TWI620933B - Method of detecting carbohydrate metabolism disorder and the prevention and treatment - Google Patents

Method of detecting carbohydrate metabolism disorder and the prevention and treatment Download PDF

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TWI620933B
TWI620933B TW105137112A TW105137112A TWI620933B TW I620933 B TWI620933 B TW I620933B TW 105137112 A TW105137112 A TW 105137112A TW 105137112 A TW105137112 A TW 105137112A TW I620933 B TWI620933 B TW I620933B
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gpx2
individual
diabetes
expression
performance
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TW201818074A (en
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余明隆
莊萬龍
黃志富
戴嘉言
柯佑民
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高雄醫學大學
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Abstract

本發明係有關於一種檢測醣類代謝異常的方法,包括檢測檢體中GPx2基因表現、GPx2蛋白表現量、或GPx2的蛋白活性,並與一正常個體的GPx2表現量進行比較,其中該個體的GPx2表現量顯著低於該正常個體的GPx2表現量代表該個體具有醣類代謝異常的狀態。本發明另提供一種GPx2用於製備治療及預防第二型糖尿病之醫藥組合物的用途。 The present invention relates to a method for detecting abnormal glucose metabolism, comprising detecting GPx2 gene expression, GPx2 protein expression amount, or GPx2 protein activity in a sample, and comparing the GPx2 expression amount of a normal individual, wherein the individual The amount of GPx2 expressed significantly lower than the GPx2 performance of the normal individual represents a state in which the individual has abnormal carbohydrate metabolism. The invention further provides a use of GPx2 for the preparation of a pharmaceutical composition for the treatment and prevention of type 2 diabetes.

Description

一種檢測醣類代謝異常的方法及其預防及治療 Method for detecting abnormal glucose metabolism and its prevention and treatment

本發明係有關於利用麩胱甘肽過氧化酶2(glutathione peroxidase 2,GPx2)檢測醣類代謝異常的方法,以及用於預防及治療第二型糖尿病的領域。 The present invention relates to a method for detecting abnormal glucose metabolism using glutathione peroxidase 2 (GPx2), and a field for preventing and treating type 2 diabetes.

隨著飲食習慣改變,全世界糖尿病好發率逐年升高,其併發症已成為人類主要的死因之一。近來許多證據顯示C型肝炎病毒為糖尿病的危險因子之一,會加速及增加病人糖尿病的風險。 With the change of eating habits, the prevalence of diabetes in the world has increased year by year, and its complications have become one of the main causes of death in humans. Recently, there is a lot of evidence that hepatitis C virus is one of the risk factors for diabetes, which will accelerate and increase the risk of diabetes in patients.

糖尿病是全世界越來越嚴重的課題,目前並無有效的藥物可以預防糖尿病的發生。而在治療方面,雖已有降血糖藥物,但使用上仍有其限制;有些藥物療效有限,有些則容易造成低血糖的併發症。 Diabetes is a growing problem worldwide, and there are no effective drugs to prevent diabetes. In terms of treatment, although there are hypoglycemic drugs, there are still restrictions on their use; some drugs have limited efficacy, and some are prone to complications of hypoglycemia.

慢性C型肝炎患者常伴隨常隨血糖值異常偏高,甚至糖尿病的發生,但具有此現象的原因並未釐清,推測可能的原因包括TNF-α系統的活化、肝細胞的脂肪化或纖維化導致胰島素阻抗等等。先前研究顯示胰島素阻抗與C型肝炎疾病的進展,特別是肝臟纖維化程度,具有相關性。在以干擾素合併雷巴威靈治療C型肝炎的情況下,治療之前病人的胰島素阻抗已被證實為預測治療成功與否的重要指標之一。另一方面,若能清除C型肝炎病毒,則體內胰島素阻抗情況、胰島β細胞功能、以及肝臟胰島素接受器的 表現皆能獲得改善。因此,C型肝炎病毒本身對胰島素阻抗以至於第二型糖尿病的形成應扮演相當重要的角色;探討此角色及其作用方式,將有助於了解糖尿病的致病機轉,並進一步找出預防及治療糖尿病的標的。 Patients with chronic hepatitis C often have abnormally high blood glucose levels and even diabetes, but the cause of this phenomenon has not been clarified. Possible causes include activation of TNF-α system, fatification of liver cells or fibrosis. Cause insulin resistance and so on. Previous studies have shown that insulin resistance is associated with progression of hepatitis C disease, particularly liver fibrosis. In the case of interferon combined with Rebavirin for the treatment of hepatitis C, the insulin resistance of the patient before treatment has been confirmed as one of the important indicators for predicting the success of treatment. On the other hand, if the hepatitis C virus can be cleared, the insulin resistance in the body, the islet β-cell function, and the liver insulin receptor Performance can be improved. Therefore, the hepatitis C virus itself should play a very important role in insulin resistance and the formation of type 2 diabetes; exploring this role and its mode of action will help to understand the pathogenesis of diabetes and further identify prevention. And the target of treating diabetes.

故,本發明藉由調控C型肝炎引發宿主血糖代謝異常的基因與其相關因子,進而發展出預防及治療糖尿病的標的。 Therefore, the present invention develops a target for preventing and treating diabetes by regulating genes and related factors of abnormal blood glucose metabolism in the host by hepatitis C.

不管是C型肝炎病毒感染或是高脂肪飲食,均會造成GPx2表現下降,使得肝細胞對葡萄糖攝取能力下降、以及肝細胞糖質新生增加,導致血中葡萄糖濃度升高而造成糖尿病。利用基因網路分析軟體發現GPx2與脂肪酸氧化、葡萄糖耐受能力、葡萄糖攝取、及糖質新生的基因都息息相關。由於肝臟胰島素阻抗及肝細胞糖質新生增加是第二型糖尿病的重要致病機轉,故本發明利用提升肝臟GPx2的表現作為預防及治療糖尿病的新標的,且不會造成低血糖的副作用。 Whether it is hepatitis C virus infection or a high-fat diet, it will cause a decline in GPx2 performance, resulting in decreased glucose uptake capacity of hepatocytes and increased gluconeogenesis in hepatocytes, leading to an increase in blood glucose levels and diabetes. The use of gene network analysis software found that GPx2 is closely related to fatty acid oxidation, glucose tolerance, glucose uptake, and gluconeogenesis genes. Since hepatic insulin resistance and hepatocyte gluconeogenesis increase are important pathogens of type 2 diabetes, the present invention utilizes the performance of liver GPx2 as a new standard for the prevention and treatment of diabetes without causing side effects of hypoglycemia.

