构建表达人胃泌酸调节素的乳酸乳球菌及其治疗肥胖的疗效研究
发布时间：2018-07-18 11:40
【摘要】：肥胖是当今世界倍受关注的一个健康问题,世界卫生组织(WHO)已经将肥胖定义为一种疾病。肥胖人数在世界范围内一直呈现显著上升的趋势。肥胖不仅影响行动和生活质量,也与很多疾病有着密不可分的关系,如糖尿病、高血压、心血管疾病、睡眠呼吸终止症以及癌症等等。导致肥胖的原因有很多,但高热量食物导致的饮食结构改变是绝大多数肥胖产生的原因。胃泌酸调节素(OXM)是一种由肠上皮细胞分泌的肽类激素。人OXM含37个氨基酸残基,具有抑制食欲和食物吸收、动员脂肪的作用。目前肽类作为药物主要是通过化学合成或生物工程生产,产品多为粉针剂,主要是通过注射的途径给药,成本高而且不方便。因此迫切需要一种载体系统,能够在人体内稳定表达OXM,并发挥其生物功能。乳酸乳球菌是益生菌,长期存在于肠道内,并且被认为是安全的(GRAS generally recognized as safe)[1]。目前已经有相当多的文献将乳酸乳球菌作为基因工程受体菌用于治疗肠道炎症[2]。我们利用基因工程的方法在乳酸乳球菌中表达人OXM,希望在人体肠道中稳定,持续地表达OXM,使其能够成为一种新型的药物来治疗肥胖症。为了验证这一假设,本论文构建了表达人OXM的乳酸乳球菌NZ3900,命名为NZ3900-OXM,同时通过Western blot等方法鉴定了菌株中OXM的表达,结果显示NZ3900-OXM能够稳定表达OXM并将其分泌至培养基中。随后,我们通过45%的高脂饮食构建了饮食诱导肥胖小鼠模型,将造模成功的肥胖小鼠分为两组,分别灌胃表达OXM的乳酸乳球菌和对照菌株,观察表达OXM的乳酸乳球菌对饮食诱导肥胖小鼠的体重、体脂、血糖、血脂和进食量等指标的影响。结果发现,灌胃表达OXM的乳酸乳球菌8周后,饮食诱导肥胖小鼠的体重显著下降至与普通饮食对照小鼠相一致,而且体脂和血脂也显著下降。有趣的是,灌胃对照乳酸菌后肥胖小鼠的血糖也显著下降,提示乳酸菌本身就足以使血糖的下降,但只有表达OXM的乳酸乳球菌才能够显著降低饮食诱导肥胖小鼠的体重、体脂和血脂。我们进一步在db/db小鼠上进行了验证。结果发现,经过三周的灌胃给药,NZ3900-OXM组的糖尿病小鼠的体重得到了很好的控制,而且糖化血红蛋白含量、血脂含量显著下降。本论文通过以上研究,旨在通过构建表达人胃泌酸调节素的乳酸乳球菌,有效地降低小鼠进食量、缓解二型糖尿病症状,从而为更安全、方便地治疗肥胖症和二型糖尿病找到一种可能的新型口服药物,并值得进一步开发和完善。
[Abstract]:Obesity is a major concern in the world today. The WHO (WHO) has defined obesity as a disease. The number of obesity has been increasing worldwide. Obesity not only affects the quality of action and life, but also has a close relationship with many diseases, such as diabetes, hypertension, and cardiovascular disease. Disease, sleep apnea and cancer and so on. There are many causes of obesity, but the diet structure changes caused by high calorie foods are the cause of most obesity. OXM is a peptide hormone secreted by intestinal epithelial cells. Human OXM contains 37 amino acid residues, which can inhibit appetite and food absorption and activity. At present, the peptide as a drug is mainly produced by chemical synthesis or Bioengineering, and the products are mostly powder, mainly by injection, which is highly cost and inconvenient. Therefore, a carrier system is urgently needed to stabilize the expression of OXM in human body and play its biological function. Lactococcus lactis is a probiotic. GRAS generally recognized as safe [1]. is currently in the intestinal tract and is considered to be safe. There have been quite a lot of literature on the use of Lactococcus Lactococcus as gene engineering receptor bacteria for the treatment of intestinal inflammation [2].. We use genetic engineering to express human OXM in Lactococcus Lactobacillus in the hope of stabilizing the human intestinal tract. In order to verify this hypothesis, this paper constructs a NZ3900 Lactococcus lactis expressing human OXM, named NZ3900-OXM, and identified the expression of OXM in the strain by Western blot, and the results show that NZ3900-OXM can express OXM and secrete it to OXM in order to verify this hypothesis. In the medium. Then, we constructed a diet induced obesity mouse model by 45% high fat diet. The obese mice were divided into two groups, and the OXM Lactococcus Lactococcus and control strains were administered respectively. The weight, body fat, blood sugar, blood lipid and food intake of the OXM Lactococcus lactis in the diet induced obese rats were observed. The results showed that after 8 weeks of gavage of OXM Lactococcus lactis, the weight of the diet induced obese mice decreased significantly to the normal diet control mice, and the body fat and blood lipid decreased significantly. Interestingly, the blood sugar of the obese mice was also significantly decreased after the gavage of lactic acid bacteria, suggesting that lactic acid bacteria itself was sufficient to make blood sugar. But only OXM Lactococcus Lactococcus could significantly reduce the weight, body fat and blood lipid in the diet induced obese mice. We further tested the db/db mice. The results showed that after three weeks of gavage, the body weight of the diabetic mice in group NZ3900-OXM was well controlled and the glycated hemoglobin was contained. In this paper, the aim of this study is to establish a new type of possible new oral medicine for the treatment of obesity and type two diabetes by constructing the Lactococcus lactis expressing human Gastro secreting hormone, reducing the intake of mice and alleviating the symptoms of type two diabetes. Development and improvement.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589.2
