羅格列酮對OLETF大鼠肝臟組織病理變化及IL-17、Bcl-xl表達(dá)的影響
發(fā)布時間：2018-08-28 06:59
【摘要】：目的糖尿病(diabetes mellitus DM)是一組代謝性疾病,由于胰島素分泌缺陷和/或障礙引起的以慢性高血糖為主的疾病。長期持續(xù)的高血糖和代謝紊亂,可導(dǎo)致全身組織器官損傷和功能障礙。肝臟在2型糖尿病(T2DM)的致病中有著重要的作用,作為一個靶器官,肝臟在糖尿病狀態(tài)下也會受到損害。持續(xù)的高血糖使得晚期糖基化產(chǎn)物增加,肝小葉結(jié)構(gòu)破壞,纖維化增加,肝細(xì)胞結(jié)構(gòu)和功能發(fā)生變化,肝細(xì)胞炎癥增加,細(xì)胞凋亡加劇等一系列變化。本實(shí)驗(yàn)以與OLETF大鼠其同系的LETO鼠作為對照組,OLETF大鼠以及經(jīng)羅格列酮治療大鼠均為實(shí)驗(yàn)組,觀察各組大鼠肝臟組織的病理改變。并測定肝臟細(xì)胞炎癥因子IL-17以及凋亡因子Bcl-xl的表達(dá)變化。進(jìn)一步來探討羅格列酮對2型糖尿病OLETF大鼠肝臟組織的影響。方法大鼠單籠飼養(yǎng),標(biāo)準(zhǔn)飼料喂養(yǎng),12/12h循環(huán)光照。LETO為正常組12只,給予普通飼料喂養(yǎng),僅注射緩沖溶液。飼養(yǎng)一個月后,每兩周做一次糖耐量實(shí)驗(yàn),大鼠禁食16h后灌胃給予單劑的糖(25%葡萄糖溶液1mL/100g)2.5g/kg作為進(jìn)食,在給藥前及給藥后30、60、120min分別從尾靜脈取血,以血糖儀測得血糖濃度。以血糖峰值16.7mmol/L和負(fù)荷后120min血糖11.1mmol/L診為糖尿病,若只具備其中一個條件則為糖耐量減低。到30周時,共有16只OLETF大鼠作為實(shí)驗(yàn)組模型。之后隨機(jī)分為兩組(n=8),RGZ組灌以羅格列酮稀釋液,劑量為3mg/kg·d。DM組和NC組灌以等量蒸餾水,每日1次灌胃12周。三組大鼠分別采集血液進(jìn)行測量,之后三組大鼠同時處死,分別取肝臟組織。放血后以無菌器械迅速取肝臟組織3塊,其中2塊分別放入DEPC處理過的凍存管中,丟入液氮保存?zhèn)溆?1塊置于10%的福爾馬林中,行石蠟包埋切片。之后進(jìn)行組織學(xué)染色,包括:HE染色、AO染色、Schiff實(shí)驗(yàn)、Masson三色染色、氯化金染色和免疫組織化學(xué)染色。觀察大鼠肝臟組織學(xué)變化、測定肝肝巨噬細(xì)胞和肝儲脂細(xì)胞數(shù)量的變化、觀察肝臟組織細(xì)胞RNA變化、膠原纖維的變化以及IL-17和Bcl-x L的陽性表達(dá)改變。另外,用置于-70℃的肝臟組織制成組織勻漿來,羥脯氨酸法測定膠原蛋白的含量。所有數(shù)據(jù)均采用SPSS13.0軟件進(jìn)行處理。結(jié)果1.大鼠一般狀況比較:與leto大鼠相比,oletf大鼠體型較為肥胖,神經(jīng)較為敏感易激怒,懶動畏寒,精神萎靡、焦慮,且肝臟體積明顯變大。2.生化指標(biāo)的改變:處死大鼠之前分別測得oletf大鼠餐后兩小時血糖,甘油三酯、谷丙轉(zhuǎn)氨酶和谷草轉(zhuǎn)氨酶以及肝指數(shù)。與nc組相比,dm組餐后兩小時血糖,甘油三酯、谷丙轉(zhuǎn)氨酶、天冬氨酸轉(zhuǎn)移酶和肝指數(shù)指標(biāo)均有明顯升高,rzg組與dm組相比,dm組餐后兩小時血糖,甘油三酯、谷丙轉(zhuǎn)氨酶有顯著降低,但谷草轉(zhuǎn)氨酶無明顯差異。3.肝臟組織學(xué)變化:nc組大鼠肝小葉規(guī)則,肝細(xì)胞索呈放射狀整齊排列,肝血竇結(jié)構(gòu)清晰,肝細(xì)胞核圓居中,胞質(zhì)均勻,沒有炎性細(xì)胞浸潤。dm組肝小葉失去正常結(jié)構(gòu),肝細(xì)胞索變窄,肝血竇界限不清,肝細(xì)胞腫脹、脂肪變,壞死,偶見點(diǎn)狀炎性灶。rgz肝細(xì)胞結(jié)構(gòu)尚清晰,肝細(xì)胞索結(jié)構(gòu)排列尚整齊,雖然也存在脂性空泡和炎性細(xì)胞,但較糖尿病對照組明顯減少4.肝臟肝巨噬細(xì)胞的比較:通過觀察he染色結(jié)果,計數(shù)各組織中肝巨噬細(xì)胞的數(shù)量,nc組肝巨噬細(xì)胞計數(shù)結(jié)果為17.05±0.89,dm組肝巨噬細(xì)胞數(shù)量為25.31±1.23,rgz組結(jié)果為38.61±4.02。結(jié)果顯示dm組較nc組相對增多,但rgz組肝巨噬細(xì)胞結(jié)果較nc組和dm組都明顯增加(p0.01)。5.肝臟肝細(xì)胞糖原含量的變化:nc組肝糖原定位于胞漿,分布均勻密集,糖原顆粒染色均一呈粉紅色。dm組肝細(xì)胞腫脹、擁擠,糖原分布疏松,糖原顆粒不明顯。rgz組肝糖原較糖尿病組密布,顆粒相對均一。測定肝細(xì)胞糖原顆�；叶�,nc組肝細(xì)胞糖原的灰度值為62.38±10.16,dm組(109.54±11.18)與nc組相比明顯增高,具有統(tǒng)計學(xué)意義(p0.01),rgz組(79.36±9.98)與nc組相比無統(tǒng)計學(xué)意義,與dm組相比明顯降低(p0.05)。6.肝臟組織rna含量變化:吖啶橙熒光染色使正常細(xì)胞核dna呈綠色或黃綠色均勻熒光,細(xì)胞質(zhì)和核仁的rna染為桔黃(紅)色熒光,測定胞質(zhì)中rna熒光染色灰度,與nc組(47.21±8.72)相比,dm組(89.84±10.71)和rgz組(61.25±8.13)灰度值增高,差異均有統(tǒng)計學(xué)意義(p0.01),與dm組相比,rgz組灰度值降低(p0.05)。7.肝臟儲脂細(xì)胞(hsc)的觀察:肝臟儲脂細(xì)胞位于狄氏間隙和肝細(xì)胞之間,經(jīng)氯化金染色使得肝儲脂細(xì)胞呈紫褐色,背景淡灰色。通過高倍鏡下儲脂細(xì)胞計數(shù),與NC組肝儲脂細(xì)胞(2.75±0.21)相比DM組(7.19±0.45)升高(P0.01),與DM組相比RGZ組儲脂細(xì)胞數(shù)值(4.93±0.48)降低(P0.01)。8.膠原纖維的變化:NC組大鼠肝小葉中央靜脈管壁薄,門管區(qū)有少量結(jié)締組織,其所占面積的百分比為[(3.19±0.75)%],與NC組相比,DM組[(15.87±0.63)%]肝小葉中央靜脈管壁增厚,門管區(qū)結(jié)締組織增多,膠原沉積(P0.01),與DM組相比,RGZ組[(10.06±0.54)%]在門管區(qū)可見纖維間隔,較DM組膠原纖維減少(P0.05)。9.膠原蛋白含量的比較:高效液相色譜法測定每克肝臟組織中的膠原蛋白的含量,與NC組(64.52±21.51 mg/g)相比,DM組(134.54±18.78 mg/g)膠原蛋白含量明顯增加(P0.01),RGZ組(91.59±10.75 mg/g)低于DM組(P0.01),有統(tǒng)計學(xué)意義。10.肝臟組織IL-17表達(dá)變化:與NC組成肝臟IL-17陽性表達(dá)吸光度值(0.129±0.045)相比,DM組(0.520±0.021)升高(P0.01),而RGZ組(0.235±0.034)低于DM組(P0.01)。11.肝臟組織Bcl-xl表達(dá)變化:與NC組肝臟組織Bcl-x L陽性表達(dá)吸光度值(0.613±0.014),DM組(0.242±0.041)降低(P0.05),RGZ組肝臟組織Bcl-x L陽性表達(dá)(0.369±0.048)較DM組升高(P0.01)。結(jié)論1.糖尿病大鼠肝小葉結(jié)構(gòu)紊亂,肝細(xì)胞腫脹,膠原纖維沉積,膠原蛋白含量明顯增加。2.糖尿病組HSC細(xì)胞活化增生,導(dǎo)致肝膠原纖維含量增加。3.糖尿病組肝小葉和門管區(qū)炎性細(xì)胞增多,可能與肝巨噬細(xì)胞數(shù)量增加有關(guān);過多肝巨噬細(xì)胞加重糖尿病肝組織損傷。4.糖尿病組IL-17陽性表達(dá)增加,Bcl-xl表達(dá)降低,且以中央靜脈為中心向周圍發(fā)射狀分布,可能與血液供給有關(guān)。5.羅格列酮對OLETF 2型糖尿病大鼠肝臟組織的有一定的保護(hù)作用,能夠減輕糖尿病對肝臟組織損害程度。