因此,本發明提供一種檢測一個體醣類代謝異常的方法,包括檢測該個體的檢體中GPx2基因(DNA或RNA)表現、GPx2蛋白表現量、或GPx2的蛋白活性,並與一正常個體的GPx2表現量進行比較,其中該個體的GPx2表現量顯著低於該正常個體的GPx2表現量代表該個體具有醣類代謝異常的狀態,特別是該醣類代謝異常係指使血糖濃度高於正常範圍。因此本發明可以GPx2作為醣類代謝異常的標記。 Accordingly, the present invention provides a method for detecting a metabolic abnormality of a body sugar, comprising detecting a GPx2 gene (DNA or RNA) expression, a GPx2 protein expression amount, or a GPx2 protein activity in a sample of the individual, and interacting with a normal individual The amount of GPx2 expression is compared, wherein the GPx2 expression of the individual is significantly lower than the GPx2 expression of the normal individual, indicating that the individual has a state of abnormal glucose metabolism, and in particular, the abnormal glucose metabolism means that the blood glucose concentration is higher than the normal range. Therefore, the present invention can use GPx2 as a marker for abnormal carbohydrate metabolism.

本發明另提供一種GPx2用於製備治療及預防第二型糖尿病之醫藥組合物的用途。在高脂飲食一開始給予GPx2可使糖質新生相關蛋白的表現量下降、GLUT(葡萄糖轉運蛋白)表現上升,因此GPx2具有預防 第二型糖尿病的功效。而在高脂飲食誘發產生第二型糖尿病後給予GPx2亦可使糖質新生相關蛋白的表現量下降、GLUT表現上升,因此GPx2具有治療第二型糖尿病的功效。 The invention further provides a use of GPx2 for the preparation of a pharmaceutical composition for the treatment and prevention of type 2 diabetes. Giving GPx2 at the beginning of a high-fat diet can reduce the expression of gluconeogenesis-related proteins and increase the expression of GLUT (glucose transporter), so GPx2 has prevention. The efficacy of type 2 diabetes. However, the administration of GPx2 after the second type of diabetes induced by the high-fat diet can also reduce the expression of gluconeogenesis-related proteins and increase the GLUT performance, so GPx2 has the effect of treating type 2 diabetes.

本文中所述之「檢體」係收集自生物個體,其中該檢體係選自由以下組成之群:組織、糞便、尿液、細胞均質物、血液、血清、血漿、一或多種生物流體或其任何組合。本發明中所提及之個體為動物,包括人類及哺乳類,其中較佳為人類。 The "sample" described herein is collected from a biological individual, wherein the test system is selected from the group consisting of: tissue, feces, urine, cell homogenate, blood, serum, plasma, one or more biological fluids or Any combination. The individuals referred to in the present invention are animals, including humans and mammals, of which humans are preferred.

本發明之方法特別適用於第二型糖尿病之高風險群體,如體重過重者、不愛活動者、家族因素(例如雙親一方或兄弟有人是第二型糖尿病患者)、種族因素、步入老化者(特別是過了四十五歲)、有糖尿病前期者(前期糖尿病是身體中的血糖值比正常值為高,但尚不足以歸類為第二型糖尿病,如不加以控制會發展為第二型糖尿病)、或曾有妊娠糖尿病者。 The method of the present invention is particularly suitable for high-risk groups of type 2 diabetes, such as overweight, non-active people, family factors (for example, parents or brothers are type 2 diabetes patients), ethnic factors, and aging (especially after the age of forty-five), those with pre-diabetes (pre-diabetes is a higher blood sugar level than normal, but not enough to be classified as type 2 diabetes, if not controlled, it will develop into Type 2 diabetes), or those who have had gestational diabetes.

透過本發明的實施例了解到,在具有C型肝炎病毒蛋白表現的肝細胞中(C型肝炎病毒複製子),GPx2的表現量降低、細胞的葡萄糖攝取亦會降低,且糖質新生大幅增加。經由給予抗C型肝炎病毒藥物(如Daclatasvir)可抑制C型肝炎病毒蛋白,或透過轉殖GPx2過度表現的質體,均可增加GPx2表現量,並有效增加肝細胞葡萄糖的攝取以及降低糖質新生。 It has been understood from the examples of the present invention that in hepatocytes having hepatitis C virus protein expression (hepatitis C virus replicon), the expression of GPx2 is decreased, the glucose uptake of cells is also decreased, and the gluconeogenesis is greatly increased. . Inhibition of hepatitis C virus protein by administration of anti-hepatitis C virus drugs (such as Daclatasvir), or plastids overexpressing GPx2, can increase GPx2 expression and effectively increase hepatocyte glucose uptake and reduce glycogen content. newborn.

