背景:肝纤维化的发展伴随诸多的形态学和机能学的改变,二甲基亚硝胺所致的大鼠肝纤维化模型病变类似于人类肝纤维化病变.目的:观察二甲基亚硝胺诱发大鼠肝纤维化发生过程中形态学与血清透明质酸、层粘连蛋白、Ⅳ型胶原含量的动态变化.设计:随机对照动物实验.单位:延边大学医学院病理教研室实验室.材料:选用80只3月龄Wistar雄性大鼠,清洁级,体质量175～200 g,由延边大学医学院动物中心提供.试剂:二甲基亚硝胺美国Sigma公司产品,α-平滑肌肌动蛋白为Dako公司产品,天狼猩红(Sirius red)为Aldrich chem公司产品,血清透明质酸、层粘连蛋白和Ⅳ型胶检测试剂盒为华美生物工程公司产品,兔抗鼠的一抗为Dako,Denmak生物公司的产品.仪器:日本产JEM-1200EX透射电镜;日本产酶联免疫检测仪;北京航空航天大学研制开发的CMTAS多功能真彩色病理图象分析系统.方法:实验于2004-06/2005-12在延边大学医学院完成.摸球法随机将大鼠分成2组.模型组(n=40):给予腹腔注射10 g/L二甲基亚硝胺(10μL/kg),1周连续注药3 d(1次/d),持续4周;对照组(n=40):在同一时间段内注射同样剂量的生理盐水;分别在实验开始7,14,21,28 d处死大鼠,处死前左心室取血,血清样品被冷冻在-70℃的低温冰箱中备实验用,处死后取其肝组织用于光镜和电镜的样品制备.①用ELISA法测定在肝纤维化形成过程中血清透明质酸、层粘连蛋白、Ⅳ型胶原含量的动态变化.②采用苏木精-伊红染色、组织化学的天狼猩红染色观察肝纤维化形成过程中组织形态学变化及对肝纤维化分级(肝纤维化程度分为0～4级,0级:没有肝纤维化;1级:汇管区有纤维化;2级:汇管区与汇管区之间形成纤维间隔;3级:肝小叶中出现纤维间隔,并且肝的正常结构出现病理改变;4级:形成肝硬化),同时计算胶原纤维的面密度.③采用透射电镜技术、免疫组织化学的α-平滑肌肌动蛋白染色观察肝星状细胞的转化情况及分布特点.④对肝纤维化形成过程中胶原纤维面密度的百分比与血清透明质酸、Ⅳ型胶原、层粘连蛋白含量的相关分析.主要观察指标:①肝纤维化形成过程中血清透明质酸、层粘连蛋白、Ⅳ型胶原含量的变化.②肝纤维化形成过程中形态学变化、肝纤维化的分级情况及胶原纤维的面密度.③肝星状细胞的转化情况及分布特点.④肝纤维化形成过程中胶原纤维面密度的百分比与血清透明质酸、Ⅳ型胶原、层粘连蛋白含量的相关分析结果.结果:纳入80只大鼠,实验组34只造模成功,与对照组40只全部进入结果分析.①模型组第7天至28天血清透明质酸、层粘连蛋白、Ⅳ型胶原含量明显高于对照组(P＜0.05),其中第28天增加程度最大.②模型组汇管区在腹腔注射二甲基亚硝胺后第7天出现出血坏死,第14天仍可在汇管区观察到出血、坏死,并且还可见较薄的纤维间隔,第21和28天可见较厚的纤维间隔;模型组大鼠腹腔注射二甲基亚硝胺后第7,14,21,28天胶原纤维面密度的百分比明显高于对照组(P＜0 05),其中第28天增加的幅度最大;对照组大鼠纤维化病理分级与模型组大鼠各时间点相比差异显著(P＜0.01),模型组大鼠的第7天较第14,28天组有明显差异(P＜0.01).③a-平滑肌肌动蛋白染色有阳性细胞出现,并且在这一阶段在电镜下观察到正处在"转化的肝星状细胞";模型组第21和28天,在电镜下观察到典型的成纤维细胞.④胶原纤维面密度的百分比与血清透明质酸、层粘连蛋白、Ⅳ型胶原含量呈正相关(r=0.707,0.675,0.662,P＜0.01).结论:二甲基亚硝胺诱发大鼠肝纤维化发生过程中,在不同时间段内形态学与血清标志物透明质酸、层粘连蛋白、Ⅳ型胶原呈动态变化,并且在不同的时间段内胶原纤维面密度的百分比与血清透明质酸、层粘连蛋白、Ⅳ型胶原含量呈正相关.