[Abstract]:Objective Diabetes mellitus (DM) is a group of metabolic diseases, mainly caused by chronic hyperglycemia due to insulin secretion deficiencies and/or disorders. Long-term persistent hyperglycemia and metabolic disorders can lead to systemic tissue and organ damage and dysfunction. The liver plays an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). As a target organ, the liver can also be damaged in diabetic state. Persistent hyperglycemia leads to the increase of advanced glycosylation products, the destruction of hepatic lobules, the increase of fibrosis, the change of hepatocyte structure and function, the increase of hepatocyte inflammation and the increase of apoptosis. As control group, OLETF rats and rosiglitazone-treated rats were both experimental groups. The pathological changes of liver tissues were observed and the expression of IL-17 and Bcl-xl were measured. The effect of rosiglitazone on liver tissues of OLETF rats with type 2 diabetes mellitus was further investigated. Methods Rats were fed in single cage. The rats were fed with standard diet for 12/12 hours and were fed with normal diet and injected with buffer solution only. At 30 weeks, 16 OLETF rats were randomly divided into two groups (n=8). The RGZ group was given rosiglitazone dilution, and the RGZ group was given rosiglitazone. The rats in the three groups were collected blood for measurement, and then the liver tissues were taken. After bleeding, three pieces of liver tissues were quickly taken out with aseptic instruments. Two of them were put into DEPC-treated cryopreserve tubes and stored in liquid nitrogen for reserve. The rats were placed in 10% formalin and paraffin-embedded sections, then stained histologically, including HE staining, AO staining, Schiff test, Masson trichrome staining, gold chloride staining and immunohistochemical staining. Changes of collagen fibers and positive expression of IL-17 and Bcl-x L were observed. In addition, liver tissue homogenate was prepared at - 70 C and the content of collagen was determined by hydroxyproline method. All data were processed by SPSS 13.0 software. Results 1. Comparing with Leto rats, the body type of OLETF rats was more than that of Leto rats. Obesity, nervous sensitivity, irritability, laziness, chills, depression, anxiety, and liver volume significantly increased. 2. Biochemical indicators: before the execution of OLETF rats were measured two hours postprandial blood glucose, triglyceride, alanine aminotransferase and glutamic oxaloacetic transaminase and liver index. Compared with DM group, blood glucose, triglyceride and alanine aminotransferase in DM group were significantly decreased, but there was no significant difference in glutamic oxaloacetic aminotransferase. 3. histological changes of liver: the hepatic lobules of NC rats were regular, the hepatic cords were arranged in a radial fashion, and the hepatic sinusoids were in good order. In DM group, hepatic lobules lost normal structure, hepatic cords narrowed, hepatic sinusoids were unclear, hepatocytes swelled, steatosis, necrosis, occasional punctate inflammatory foci. RGz hepatocyte structure was still clear, hepatic cords were arranged regularly, although there were also lipid vacuoles and inflammation The number of hepatic macrophages in the NC group was 17.05 (+ 0.89), the number of hepatic macrophages in the DM group was 25.31 (+ 1.23) and that in the RGz group was 38.61 (+ 4.02). The content of glycogen in hepatocytes of RGz group was more than that of NC group and DM group (p0.01). 