透過不具有C型肝炎病毒複製子且GPx2表現正常的肝細胞,經由轉殖GPx2的siRNA調降GPx2的表現來觀察GPx2所影響的代謝機轉。由結果可知,添加GPx2的siRNA可以降低GPx2的表現,且同時影響胰島素的訊息途徑,降低肝細胞的葡萄糖攝取,並大幅增加肝細胞糖質新生的進行。圖15所示,圖15A為Huh7.5細胞經由GPx2 siRNA處理而抑制GPx2 表現後,經西方點墨法發現當GPx2表現量下降時,細胞內的GLUT1(葡萄糖轉運蛋白1)與GLUT2(葡萄糖轉運蛋白2)的表現量亦下降;細胞內的PEPCK與G6PC(G6Pase)表現量則明顯增加(圖15B)。;而肝細胞糖質新生的增加則是導致糖尿病的重要因素之一。由此可知,GPx2是肝細胞調控葡萄糖代謝的重要樞紐,C型肝炎病毒乃是藉由影響肝細胞GPx2的表現造成宿主的葡萄糖代謝異常而引發第二型糖尿病。因此透過矯正或增強肝細胞GPx2的低表現,可有效回復肝細胞的葡萄糖代謝異常現象。 Metabolites affected by GPx2 were observed by down-regulating the expression of GPx2 by transfecting GPx2 siRNA into hepatocytes that did not have a hepatitis C virus replicon and normal GPx2 expression. From the results, it can be seen that the addition of GPx2 siRNA can reduce the performance of GPx2, and at the same time affect the insulin signaling pathway, reduce the glucose uptake of hepatocytes, and greatly increase the gluconeogenesis of hepatocytes. As shown in Figure 15, Figure 15A shows that Huh7.5 cells inhibit GPx2 via GPx2 siRNA treatment. After performance, the expression of GLUT1 (glucose transporter 1) and GLUT2 (glucose transporter 2) in cells decreased when the expression of GPx2 decreased, and the expression of PEPCK and G6PC (G6Pase) in cells was observed by Western blotting. The amount is significantly increased (Fig. 15B). And the increase in gluconeogenesis in hepatocytes is one of the important factors leading to diabetes. It can be seen that GPx2 is an important hub for hepatocytes to regulate glucose metabolism. Hepatitis C virus causes type 2 diabetes by affecting the abnormal glucose metabolism of the host by affecting the performance of hepatocyte GPx2. Therefore, by correcting or enhancing the low performance of hepatocyte GPx2, it can effectively restore the abnormal glucose metabolism of hepatocytes.

透過建構GPx2過度表現的質體,並將質體以靜脈注射打入高脂食物飼養的小鼠體內,藉以觀察GPx2對血糖的影響。由結果可知,相較於GPx2正常表現的小鼠而言,早期給予GPx2高度表現質體的小鼠可明顯預防糖質新生的增加、有效改善葡萄糖代謝異常以及控制血糖,進而減低罹患糖尿病的風險。此外,在已引發葡萄糖代謝異常的高脂餵食糖尿病鼠中,施打過度表現GPx2質體來進行治療,亦可有效改善醣類代謝異常的情形,達到控制血糖的目的。如圖3B所示,經高脂餵食16週而成功誘導為糖尿病鼠後,藉由尾靜脈給予GPx2過度表現質體32週後可以有效改善醣類代謝異常。由以上結果可證實肝細胞GPx2是影響血糖調控的重要樞紐;矯正或增強肝細胞GPx2的表現可以有效改善葡萄糖代謝異常,進而達到控制血糖的目的。因此GPx2不但可預防糖尿病,亦可在糖尿病已發生時達到治療的效果。本發明的組合物適用於腸內或腸胃外應用的有機或無機的載劑或賦型劑。例如,可將活性成分複合通常無毒且在藥學上可接受用於片劑、丸劑、膠囊劑、栓劑、溶液、懸浮液,和任何其他適合使用的形式的載體。可以使用的載體包括葡萄糖、乳糖、阿拉伯膠、明膠、甘露糖醇、澱粉糊, 三矽酸鹽、滑石、玉米澱粉、角蛋白、膠體二氧化矽、馬鈴薯澱粉、尿素、中等鏈長甘油三酯、葡聚醣,和其他適用於製造固體、半固體或液體形式製劑的載體。此外,亦可使用輔劑、穩定劑、增稠劑和著色劑和香料。 Through the construction of GPx2 over-expressed plastids, and the plastids were injected intravenously into mice fed high-fat diets to observe the effect of GPx2 on blood glucose. From the results, it was found that mice with high GPx2 expression of plastids in early stage can significantly prevent the increase of gluconeogenesis, effectively improve glucose metabolism abnormality and control blood sugar, thereby reducing the risk of diabetes. . In addition, in the high-fat-fed diabetic rats that have caused abnormal glucose metabolism, the GPx2 plastid is over-expressed for treatment, and the abnormal glucose metabolism can be effectively improved to achieve the purpose of controlling blood sugar. As shown in Fig. 3B, after successful induction of diabetic rats by high-fat diet for 16 weeks, GPx2 over-expressed plastids for 32 weeks in the tail vein could effectively improve carbohydrate metabolism abnormalities. The above results can confirm that hepatocyte GPx2 is an important hub affecting blood glucose regulation; correcting or enhancing the performance of hepatocyte GPx2 can effectively improve glucose metabolism abnormality, and thus achieve the purpose of controlling blood sugar. Therefore, GPx2 not only prevents diabetes, but also achieves therapeutic effects when diabetes has already occurred. The compositions of the present invention are suitable for use in organic or inorganic carriers or excipients for enteral or parenteral applications. For example, the active ingredient may be complexed, usually non-toxic, and pharmaceutically acceptable for use in the compositions in the form of tablets, pills, capsules, suppositories, solutions, suspensions, and any other suitable form. Carriers which may be used include glucose, lactose, acacia, gelatin, mannitol, starch paste, Tricaprate, talc, corn starch, keratin, colloidal cerium oxide, potato starch, urea, medium chain length triglycerides, dextran, and other carriers suitable for the manufacture of solid, semi-solid or liquid form preparations. In addition, adjuvants, stabilizers, thickeners, colorants and perfumes can also be used.

本發明的組合物可為一適合口服使用的形式,例如,作為錠劑、片劑、糖錠、水性或油性懸浮液、散粉劑或顆粒劑、乳劑、硬或軟膠囊,或糖漿或酏劑。用於口服使用的組合物或混合物,可以根據製造醫藥品領域中已知的任何方法來製備。 The composition of the present invention may be in a form suitable for oral use, for example, as a tablet, a tablet, a lozenge, an aqueous or oily suspension, a powder or granule, an emulsion, a hard or soft capsule, or a syrup or elixir. . Compositions or mixtures for oral use can be prepared according to any method known in the art of manufacture of pharmaceuticals.

本發明的醫藥組合物亦可為無菌注射懸浮液。此懸浮液可根據已知的方法,使用適合的散佈劑、浸潤劑和懸浮劑來配製。無菌注射液也可以為一以無毒、胃腸道可接受的稀釋劑或溶劑來配製的無菌注射溶液或懸浮液。 The pharmaceutical compositions of the invention may also be sterile injectable suspensions. This suspension can be formulated according to known methods using suitable dispersing agents, wetting agents and suspending agents. The sterile injectable solution can also be a sterile injectable solution or suspension in the form of a non-toxic, pharmaceutically acceptable diluent or solvent.

