayer was treated with inhibitors of specific kinases or antagonist of receptor,and the expression of target protein was analyzed by WB,by comparing with W.confusa BSP201703 treatment group and LPS treatment group,it can be shown whether specific kinases or receptor are involved in the process of W.confusa BSP201703 regulating Ocld protein expression.The results showed:in the Caco-2 cell model,W.confusa BSP201703 pretreatment could effectively alleviate LPS-induced the decrease in TEER and the increase in P_app of FD-4,and protect the intestinal epithelial barrier function.Take the expression of Ocld as an indicator,also refer to the cell proliferation-toxicity test data,the experimental parameters optimization results suggested that stimulation of Caco-2 monolayer with 100μg/m L LPS for 2 h could establish a suitable model of impaired intestinal epithelial barrier,and pretreatment of the impaired intestinal epithelial barrier with W.confusa BSP201703 at the concentration of 1×10⁶CFU/m L for 6 h had the best effect for improving intercellular Ocld expression.The mechanism exploration experimental results showed W.confusa BSP201703 pretreatment inhibited LPS-induced increase in ROCK1（P<0.01）and phosphorylated p38 MAPK（P<0.01）,simultaneously enhanced cellular MLCK expression（P<0.01）,and then exerting an influence on the expression of Ocld protein.However,Caco-2 monolayer pretreated with TLR2 antagonist Cu-CPT22eliminated the positive effect of W.confusa BSP201703 on the expression of Ocld protein,suggesting that TLR2 is the receptor of W.confusa BSP201703 on intestinal epithelial cells.In summary,in vitro,this study confirmed that W.confusa BSP201703 had positive effects on the expression of Ocld protein and intestinal epithelial barrier function maintenance,and we found that ROCK/p38 MAPK/MLCK signaling pathway is one of the mechanisms by which W.confusa BSP201703 regulates the expression of tight junction protein Ocld in intestinal epithelial cells,and TLR2 is the receptor of W.confusa BSP201703 on Caco-2 cells.Our work laid a research foundation for the farther application of W.confusa BSP201703.However,the effective structural elements of W.confusa BSP201703 in action have not yet been discovered,it deserves further study.更多还原