本文選題：甘油三酯水解酶(TGH) + 敏感脂肪酶(HSL)�。� 參考：《西北農(nóng)林科技大學(xué)》2007年碩士論文

【摘要】： 脂肪細胞內(nèi)甘油三酯的水解對機體能量代謝有著重要影響。脂解的平衡不僅可以有效地為機體提供能量,而且可以防止因體脂過度沉積引發(fā)的肥胖以及胰島素抵抗等一系列代謝性疾病,因此對脂解的調(diào)控研究十分關(guān)鍵。脂肪分解酶是脂解反應(yīng)中的關(guān)鍵因素。甘油三酯水解酶(TGH)是繼激素敏感脂酶(HSL)之后發(fā)現(xiàn)的又一脂肪分解關(guān)鍵酶。以往對于TGH的功能研究主要集中于嚙齒類動物,有關(guān)哺乳動物中TGH的脂解作用研究尚未見報道;TGH是和HSL一樣是在激素刺激作用下發(fā)揮作用,還是主要介導(dǎo)基礎(chǔ)狀態(tài)下的脂解,目前尚不明確。 因此,本實驗以與人類生理生化方面最為相似的豬為模式動物,利用細胞培養(yǎng)、甘油三酯含量檢測、甘油釋放量測定,半定量RT-PCR、Western-Blot以及載體構(gòu)建等細胞生物學(xué)和分子生物學(xué)技術(shù)研究了TGH在豬原代脂肪細胞分化中的表達規(guī)律以及細胞饑餓和重新恢復(fù)營養(yǎng)狀態(tài)下的脂解及TGH的表達變化;并且通過在異丙腎上腺素刺激作用下與HSL的對比,初步探討了其在脂肪分解中的作用方式;此外,構(gòu)建了TGH的RNA干擾載體,為實驗室以后進一步研究TGH的功能作出鋪墊。所取得的結(jié)果如下: 1檢測了豬原代脂肪細胞分化過程中TGH的表達。TGH mRNA在前脂肪細胞不表達,在誘導(dǎo)分化第4天表達量最高,之后表達量相對穩(wěn)定,到第10天表達量略有下降。TGH的蛋白表達和其mRNA表達不盡相同,TGH蛋白在第0天幾乎不表達,第二天表達量較低,第六天表達量最高,隨后表達有所降低,然后基本趨于穩(wěn)定。 2檢測了細胞饑餓及重新恢復(fù)營養(yǎng)狀況下脂肪分解的變化及TGH的表達改變,分析該過程中TGH的作用。細胞缺乏營養(yǎng)狀態(tài)下,即用無糖無血清培養(yǎng)基培養(yǎng)時甘油釋放量升高,即脂解作用增強,同時TGHmRNA和蛋白表達與對照相比升高;當恢復(fù)營養(yǎng)(完全培養(yǎng)基培養(yǎng))后其表達又有所下降,但脂解下降不顯著。 3研究了異丙腎上腺素(Isoproterenol)刺激狀態(tài)下脂肪分解及TGH的表達變化,并分析該狀態(tài)下TGH的作用。低濃度的異丙腎上腺素(0.1μmol/L,1μmol/L)沒有檢測到脂肪細胞釋放到培養(yǎng)基中的甘油含量的改變,隨異丙腎上腺素濃度的升高,甘油釋放量顯著上升,同時檢測到細胞中甘油三酯含量下降,即脂解作用增強。此過程中HSL mRNA及蛋白表達均隨異丙腎上腺素濃度的升高以及作用時間的不同而呈劑量和時間依賴性改變,但TGHmRNA和蛋白的表達在此過程中沒有顯著變化。 4構(gòu)建了pSilencer-TGH載體并篩選了潮霉素的最佳致死濃度。對原質(zhì)粒進行酶切、回收,連接、轉(zhuǎn)化、質(zhì)粒提取,最后測序鑒定表明TGH RNA干擾載體已經(jīng)構(gòu)建成功。200μg/mL是潮霉素抗性篩選的最佳濃度。
[Abstract]:The hydrolysis of triglycerides in adipocytes plays an important role in energy metabolism. The balance of lipids can not only provide energy for the body, but also prevent obesity and insulin resistance caused by excessive body fat deposition. Therefore, the regulation of lipid hydrolysis is very important. Lipase is a key factor in the lipopolysis reaction. Triglyceride hydrolase (TGH) is a key lipolytic enzyme after hormone sensitive lipase (HSL). In the past, the functional studies of TGH were mainly focused on rodents. It has not been reported that whether TGH plays the same role as HSL in hormone stimulation or mainly mediates the lipid hydrolysis in basic state. It is not clear yet. Therefore, in this experiment, pig, which is the most similar to human physiology and biochemistry, was used as the model animal. The cell culture, triglyceride content and glycerol release were used in this experiment. Semi-quantitative RT-PCR Western-Blot and vector construction were used to study the expression of TGH in primary adipocyte differentiation, and the changes of lipid and TGH expression in the condition of starvation and restoration of nutrition. By comparing with HSL stimulated by isoproterenol, the mechanism of its action in adipose decomposition was preliminarily discussed. In addition, the RNA interference vector of TGH was constructed to pave the way for further study on the function of TGH in laboratory. The results were as follows: 1 the expression of TGH was detected during the differentiation of porcine primary adipocytes. TGH mRNA was not expressed in preadipocytes, but the expression level was the highest in preadipocytes on the 4th day after differentiation, and then the expression level was relatively stable. On the 10th day, the expression of TGH was slightly decreased. The expression of TGH and its mRNA were not the same. On the second day, the expression of TGH was lower, the expression of TGH was the highest on the sixth day, then the expression decreased, and then it tended to be stable. (2) the changes of lipid decomposition and TGH expression were detected under the condition of cell starvation and recovery of nutrition, and the role of TGH in the process was analyzed. Under the condition of lack of nutrition, the amount of glycerol released increased in the culture medium without glucose and serum, and the expression of TGHmRNA and protein was higher than that of the control. When the nutrition was restored (complete culture medium), its expression decreased again, but the lipolysis did not decrease significantly. 3 the changes of adipose decomposition and TGH expression in Isoproterenol stimulated by isoprenaline were studied, and the role of TGH in this state was analyzed. Low concentration of isoproterenol (0.1 渭 mol / L ~ (-1) 渭 mol / L) did not detect the change of glycerol content released by adipocytes into the medium. With the increase of isoproterenol concentration, glycerol release increased significantly, and the content of triglyceride decreased. In other words, the lipopolysis is enhanced. The expression of HSL mRNA and protein changed in a dose-and time-dependent manner with the increase of isoproterenol concentration and the time of action, but the expression of TGHmRNA and protein did not change in this process. PSilencer-TGH vector was constructed and the optimum lethal concentration of hygromycin was screened. The plasmids were digested, recovered, ligated, transformed, and the plasmid was extracted. The result of sequencing indicated that TGH RNA interference vector had been successfully constructed. 200 渭 g/mL was the best concentration for hygromycin resistance screening.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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本文編號：1777960