5. the content of glycogen in hepatocytes of RGz group was higher than that of NC group and DM group. The gray level of hepatocyte glycogen granules in NC group was 62.38 (+ 10.16), while that in DM group (109.54 (+ 11.18) was significantly higher than that in NC group (p0.01), and that in RGz group (79.36 (+ 9.98) was significantly lower than that in NC group (p0.05). Acridine orange fluorescence staining made the normal nuclear DNA show green or yellow-green uniform fluorescence, cytoplasm and nucleolus RNA stained orange (red) fluorescence, and measured the gray level of cytoplasmic RNA fluorescence staining, compared with NC group (47.21 + 8.72), DM group (89.84 + 10.71) and RGz group (61.25 + 8.13) gray level increased, the difference was statistically significant (p0.01), compared with DM group (61.25 + 8.13). The observation of hepatic fat storage cells (hsc) in the dieldrin space and between the hepatocytes, the liver fat storage cells were stained with gold chloride to appear purple brown, and the background was light gray. The number of fat storage cells in the high power microscope was higher in the DM group (7.19 + 0.45) than that in the NC group (2.75 + 0.21). Compared with DM group, the number of fat storage cells in RGZ group decreased (P 0.01). 8. Changes of collagen fibers: the wall of hepatic lobule central vein was thin and there were a few connective tissues in portal area in NC group. The percentage of connective tissue in portal area was [(3.19 [0.75)%]. Compared with NC group, the wall of hepatic lobule central vein was thickened and the connective tissue in portal area was thickened in DM group [(15.87 [(0.63)%)]. Collagen deposition increased (P 0.01). Compared with DM group, fibrous septum was observed in RGZ group [(10.06.54)%] and decreased in DM group ((P 0.05). 9. Comparison of collagen content: Collagen content in every gram of liver tissue was determined by high performance liquid chromatography, and collagen content in DM group ((134.54 18.78 mg/g) was compared with NC group (64.52 21.51 mg/g). The expression of IL-17 in liver tissue was significantly higher in DM group than in DM group (P 0.01). The expression of Bcl-xl in RGZ group was significantly lower than that in DM group (P 0.01). Changes: Compared with NC group, the absorbance value of Bcl-x L positive expression in liver tissue (0.613+0.014), DM group (0.242+0.041) decreased (P 0.05), RGZ group liver tissue Bcl-x L positive expression (0.369+0.048) increased (P 0.01). Conclusion 1. Diabetic rats liver lobule structure disorder, hepatocyte swelling, collagen fibril deposition, collagen content increased significantly.2. Activation and proliferation of HSC cells in diabetic group lead to the increase of hepatic collagen fiber content. 3. Increase of inflammatory cells in hepatic lobules and portal tract may be related to the increase of hepatic macrophages; excessive hepatic macrophages aggravate liver injury in diabetic group. 4. Increase of IL-17 positive expression and decrease of Bcl-xl expression in diabetic group, and the central vein as the center. Peripheral emission distribution may be related to blood supply. 5. Rosiglitazone has a protective effect on liver tissue of OLETF type 2 diabetic rats, and can reduce the degree of liver damage caused by diabetes mellitus.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1
本文編號：2208638