圖1所示為經qPCR分析的GPx2 Log2蛋白表現量;1A:GPx2在葡萄糖耐受異常組的表現量;1B:GPx2在驗證組的表現量。 Figure 1 shows the GPx2 Log2 protein expression by qPCR analysis; 1A: GPx2 performance in the glucose tolerance abnormal group; 1B: GPx2 performance in the validation group.

圖2所示為利用2-△CT方法計算24週高脂飲食的小鼠肝臟GPx2、PEPCK、G6PC(G6Pase)表現量(與GAPDH比較,以倍數呈現)(與控制組相比,*:p<0.05;**:p<0.01;***:p<0.001;與空載體vehicle相比,###:p<0.001)。 Figure 2 shows the expression of GPx2, PEPCK, and G6PC (G6Pase) in the liver of mice fed a 24-week high-fat diet by 2 -ΔCT method (compared with GAPDH, in multiples) (*:p compared with control group) <0.05; **: p<0.01; ***: p<0.001; compared with empty vector vehicle, ###: p<0.001).

圖3所示為高脂飲食後小鼠的IPGTT結果;3A:24週高脂飲食並給予GPx2過度表現質體的IPGTT結果;3B:16週高脂飲食誘發第二型糖尿病後給予GPx2過度表現質體的IPGTT結果(**:與控制組相比p<0.01;##:與空載體vehicle相比p<0.01)。 Figure 3 shows the IPGTT results of mice after high-fat diet; 3A: 24-week high-fat diet and GPx2 over-expressed plastid IPGTT results; 3B: 16-week high-fat diet induced GPx2 overexpression after type 2 diabetes Peptide IPGTT results (**: p<0.01 compared to control group; ##: p<0.01 compared to empty vector vehicle).

圖4所示為C型肝炎病患之肝臟組織切片之RNA及cDNA完整性確認圖;4A:RNA濃度及完整性確認圖;4B:cDNA完整性確認圖。 Figure 4 shows the RNA and cDNA integrity confirmation map of liver tissue sections of patients with hepatitis C; 4A: RNA concentration and integrity confirmation map; 4B: cDNA integrity confirmation map.

圖5所示為微陣列晶片實驗進行全基因表現偵測結果圖。 Figure 5 shows the results of the whole gene performance detection in the microarray wafer experiment.

圖6所示為GPx2分別在HepG2、Con1、Huh7.5細胞株中的表現量。 Figure 6 shows the expression levels of GPx2 in HepG2, Con1, Huh7.5 cell lines, respectively.

圖7所示為給予GPx2過度表現載體之Con1細胞株的GLUT1及GLUT2表現量(*:與空載體vehicle相比p<0.05;**:與空載體vehicle相比p<0.01;#:與控制組相比p<0.05)。 Figure 7 shows the GLUT1 and GLUT2 expression levels of Con1 cell lines given GPx2 overexpression vector (*: p<0.05 compared to empty vector vehicle; **: p<0.01 compared to empty vector vehicle; #: and control Group vs. p<0.05).

圖8所示為給予GPx2過度表現載體之Con1細胞株的G6Pase及PEPCK表現量(*:與空載體vehicle相比p<0.05;**:與空載體vehicle相比p<0.01;##:與控制組相比p<0.01;###:與控制組相比p<0.001)。 Figure 8 shows the G6Pase and PEPCK expression levels of Con1 cell lines given GPx2 overexpression vector (*: p<0.05 compared to empty vector vehicle; **: p<0.01 compared to empty vector vehicle; ##: with The control group was p<0.01; ###: p<0.001 compared with the control group.

圖9所示為給予不同濃度之HCV專一性病毒抑制劑(Daclatasvir)的Con1細胞株的GPx2、GLUT1及GLUT2表現量(與DMSO(Daclatasvir濃度0pM)相比,*:p<0.05;**:p<0.01;***:p<0.001;與Daclatasivir相異濃度作用相比,#:p<0.05;##:p<0.01)。 Figure 9 shows the GPx2, GLUT1 and GLUT2 expression levels of Con1 cell lines administered with different concentrations of HCV-specific viral inhibitors (Daclatasvir) (*: p<0.05 compared to DMSO (Daclatasvir concentration 0 pM); **: p < 0.01; ***: p < 0.001; compared with the effect of Daclatasivir different concentrations, #: p < 0.05; ##: p < 0.01).

圖10所示為給予不同濃度之HCV專一性病毒抑制劑(Daclatasvir)的Con1細胞株的G6PC(G6Pase)及PEPCK表現量(與DMSO(Daclatasvir濃度0pM)相比,*:p<0.05;**:p<0.01;***:p<0.001;與Daclatasivir相異濃度作用相比,#:p<0.05;##:p<0.01)。 Figure 10 shows the G6PC (G6Pase) and PEPCK expression levels of Con1 cell lines administered with different concentrations of HCV-specific viral inhibitors (Daclatasvir) (*: p<0.05 compared to DMSO (Daclatasvir concentration 0 pM); : p < 0.01; ***: p < 0.001; compared with the effect of Daclatasivir different concentrations, #: p < 0.05; ##: p < 0.01).

圖11所示為24週高脂飲食之小鼠肝臟GPx2表現量。 Figure 11 shows the amount of liver GPx2 expression in mice on a 24-week high-fat diet.

圖12所示為為48週高脂飲食之小鼠肝臟GPx2表現量。 Figure 12 shows the amount of liver GPx2 expression in mice on a 48-week high-fat diet.

圖13所示為48週高脂飲食之小鼠肝臟GLUT表現量。 Figure 13 shows the liver GLUT expression in mice on a 48-week high-fat diet.

圖14所示為48週高脂飲食之小鼠肝臟糖質新生相關蛋白表現量。 Figure 14 shows the amount of liver gluconeogenesis-related protein expression in mice on a 48-week high-fat diet.

