【摘要】：肥胖是當(dāng)今世界倍受關(guān)注的一個(gè)健康問(wèn)題,世界衛(wèi)生組織(WHO)已經(jīng)將肥胖定義為一種疾病。肥胖人數(shù)在世界范圍內(nèi)一直呈現(xiàn)顯著上升的趨勢(shì)。肥胖不僅影響行動(dòng)和生活質(zhì)量,也與很多疾病有著密不可分的關(guān)系,如糖尿病、高血壓、心血管疾病、睡眠呼吸終止癥以及癌癥等等。導(dǎo)致肥胖的原因有很多,但高熱量食物導(dǎo)致的飲食結(jié)構(gòu)改變是絕大多數(shù)肥胖產(chǎn)生的原因。胃泌酸調(diào)節(jié)素(OXM)是一種由腸上皮細(xì)胞分泌的肽類激素。人OXM含37個(gè)氨基酸殘基,具有抑制食欲和食物吸收、動(dòng)員脂肪的作用。目前肽類作為藥物主要是通過(guò)化學(xué)合成或生物工程生產(chǎn),產(chǎn)品多為粉針劑,主要是通過(guò)注射的途徑給藥,成本高而且不方便。因此迫切需要一種載體系統(tǒng),能夠在人體內(nèi)穩(wěn)定表達(dá)OXM,并發(fā)揮其生物功能。乳酸乳球菌是益生菌,長(zhǎng)期存在于腸道內(nèi),并且被認(rèn)為是安全的(GRAS generally recognized as safe)[1]。目前已經(jīng)有相當(dāng)多的文獻(xiàn)將乳酸乳球菌作為基因工程受體菌用于治療腸道炎癥[2]。我們利用基因工程的方法在乳酸乳球菌中表達(dá)人OXM,希望在人體腸道中穩(wěn)定,持續(xù)地表達(dá)OXM,使其能夠成為一種新型的藥物來(lái)治療肥胖癥。為了驗(yàn)證這一假設(shè),本論文構(gòu)建了表達(dá)人OXM的乳酸乳球菌NZ3900,命名為NZ3900-OXM,同時(shí)通過(guò)Western blot等方法鑒定了菌株中OXM的表達(dá),結(jié)果顯示NZ3900-OXM能夠穩(wěn)定表達(dá)OXM并將其分泌至培養(yǎng)基中。隨后,我們通過(guò)45%的高脂飲食構(gòu)建了飲食誘導(dǎo)肥胖小鼠模型,將造模成功的肥胖小鼠分為兩組,分別灌胃表達(dá)OXM的乳酸乳球菌和對(duì)照菌株,觀察表達(dá)OXM的乳酸乳球菌對(duì)飲食誘導(dǎo)肥胖小鼠的體重、體脂、血糖、血脂和進(jìn)食量等指標(biāo)的影響。結(jié)果發(fā)現(xiàn),灌胃表達(dá)OXM的乳酸乳球菌8周后,飲食誘導(dǎo)肥胖小鼠的體重顯著下降至與普通飲食對(duì)照小鼠相一致,而且體脂和血脂也顯著下降。有趣的是,灌胃對(duì)照乳酸菌后肥胖小鼠的血糖也顯著下降,提示乳酸菌本身就足以使血糖的下降,但只有表達(dá)OXM的乳酸乳球菌才能夠顯著降低飲食誘導(dǎo)肥胖小鼠的體重、體脂和血脂。我們進(jìn)一步在db/db小鼠上進(jìn)行了驗(yàn)證。結(jié)果發(fā)現(xiàn),經(jīng)過(guò)三周的灌胃給藥,NZ3900-OXM組的糖尿病小鼠的體重得到了很好的控制,而且糖化血紅蛋白含量、血脂含量顯著下降。本論文通過(guò)以上研究,旨在通過(guò)構(gòu)建表達(dá)人胃泌酸調(diào)節(jié)素的乳酸乳球菌,有效地降低小鼠進(jìn)食量、緩解二型糖尿病癥狀,從而為更安全、方便地治療肥胖癥和二型糖尿病找到一種可能的新型口服藥物,并值得進(jìn)一步開(kāi)發(fā)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R589.2

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本文編號(hào)：2131793