[Abstract]:Objective Diabetes mellitus (DM) is a group of metabolic diseases, mainly caused by chronic hyperglycemia due to insulin secretion deficiencies and/or disorders. Long-term persistent hyperglycemia and metabolic disorders can lead to systemic tissue and organ damage and dysfunction. The liver plays an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). As a target organ, the liver can also be damaged in diabetic state. Persistent hyperglycemia leads to the increase of advanced glycosylation products, the destruction of hepatic lobules, the increase of fibrosis, the change of hepatocyte structure and function, the increase of hepatocyte inflammation and the increase of apoptosis. As control group, OLETF rats and rosiglitazone-treated rats were both experimental groups. The pathological changes of liver tissues were observed and the expression of IL-17 and Bcl-xl were measured. The effect of rosiglitazone on liver tissues of OLETF rats with type 2 diabetes mellitus was further investigated. Methods Rats were fed in single cage. The rats were fed with standard diet for 12/12 hours and were fed with normal diet and injected with buffer solution only. At 30 weeks, 16 OLETF rats were randomly divided into two groups (n=8). The RGZ group was given rosiglitazone dilution, and the RGZ group was given rosiglitazone. The rats in the three groups were collected blood for measurement, and then the liver tissues were taken. After bleeding, three pieces of liver tissues were quickly taken out with aseptic instruments. Two of them were put into DEPC-treated cryopreserve tubes and stored in liquid nitrogen for reserve. The rats were placed in 10% formalin and paraffin-embedded sections, then stained histologically, including HE staining, AO staining, Schiff test, Masson trichrome staining, gold chloride staining and immunohistochemical staining. Changes of collagen fibers and positive expression of IL-17 and Bcl-x L were observed. In addition, liver tissue homogenate was prepared at - 70 C and the content of collagen was determined by hydroxyproline method. All data were processed by SPSS 13.0 software. Results 1. Comparing with Leto rats, the body type of OLETF rats was more than that of Leto rats. Obesity, nervous sensitivity, irritability, laziness, chills, depression, anxiety, and liver volume significantly increased. 2. Biochemical indicators: before the execution of OLETF rats were measured two hours postprandial blood glucose, triglyceride, alanine aminotransferase and glutamic oxaloacetic transaminase and liver index. Compared with DM group, blood glucose, triglyceride and alanine aminotransferase in DM group were significantly decreased, but there was no significant difference in glutamic oxaloacetic aminotransferase. 3. histological changes of liver: the hepatic lobules of NC rats were regular, the hepatic cords were arranged in a radial fashion, and the hepatic sinusoids were in good order. In DM group, hepatic lobules lost normal structure, hepatic cords narrowed, hepatic sinusoids were unclear, hepatocytes swelled, steatosis, necrosis, occasional punctate inflammatory foci. RGz hepatocyte structure was still clear, hepatic cords were arranged regularly, although there were also lipid vacuoles and inflammation The number of hepatic macrophages in the NC group was 17.05 (+ 0.89), the number of hepatic macrophages in the DM group was 25.31 (+ 1.23) and that in the RGz group was 38.61 (+ 4.02). The content of glycogen in hepatocytes of RGz group was more than that of NC group and DM group (p0.01). 