圖15所示為GPx2 siRNA處理而抑制GPx2表現後之GLUT2、G6PC(G6Pase)及PEPCK的表現量。 Figure 15 shows the expression levels of GLUT2, G6PC (G6Pase) and PEPCK after GPx2 siRNA treatment and inhibition of GPx2 expression.

細胞實驗Cell experiment

將複製子細胞Huh7.5及Huh7.5/Con1(基因型1b)培養於高葡萄糖的Dulbecco’s Modified Eagle’s培養基(DMEM),培育於5%二氧化碳/37℃孵育箱中,並補充10%熱滅活性的胎牛血清(heat-inactivated fetal bovine serum,heat-inactivated FBS)、5%抗生素-抗黴菌素(antibiotic-antimycotic)溶液、100U/mL盤尼西林、100μg/mL鏈黴素、及5%非必需胺基酸溶液。細胞株需先飢餓處理24小時後,再加入含有10% FBS及annonacin的新鮮培養基並進行細胞實驗。。Con1細胞株持續養育於完全培養基(含有0.5mg/mL的G418)。 Replicon cells Huh7.5 and Huh7.5/Con1 (genotype 1b) were cultured in Dulbecco's Modified Eagle's medium (DMEM) with high glucose, incubated in a 5% carbon dioxide/37 °C incubator, and supplemented with 10% heat-killing activity. Heat-inactivated fetal bovine serum (heat-inactivated FBS), 5% antibiotic-antibiotic-antimycotic solution, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% non-essential amine Base acid solution. The cell line was first starved for 24 hours, then fresh medium containing 10% FBS and annanacin was added and cell experiments were performed. . The Con1 cell line was continuously reared in complete medium (containing 0.5 mg/mL of G418).

西方墨點法Western ink point method

將30μg的細胞溶解物樣本置於10% SDS-polyacrylamide膠體上,跑電泳,並轉漬至PVDF膜。阻抗後,再將該膜分別浸於含有目標蛋白GPx2、GLUT1、PCK1(PEPCK)、GLUT2、與G6PC(G6Pase)抗體(一級抗體)的溶液中,並培育兩小時,再用含有0.1% tween 20的PBS洗五分鐘。接著該膜培育於HRP複合二級抗體一小時。將增強化學發光試劑(ECL)加在膜上照光,藉以觀測蛋白質分布。 30 μg of the cell lysate sample was placed on a 10% SDS-polyacrylamide colloid, run for electrophoresis, and transferred to a PVDF membrane. After the impedance, the membrane was separately immersed in a solution containing the target protein GPx2, GLUT1, PCK1 (PEPCK), GLUT2, and G6PC (G6Pase) antibody (primary antibody), and incubated for two hours, and then contained 0.1% tween 20 Wash the PBS for five minutes. The membrane was then incubated in HRP complex secondary antibody for one hour. An enhanced chemiluminescence reagent (ECL) was applied to the film to observe the protein distribution.

質體準備及短暫轉染Plastid preparation and transient transfection

將細胞以每格1.2x105顆的數量置於六孔盤培養基裡,並使之成長一晚。在特定的指定期間利用LipofectAMINE®將GPx2過度表現的質體及siGPx2(GPx2 siRNA)轉染至該細胞。另外,載體僅轉染pcDNA(空載體,vihecle)或siNC(control siRNA)作為控制組。 The number of cells per division pieces of 1.2x10 5 was placed in six-well culture plate and allowed to grow overnight. Using LipofectAMINE ® during a particular plastid specify GPX2 over-represented and siGPx2 (GPx2 siRNA) transfected into the cells. In addition, the vector was only transfected with pcDNA (vihecle) or siNC (control siRNA) as a control group.

動物實驗Animal experiment

24週:以高脂餵食誘導醣類代謝異常的小鼠:將六週大的C57BL/6公小鼠飼養於無病原的環境兩週。之後所有的小鼠隨機分成六組,每組至少有五隻小鼠。組別1餵食正常食物;組別2餵食正常食物,且每個禮拜一次自小鼠尾部靜脈注射質體載體(50μg/plasmid/小鼠/週,溶於Turbofect);組別3餵食正常食物,且每個禮拜一次自小鼠尾部靜脈注射GPx2過度表現質體(50μg/plasmid/小鼠/週,溶於Turbofect);組別4餵食比正常食物多出30%高脂肪食物;組別5餵食比正常食物多出30%高脂肪食物,且每個禮拜一次自小鼠尾部靜脈注射質體載體(50μg/plasmid/小鼠/週,溶於Turbofect);組別6餵食比正常食物多出30%高脂肪食物,且每個禮拜一次自小鼠尾部靜脈注射GPx2過度表現質體(50μg/plasmid/小鼠/週,溶於Turbofect)。所有小鼠24週後犧牲,收取其組織並分成三部分:第一部分以4%甲醛固定,石蠟包埋,作為組織切片研究;第二部分的組織儲存於組織RNA樣品保存液(RNAlater)並置於-80℃環境,以備之後基因表現檢測之用;第三部分的組織則儲存於液態氮。 24 weeks: mice fed with high fat feeding to induce abnormal glucose metabolism: Six-week old C57BL/6 male mice were housed in a pathogen free environment for two weeks. All mice were then randomized into six groups of at least five mice each. Group 1 was fed normal food; group 2 was fed normal food, and once every week, the plastid carrier (50 μg/plasmid/mouse/week, dissolved in Turbofect) was injected from the tail of the mouse; group 3 was fed normal food. And once every week, GPx2 overexpressed plastids (50μg/plasmid/mouse/week, dissolved in Turbofect) from the tail of the mouse; group 4 fed 30% more fatty food than normal food; group 5 fed 30% more fatty food than normal food, and once every week from the tail of the mouse intravenous plastid carrier (50μg/plasmid/mouse/week, dissolved in Turbofect); group 6 fed 30 times more than normal food % high-fat food, and GPx2 overexpressing plastids (50 μg/plasmid/mouse/week, dissolved in Turbofect) from the tail vein of the mouse once a week. All mice sacrificed after 24 weeks, their tissues were collected and divided into three parts: the first part was fixed with 4% formaldehyde, embedded in paraffin, as a tissue section study; the second part of the tissue was stored in tissue RNA sample preservation solution (RNAlater) and placed -80 ° C environment for later detection of gene expression; the third part of the tissue is stored in liquid nitrogen.