本文编号：2131793
[Abstract]:Obesity is a major concern in the world today. The WHO (WHO) has defined obesity as a disease. The number of obesity has been increasing worldwide. Obesity not only affects the quality of action and life, but also has a close relationship with many diseases, such as diabetes, hypertension, and cardiovascular disease. Disease, sleep apnea and cancer and so on. There are many causes of obesity, but the diet structure changes caused by high calorie foods are the cause of most obesity. OXM is a peptide hormone secreted by intestinal epithelial cells. Human OXM contains 37 amino acid residues, which can inhibit appetite and food absorption and activity. At present, the peptide as a drug is mainly produced by chemical synthesis or Bioengineering, and the products are mostly powder, mainly by injection, which is highly cost and inconvenient. Therefore, a carrier system is urgently needed to stabilize the expression of OXM in human body and play its biological function. Lactococcus lactis is a probiotic. GRAS generally recognized as safe [1]. is currently in the intestinal tract and is considered to be safe. There have been quite a lot of literature on the use of Lactococcus Lactococcus as gene engineering receptor bacteria for the treatment of intestinal inflammation [2].. We use genetic engineering to express human OXM in Lactococcus Lactobacillus in the hope of stabilizing the human intestinal tract. In order to verify this hypothesis, this paper constructs a NZ3900 Lactococcus lactis expressing human OXM, named NZ3900-OXM, and identified the expression of OXM in the strain by Western blot, and the results show that NZ3900-OXM can express OXM and secrete it to OXM in order to verify this hypothesis. In the medium. Then, we constructed a diet induced obesity mouse model by 45% high fat diet. The obese mice were divided into two groups, and the OXM Lactococcus Lactococcus and control strains were administered respectively. The weight, body fat, blood sugar, blood lipid and food intake of the OXM Lactococcus lactis in the diet induced obese rats were observed. The results showed that after 8 weeks of gavage of OXM Lactococcus lactis, the weight of the diet induced obese mice decreased significantly to the normal diet control mice, and the body fat and blood lipid decreased significantly. Interestingly, the blood sugar of the obese mice was also significantly decreased after the gavage of lactic acid bacteria, suggesting that lactic acid bacteria itself was sufficient to make blood sugar. But only OXM Lactococcus Lactococcus could significantly reduce the weight, body fat and blood lipid in the diet induced obese mice. We further tested the db/db mice. The results showed that after three weeks of gavage, the body weight of the diabetic mice in group NZ3900-OXM was well controlled and the glycated hemoglobin was contained. In this paper, the aim of this study is to establish a new type of possible new oral medicine for the treatment of obesity and type two diabetes by constructing the Lactococcus lactis expressing human Gastro secreting hormone, reducing the intake of mice and alleviating the symptoms of type two diabetes. Development and improvement.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589.2
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