5. the content of glycogen in hepatocytes of RGz group was higher than that of NC group and DM group. The gray level of hepatocyte glycogen granules in NC group was 62.38 (+ 10.16), while that in DM group (109.54 (+ 11.18) was significantly higher than that in NC group (p0.01), and that in RGz group (79.36 (+ 9.98) was significantly lower than that in NC group (p0.05). Acridine orange fluorescence staining made the normal nuclear DNA show green or yellow-green uniform fluorescence, cytoplasm and nucleolus RNA stained orange (red) fluorescence, and measured the gray level of cytoplasmic RNA fluorescence staining, compared with NC group (47.21 + 8.72), DM group (89.84 + 10.71) and RGz group (61.25 + 8.13) gray level increased, the difference was statistically significant (p0.01), compared with DM group (61.25 + 8.13). The observation of hepatic fat storage cells (hsc) in the dieldrin space and between the hepatocytes, the liver fat storage cells were stained with gold chloride to appear purple brown, and the background was light gray. The number of fat storage cells in the high power microscope was higher in the DM group (7.19 + 0.45) than that in the NC group (2.75 + 0.21). Compared with DM group, the number of fat storage cells in RGZ group decreased (P 0.01). 8. Changes of collagen fibers: the wall of hepatic lobule central vein was thin and there were a few connective tissues in portal area in NC group. The percentage of connective tissue in portal area was [(3.19 [0.75)%]. Compared with NC group, the wall of hepatic lobule central vein was thickened and the connective tissue in portal area was thickened in DM group [(15.87 [(0.63)%)]. Collagen deposition increased (P 0.01). Compared with DM group, fibrous septum was observed in RGZ group [(10.06.54)%] and decreased in DM group ((P 0.05). 9. Comparison of collagen content: Collagen content in every gram of liver tissue was determined by high performance liquid chromatography, and collagen content in DM group ((134.54 18.78 mg/g) was compared with NC group (64.52 21.51 mg/g). The expression of IL-17 in liver tissue was significantly higher in DM group than in DM group (P 0.01). The expression of Bcl-xl in RGZ group was significantly lower than that in DM group (P 0.01). Changes: Compared with NC group, the absorbance value of Bcl-x L positive expression in liver tissue (0.613+0.014), DM group (0.242+0.041) decreased (P 0.05), RGZ group liver tissue Bcl-x L positive expression (0.369+0.048) increased (P 0.01). Conclusion 1. Diabetic rats liver lobule structure disorder, hepatocyte swelling, collagen fibril deposition, collagen content increased significantly.2. Activation and proliferation of HSC cells in diabetic group lead to the increase of hepatic collagen fiber content. 3. Increase of inflammatory cells in hepatic lobules and portal tract may be related to the increase of hepatic macrophages; excessive hepatic macrophages aggravate liver injury in diabetic group. 4. Increase of IL-17 positive expression and decrease of Bcl-xl expression in diabetic group, and the central vein as the center. Peripheral emission distribution may be related to blood supply. 5. Rosiglitazone has a protective effect on liver tissue of OLETF type 2 diabetic rats, and can reduce the degree of liver damage caused by diabetes mellitus.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1
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，本文編號：2208638
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