48週:將六週大的C57BL/6公小鼠飼養於無病原的環境兩週。之後所有的小鼠隨機分成四組,每組至少有五隻小鼠。組別1餵食正常食物;組別2餵食比正常食物多出30%高脂肪食物;組別3餵食比正常食物多 出30%高脂肪食物,且在實驗開始餵食的16週後每個禮拜一次自小鼠尾部靜脈注射質體載體(50μg/plasmid/小鼠/週,溶於Turbofect);組別4餵食比正常食物多出30%高脂肪食物,且在實驗開始餵食的16週後每個禮拜一次自小鼠尾部靜脈注射GPx2過度表現質體(50μg/plasmid/小鼠/週,溶於Turbofect)。所有小鼠48週後犧牲,收取其組織並分成三部分:第一部分以4%甲醛固定,石蠟包埋,作為組織切片研究;第二部分的組織儲存於組織RNA樣品保存液(RNAlater)並置於-80℃環境,以備之後基因表現檢測之用;第三部分的組織則儲存於液態氮。 Week 48: Six-week old C57BL/6 male mice were housed in a pathogen free environment for two weeks. All mice were then randomized into four groups of at least five mice each. Group 1 fed normal food; group 2 fed 30% more fatty food than normal food; group 3 fed more than normal food 30% high-fat food, and the plastid carrier (50μg/plasmid/mouse/week, dissolved in Turbofect) was injected from the tail of the mouse once every week after the start of the experiment. The group 4 was fed more than normal. Food was 30% more fatty, and GPx2 overexpressed plastids (50 μg/plasmid/mouse/week, dissolved in Turbofect) from the tail of the mouse once every week after 16 weeks of feeding. All mice were sacrificed 48 weeks later and their tissues were harvested and divided into three sections: the first part was fixed with 4% formaldehyde and embedded in paraffin as a tissue section study; the second part of the tissue was stored in tissue RNA sample storage (RNAlater) and placed -80 ° C environment for later detection of gene expression; the third part of the tissue is stored in liquid nitrogen.

以高脂餵食誘導醣類代謝異常的小鼠,其肝臟GPx2的表現量明顯降低。 In mice fed with high-fat diets that induced abnormal glucose metabolism, the amount of GPx2 in the liver was significantly reduced.

以高脂餵食小鼠並同時經由尾靜脈給予GPx2過度表現之質體,經由qPCR檢測後發現在具有GPx2過度表現質體的小鼠其肝臟的GPx2表現明顯增加且可有效降低糖質新生相關基因(PEPCK與G6PC(G6Pase))的表現,如圖2所示。 The mice were fed with high fat and simultaneously administered GPx2 overexpressing plastids via the tail vein. After qPCR detection, it was found that the GPx2 expression in the liver of mice with GPx2 overexpressing plastids was significantly increased and the gluconeogenesis related genes were effectively reduced. (PEPCK and G6PC (G6Pase)) performance, as shown in Figure 2.

葡萄糖耐量試驗(Glucose tolerance test,GTT)Glucose tolerance test (GTT)

所有小鼠禁食12小時,並透過腹膜內注射葡萄糖(2g葡萄糖/公斤體重),接著在葡萄糖注射後0分、30分、60分、及120分的時間點自尾部採血,利用血糖檢測儀檢測血糖濃度。 All mice were fasted for 12 hours and injected intraperitoneally with glucose (2 g glucose/kg body weight), followed by blood sampling from the tail at 0, 30, 60, and 120 minutes after glucose injection. Check blood sugar concentration.

結果如圖3A所示,在高脂餵食的小鼠並同時經由尾靜脈給予GPx2過度表現之質體24週後,測試糖類的耐受性,給予GPx2過度表現質體的小鼠具有較佳的醣類代謝情形。如圖3B所示,經高脂餵食16週而成功誘導為糖尿病鼠後,藉由尾靜脈給予GPx2過度表現質體32週後,透過IPGTT (Intraperitoneal glucose tolerance test,腹腔葡萄糖耐量試驗)結果顯示GPx2可以有效改善醣類代謝異常,給予GPx2過度表現質體的小鼠具有較佳的醣類代謝情形,與正常飲食之無糖尿病鼠相當。 As a result, as shown in Fig. 3A, in the high-fat-fed mice and simultaneously administered to the plastids of GPx2 overexpressed via the tail vein for 24 weeks, the tolerance of the saccharide was tested, and the mice to which the GPx2 overexpressed the plastids were better. Sugar metabolism. As shown in Fig. 3B, after being successfully induced into diabetic rats by high-fat feeding for 16 weeks, GPx2 was overexpressed by tail vein for 32 weeks, and then passed through IPGTT. (Intraperitoal glucose tolerance test, abdominal glucose tolerance test) results show that GPx2 can effectively improve carbohydrate metabolism abnormalities, mice given GPx2 over-expressed plastids have better carbohydrate metabolism, comparable to normal diet-free diabetic rats.

免疫組織化學分析Immunohistochemical analysis

將石蠟包埋的肝臟組織切成4μm大小的切片,以100℃微波30分鐘,並阻斷非特異性反應。以一級抗體培養切片於4℃整夜,之後以含有0.2% Tween 20的PBS沖洗10分鐘兩次。以生物素標記二級抗體培養該切片一小時,之後以含有0.2% Tween 20的PBS沖洗10分鐘兩次。最後將該切片染色,觀察各種蛋白的表現。 The paraffin-embedded liver tissue was cut into 4 μm-sized sections, microwaved at 100 ° C for 30 minutes, and the non-specific reaction was blocked. Sections were cultured with primary antibody at 4 ° C overnight, followed by rinsing with PBS containing 0.2% Tween 20 for 10 minutes twice. The sections were incubated with biotinylated secondary antibody for one hour and then washed twice with PBS containing 0.2% Tween 20 for 10 minutes. Finally, the sections were stained to observe the performance of various proteins.

