乳杆菌通过调节肠道菌群抑制肥胖小鼠骨吸收作用的研究
本文选题:乳杆菌 + 肥胖 ; 参考:《哈尔滨工业大学》2017年硕士论文
【摘要】:高脂膳食不仅能导致超重,还会诱发骨代谢和吸收的异常,导致骨量减少和骨强度降低,从而导致自发和创伤性骨损伤的风险增加,严重危害骨健康。骨代谢异常或骨吸收受多方面因素影响,其中营养膳食因素占据重要地位。但是,长期高脂膳食与骨性能降低的关系目前并未有确切解释。益生菌对骨健康状态的改善主要是通过对免疫系统的调控来影响破骨细胞的活性,达到调节骨吸收与骨重建平衡的作用。因此,我们推测肠道菌群可能是治疗缓解骨质疏松的一个新靶点。但是益生菌与免疫系统及骨吸收之间具体的关系仍需进一步探讨。本课题拟从两株乳酸菌中筛选出对高脂膳食诱导Balb/c小鼠肥胖有明显抑制作用的益生菌株,研究其对肥胖小鼠骨性能的影响;探究肥胖小鼠肠道菌群变化、结肠免疫因子及粪便短链脂肪酸的关系,进而推断乳杆菌促进肥胖小鼠骨健康的机制。(1)采用灌胃的方式,将本实验室分离鉴定得到的Lactobacillus paracasei subsp.Paracasei M5(M5)及Lactobacillus coryniformis subsp.torquens T3L(T3L)乳杆菌以109 CFU/mL的剂量给予造模成功的肥胖Balb/c小鼠,4周末,T3L、M5均显著降低小鼠体重,体脂率及Lee’s指数(p0.05)。通过测定小鼠血清甘油三酯(TG)和总胆固醇(TC),结果表明M5及T3L均显著降低血清TG、TC含量(p0.05)。通过对各组小鼠的肝脏进行HE染色切片发现,经T3L、M5乳杆菌干预后,高脂膳食小鼠肝脏脂肪病变的程度有所改善,脂肪空泡变小而且变少。结果显示T3L菌株效果较显著。综上结果提示,M5及T3L菌株能抑制高脂饮食导致的小鼠肥胖及血脂上升。(2)乳杆菌对肥胖小鼠骨健康具有保护作用:测定小鼠血清ALP、TRAP及RANKL的含量,结果显示灌胃T3L乳杆菌4周后,血清中这三者的水平得到显著降低(p0.05),说明高脂膳食使得破骨细胞增生活跃,机体骨吸收增加,灌胃乳杆菌后得到改善。经T3L乳杆菌干预后,肥胖小鼠股骨生物力学性能增强、骨矿含量增加,且通过Micro-CT扫描发现股骨微结构均得到有效地改善。RT-qPCR法检测小鼠股骨组织中相关因子的表达,结果显示高脂膳食使股骨中RANKL和CTSK mRNA表达水平显著上调(p0.05),经T3L灌胃4周显著降低了其表达量;同时,T3L乳杆菌的摄入显著地提高了COL1α1的表达(p0.05)。(3)通过高通量测序法对小鼠肠道菌群多样性进行分析。高脂膳食诱导的肥胖使小鼠肠道菌群出现差异,灌胃乳杆菌T3L后,小鼠肠道菌群的多样性发生改变,产短链脂肪酸菌属增多。通过RT-qPCR检测结肠组织中IL-10、TGF-β1及Foxp3的表达,发现T3L的补充显著上调了高脂膳食小鼠肠内IL-10的表达,且表达水平接近对照组,p0.05。同时,灌胃T3L乳杆菌后粪便中短链脂肪酸的含量增多。益生菌可能通过调节肠道菌群结构,使产短链脂肪酸菌比例增加,从而利用短链脂肪酸影响结肠免疫功能进而干预骨重塑机制来完成对骨的保护作用。
[Abstract]:High fat diet can not only lead to overweight, but also induce abnormal metabolism and absorption of bone, reduce bone mass and bone strength, thus lead to increased risk of spontaneous and traumatic bone injury, and seriously endanger bone health. Abnormal bone metabolism or bone resorption is affected by many factors, among which nutritional and dietary factors play an important role. However, the relationship between long-term high-fat diet and bone loss has not been explained. The improvement of bone health by probiotics is mainly through the regulation of the immune system to affect the activity of osteoclasts and to regulate the balance between bone resorption and bone reconstruction. Therefore, we speculate that intestinal flora may be a new target for the treatment of osteoporosis. But the specific relationship between probiotics and immune system and bone resorption needs to be further explored. In this study, two strains of lactic acid bacteria were screened out to inhibit obesity induced by high fat diet in Balb/c mice, to study the effect of probiotics on the bone properties of obese mice, and to explore the changes of intestinal flora in obese mice. The relationship between colon immune factors and faecal short chain fatty acids, and then infer the mechanism of Lactobacillus to promote bone health in obese mice. Lactobacillus paracasei subsp.Paracasei M5 (M5) and Lactobacillus coryniformis subsp.torquens T3 LnT3L) were isolated and identified in our laboratory. Lactobacillus T3L5 and Lactobacillus coryniformis subsp.torquens T3 LnT3L) were given to obese Balb/c mice at the dose of 109 CFU/mL. The weight, body fat rate and Lee's index of the obese Balb/c mice were significantly decreased at the end of the 4th week. The results showed that both M5 and T3L significantly decreased the serum TGN TC content in mice. The liver sections of each group of mice were stained with HE staining. It was found that after the intervention of Lactobacillus T3L5, the degree of fatty lesions in liver of high-fat diet mice was improved, and the fat vacuoles became smaller and fewer. The results showed that the effect of strain T 3 L was significant. The results showed that strain M5 and T3L could inhibit obesity and increase of blood lipid in mice induced by high fat diet. Lactobacillus lactis had protective effect on bone health of obese mice. Serum levels of ALP trap and RANKL were measured. The results showed that Lactobacillus sp. T3L was perfused 4 weeks after oral administration of Lactobacillus t3L. The serum levels of these three factors were significantly decreased (P 0.05), which indicated that high fat diet made osteoclast proliferation active, bone resorption increased, and Lactobacillus gastric administration improved. After the intervention of Lactobacillus T3L, the biomechanical properties and bone mineral content in the femur of obese mice were enhanced, and the expression of related factors in the femur of obese mice was detected by RT-qPCR, and the microstructures of femur were all improved by Micro-CT scanning. The results showed that high fat diet significantly upregulated the expression of RANKL and CTSK mRNA in femur. At the same time, the uptake of Lactobacillus sp. T3L significantly increased the expression of COL1 伪 1. The intestinal flora of mice induced by high-fat diet was different. The diversity of intestinal flora of mice was changed and the short-chain fatty acid producing bacteria was increased after Lactic bacteria T3L was infused into the stomach. The expression of IL-10 TGF- 尾 1 and Foxp3 in colon tissue was detected by RT-qPCR. It was found that T3L supplementation significantly upregulated the expression of IL-10 in the intestine of high-fat diet mice, and the expression level was close to that of control group (p0.05. At the same time, the content of short chain fatty acids in feces increased after gastric administration of Lactobacillus. Probiotics may increase the proportion of short-chain fatty acid producing bacteria by adjusting the structure of intestinal flora, so that the short chain fatty acids can affect the immune function of colon and interfere with the mechanism of bone remodeling to protect the bone.
【学位授予单位】:哈尔滨工业大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R580;TS201.3
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