病患募集Patient recruitment

募集48位C型肝炎患者,按照個人口服葡萄糖耐受度測試及糖化血色素進行分類(如表1),19位為正常血糖患者、11位為葡萄糖耐受異常患者、18位為第二型糖尿病患者。 48 patients with hepatitis C were recruited according to individual oral glucose tolerance test and glycated hemoglobin (Table 1), 19 were normal blood glucose patients, 11 were glucose tolerance patients, and 18 were type 2 diabetes. patient.

病毒定型分析Virus typing analysis

每位病患皆進行病毒定型分析,確定病毒基因型和病毒定量分析,並隨機選取5位正常血糖患者、3位葡萄糖耐受異常患者、3位第二型 糖尿病患者且未曾服用胰島素或口服降血糖藥物者為樣本進行基因晶片實驗以篩選候選基因,其他樣本則用作驗證組,用以驗證候選基因使用。兩個樣本組的基本資料如表2。 Each patient underwent virus typing analysis to determine viral genotype and virus quantitative analysis, and randomly selected 5 patients with normal blood glucose, 3 patients with abnormal glucose tolerance, and 3 patients with type 2 Patients with diabetes who have not taken insulin or oral hypoglycemic agents are genetically tested to select candidate genes, and other samples are used as validation groups to validate candidate gene use. The basic information of the two sample groups is shown in Table 2.

肝臟切片Liver section

收集各患者的肝臟切片,個別萃取其RNA,測RNA濃度,再個別轉為cDNA以確認完整性。結果如圖4所示。 Liver sections of each patient were collected, RNA was extracted individually, RNA concentration was measured, and then individually converted to cDNA to confirm integrity. The result is shown in Figure 4.

全基因表現偵測Whole gene performance detection

利用微陣列晶片實驗進行全基因表現偵測,將所有晶片結果進行正規化,如圖5所示。 Whole-genome performance detection was performed using microarray wafer experiments to normalize all wafer results, as shown in Figure 5.

接著分析基因表現差異。以1.5倍差異為門檻值,選取有表現差異的基因。在葡萄糖耐受異常與正常血糖患者的比較中,有81個基因表現上升,77個基因表現下降;在第二型糖尿病患者與正常血糖患者的比較中,有161個基因表現上升,99個基因表現下降。 Then analyze the difference in gene expression. Using a 1.5-fold difference as a threshold, select genes with differences in performance. In the comparison of patients with abnormal glucose tolerance and normal blood glucose, 81 genes showed an increase in performance, and 77 genes showed decreased performance. In the comparison between patients with type 2 diabetes and normal blood glucose, 161 genes showed an increase in 99 genes. Performance has declined.

進行基因網路分析:在葡萄糖耐受異常與第二型糖尿病患者的比較中,有表現差異的基因功能集中於脂質代謝(Lipid metabolism)、分子輸送(molecular transport)、小分子生物化學(small molecule biochemistry)、及維他命和礦物質代謝(vitamin and mineral metabolism)等功能。其結果如表3。 Gene network analysis: In the comparison of glucose tolerance abnormalities with patients with type 2 diabetes, the differentially expressed genes function in Lipid metabolism, molecular transport, and small molecule biochemistry (small molecule). Biochemistry), and functions such as vitamin and mineral metabolism. The results are shown in Table 3.

將葡萄糖耐受異常及第二型糖尿病兩組比較結果合併得到顯著差異的候選基因GPx2:以qPCR方式分析GPx2,發現隨著不同的糖耐 受異常程度,GPx2也有不同程度改變,如圖1A所示。而以qPCR方式測試驗證組樣本,顯示GPx2仍維持顯著差異,且隨著糖耐受性的變差,GPx2基因的表現量亦降低,如圖1B所示。 The candidate gene GPx2 was significantly combined with the results of glucose tolerance abnormality and type 2 diabetes: GPx2 was analyzed by qPCR and found to be resistant to different sugars. Due to the degree of abnormality, GPx2 also changes to varying degrees, as shown in Figure 1A. The test group samples were tested by qPCR, showing that GPx2 still maintained significant differences, and as the glucose tolerance deteriorated, the expression of GPx2 gene also decreased, as shown in Figure 1B.

基因網路分析Gene network analysis

由基因網路分析推估,GPx2與脂肪酸氧化、葡萄糖耐受能力、葡萄糖攝取及糖質新生的基因都息息相關。 Estimated by gene network analysis, GPx2 is closely related to fatty acid oxidation, glucose tolerance, glucose uptake and gluconeogenesis genes.

GPx2在醣類代謝的角色The role of GPx2 in carbohydrate metabolism

在細胞實驗中驗證GPx2在醣類代謝的角色。C型肝炎病毒(Hepatitis C virus,HCV)經由抑制GPx2的表現可使HCV導致肝臟細胞醣類代謝異常(降低葡萄糖運輸與增加糖質新生),而經由增加GPx2的表現可明顯改善HCV所導致的醣類代謝異常現象。 The role of GPx2 in carbohydrate metabolism was verified in cell experiments. Hepatitis C virus (HCV) can cause HCV to cause abnormal glucose metabolism in liver cells (reducing glucose transport and increasing gluconeogenesis) by inhibiting the expression of GPx2, and significantly improving HCV by increasing the performance of GPx2. Abnormal glucose metabolism.

透過質體轉染及西方墨點法的實驗發現,相較於不具有HCV相關基因的細胞(HepG2與Huh7.5),GPx2的表現量在具有HCV type 1b複製子相關基因的細胞(Con1細胞)中明顯降低,如圖6所示。 Through plastid transfection and Western blotting experiments, GPx2 was expressed in cells with HCV type 1b replicon-related genes (Con1 cells) compared to cells without HCV-related genes (HepG2 and Huh7.5). Significantly reduced, as shown in Figure 6.

已有文獻指出,在具有HCV相關基因表現的細胞中(Con1),HCV可減少GLUT的表現量。故本發明透過給予GPx2過度表現的載體增加GPx2的表現量後發現,GLUT的表現量明顯增加,顯示GPx2可改善細胞對於葡萄糖的運輸能力,如圖7所示。 It has been reported in the literature that in cells with HCV-related gene expression (Con1), HCV can reduce the amount of GLUT expression. Therefore, in the present invention, by increasing the expression level of GPx2 by giving a GPx2 over-expressing vector, it was found that the expression amount of GLUT was significantly increased, indicating that GPx2 can improve the cell's ability to transport glucose, as shown in FIG.

已有文獻指出,在具有HCV相關基因表現的細胞(Con1)中,HCV可增加糖質新生相關基因(G6PC(G6Pase)與PEPCK)的表現量。故本發明透過給予GPx2過度表現的載體增加GPx2的表現量後發現,G6PC(G6Pase)與PEPCK的表現明顯降低,抑制細胞內的糖質新生作用,如圖8 所示。 It has been reported that HCV can increase the expression of gluconeogenesis-related genes (G6PC (G6Pase) and PEPCK) in cells with HCV-related gene expression (Con1). Therefore, the present invention increases the expression level of GPx2 by giving a GPx2 over-expression vector, and finds that the expression of G6PC (G6Pase) and PEPCK is significantly reduced, and the gluconeogenesis in the cell is inhibited, as shown in FIG. Shown.

Con1細胞經由具HCV專一性病毒抑制劑(Daclatasvir)處理,經由西方墨點法檢測後發現,隨著抑制劑濃度的增加,病毒量會減少,進而使得GPx2以及GLUT表現量上升,具有濃度依賴的現象,如圖9所示。而Daclatasvir亦會下調糖質新生的相關蛋白G6PC(G6Pase)及PEPCK,具有濃度依賴的現象,如圖10所示。 Con1 cells were treated with HCV-specific virus inhibitor (Daclatasvir) and detected by Western blotting. As the concentration of inhibitor increased, the amount of virus decreased, which led to an increase in the expression of GPx2 and GLUT, which was concentration-dependent. The phenomenon is shown in Figure 9. Daclatasvir also down-regulated the glycoprotein-associated proteins G6PC (G6Pase) and PEPCK, which are concentration-dependent, as shown in Figure 10.

透過免疫組織染色圖可得知,高脂飲食誘導的糖尿病小鼠,其GPx2表現量下降;而給予GPx2過度表現質體後,其GPx2表現量即有回升,如圖11所示。在48週高脂飲食的實驗中,亦可顯著增加GPx2的表現,如圖12所示。高脂飲食誘導的糖尿病小鼠,其GLUT表現量下降;而給予GPx2過度表現質體後,其GLUT表現量即有回升,如圖13。高脂飲食誘導的糖尿病小鼠,其G6PC(G6Pase)及PEPCK的表現量上升;而給予GPx2過度表現質體後,其G6PC(G6Pase)及PEPCK表現量即有回降,如圖14。 Through immunostaining staining, it can be seen that the GPx2 expression decreased in diabetic mice induced by high-fat diet, and the GPx2 expression increased after GPx2 overexpressed plastids, as shown in Figure 11. In the 48-week high-fat diet, the performance of GPx2 was also significantly increased, as shown in Figure 12. The GLUT expression decreased in diabetic mice induced by high-fat diet, and the GLUT expression increased after GPx2 overexpressed plastids, as shown in Figure 13. The expression of G6PC (G6Pase) and PEPCK in diabetic mice induced by high-fat diet increased, while the expression of G6PC (G6Pase) and PEPCK decreased after GPx2 overexpressed plastids, as shown in Fig. 14.

Claims (7)

一種檢測一所需的個體之醣類代謝異常的方法,包括(a)檢測該所需個體的檢體中GPx2基因表現、GPx2蛋白表現量、或GPx2的蛋白活性;及(b)與一正常個體的GPx2表現量進行比較,其中該所需個體的GPx2表現量顯著低於該正常個體的GPx2表現量代表該所需個體具有醣類代謝異常的狀態;其中該所需個體為C型肝炎患者或高脂肪飲食族群。 A method for detecting a glucose abnormality in a desired individual, comprising: (a) detecting a GPx2 gene expression, a GPx2 protein expression amount, or a GPx2 protein activity in a sample of the desired individual; and (b) The individual's GPx2 performance is compared, wherein the GPx2 performance of the desired individual is significantly lower than the GPx2 performance of the normal individual, indicating that the desired individual has a state of abnormal glucose metabolism; wherein the desired individual is a hepatitis C patient Or a high-fat diet group. 如申請專利範圍第1項之方法,其中該檢體係選自由以下組成之群:組織、糞便、尿液、細胞均質物、血液、血清、血漿、一或多種生物流體或其任何組合。 The method of claim 1, wherein the test system is selected from the group consisting of: tissue, feces, urine, cell homogenate, blood, serum, plasma, one or more biological fluids, or any combination thereof. 如申請專利範圍第1項之方法,其中該個體為動物,包括人類及哺乳類。 The method of claim 1, wherein the individual is an animal, including humans and mammals. 如申請專利範圍第1項之方法,其中該個體為人類。 The method of claim 1, wherein the individual is a human. 如申請專利範圍第1項之方法,其中該所需個體為第二型糖尿病之高風險群。 The method of claim 1, wherein the desired individual is a high risk group of type 2 diabetes. 一種利用GPx2製備預防或治療第二型糖尿病之醫藥組合物的用途,其中該組合物包含一有效劑量的GPx2及醫藥上可接受之載體,其中該第二型糖尿病係由C型肝炎病毒感染或高脂肪飲食所導致。 A use of GPx2 for the preparation of a pharmaceutical composition for preventing or treating type 2 diabetes, wherein the composition comprises an effective amount of GPx2 and a pharmaceutically acceptable carrier, wherein the second type of diabetes is infected with hepatitis C virus or Caused by a high-fat diet. 如申請專利範圍第6項之用途,其中該有效劑量至少為可使一投藥對象之GPx2回復至正常表現量之劑量。 The use of the sixth aspect of the patent application, wherein the effective dose is at least a dose which restores GPx2 of a subject to a normal performance amount.
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2005年04月12日,Kidney expression of glutathione peroxidase-1 is not protective against streptozotocin-induced diabetic nephropathy,Am J Physiol Renal Physiol. 2005 Sep;289(3):F544-51. *
2008年12月12日, Gene expression analysis of hepatic roles in cause and development of diabetes in Goto-Kakizaki rats,J Endocrinol. 2009 Mar;200(3):331-46 *

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