揹景:肝纖維化的髮展伴隨諸多的形態學和機能學的改變,二甲基亞硝胺所緻的大鼠肝纖維化模型病變類似于人類肝纖維化病變.目的:觀察二甲基亞硝胺誘髮大鼠肝纖維化髮生過程中形態學與血清透明質痠、層粘連蛋白、Ⅳ型膠原含量的動態變化.設計:隨機對照動物實驗.單位:延邊大學醫學院病理教研室實驗室.材料:選用80隻3月齡Wistar雄性大鼠,清潔級,體質量175～200 g,由延邊大學醫學院動物中心提供.試劑:二甲基亞硝胺美國Sigma公司產品,α-平滑肌肌動蛋白為Dako公司產品,天狼猩紅(Sirius red)為Aldrich chem公司產品,血清透明質痠、層粘連蛋白和Ⅳ型膠檢測試劑盒為華美生物工程公司產品,兔抗鼠的一抗為Dako,Denmak生物公司的產品.儀器:日本產JEM-1200EX透射電鏡;日本產酶聯免疫檢測儀;北京航空航天大學研製開髮的CMTAS多功能真綵色病理圖象分析繫統.方法:實驗于2004-06/2005-12在延邊大學醫學院完成.摸毬法隨機將大鼠分成2組.模型組(n=40):給予腹腔註射10 g/L二甲基亞硝胺(10μL/kg),1週連續註藥3 d(1次/d),持續4週;對照組(n=40):在同一時間段內註射同樣劑量的生理鹽水;分彆在實驗開始7,14,21,28 d處死大鼠,處死前左心室取血,血清樣品被冷凍在-70℃的低溫冰箱中備實驗用,處死後取其肝組織用于光鏡和電鏡的樣品製備.①用ELISA法測定在肝纖維化形成過程中血清透明質痠、層粘連蛋白、Ⅳ型膠原含量的動態變化.②採用囌木精-伊紅染色、組織化學的天狼猩紅染色觀察肝纖維化形成過程中組織形態學變化及對肝纖維化分級(肝纖維化程度分為0～4級,0級:沒有肝纖維化;1級:彙管區有纖維化;2級:彙管區與彙管區之間形成纖維間隔;3級:肝小葉中齣現纖維間隔,併且肝的正常結構齣現病理改變;4級:形成肝硬化),同時計算膠原纖維的麵密度.③採用透射電鏡技術、免疫組織化學的α-平滑肌肌動蛋白染色觀察肝星狀細胞的轉化情況及分佈特點.④對肝纖維化形成過程中膠原纖維麵密度的百分比與血清透明質痠、Ⅳ型膠原、層粘連蛋白含量的相關分析.主要觀察指標:①肝纖維化形成過程中血清透明質痠、層粘連蛋白、Ⅳ型膠原含量的變化.②肝纖維化形成過程中形態學變化、肝纖維化的分級情況及膠原纖維的麵密度.③肝星狀細胞的轉化情況及分佈特點.④肝纖維化形成過程中膠原纖維麵密度的百分比與血清透明質痠、Ⅳ型膠原、層粘連蛋白含量的相關分析結果.結果:納入80隻大鼠,實驗組34隻造模成功,與對照組40隻全部進入結果分析.①模型組第7天至28天血清透明質痠、層粘連蛋白、Ⅳ型膠原含量明顯高于對照組(P＜0.05),其中第28天增加程度最大.②模型組彙管區在腹腔註射二甲基亞硝胺後第7天齣現齣血壞死,第14天仍可在彙管區觀察到齣血、壞死,併且還可見較薄的纖維間隔,第21和28天可見較厚的纖維間隔;模型組大鼠腹腔註射二甲基亞硝胺後第7,14,21,28天膠原纖維麵密度的百分比明顯高于對照組(P＜0 05),其中第28天增加的幅度最大;對照組大鼠纖維化病理分級與模型組大鼠各時間點相比差異顯著(P＜0.01),模型組大鼠的第7天較第14,28天組有明顯差異(P＜0.01).③a-平滑肌肌動蛋白染色有暘性細胞齣現,併且在這一階段在電鏡下觀察到正處在"轉化的肝星狀細胞";模型組第21和28天,在電鏡下觀察到典型的成纖維細胞.④膠原纖維麵密度的百分比與血清透明質痠、層粘連蛋白、Ⅳ型膠原含量呈正相關(r=0.707,0.675,0.662,P＜0.01).結論:二甲基亞硝胺誘髮大鼠肝纖維化髮生過程中,在不同時間段內形態學與血清標誌物透明質痠、層粘連蛋白、Ⅳ型膠原呈動態變化,併且在不同的時間段內膠原纖維麵密度的百分比與血清透明質痠、層粘連蛋白、Ⅳ型膠原含量呈正相關.
배경:간섬유화적발전반수제다적형태학화궤능학적개변,이갑기아초알소치적대서간섬유화모형병변유사우인류간섬유화병변.목적:관찰이갑기아초알유발대서간섬유화발생과정중형태학여혈청투명질산、층점련단백、Ⅳ형효원함량적동태변화.설계:수궤대조동물실험.단위:연변대학의학원병리교연실실험실.재료:선용80지3월령Wistar웅성대서,청길급,체질량175～200 g,유연변대학의학원동물중심제공.시제:이갑기아초알미국Sigma공사산품,α-평활기기동단백위Dako공사산품,천랑성홍(Sirius red)위Aldrich chem공사산품,혈청투명질산、층점련단백화Ⅳ형효검측시제합위화미생물공정공사산품,토항서적일항위Dako,Denmak생물공사적산품.의기:일본산JEM-1200EX투사전경;일본산매련면역검측의;북경항공항천대학연제개발적CMTAS다공능진채색병리도상분석계통.방법:실험우2004-06/2005-12재연변대학의학원완성.모구법수궤장대서분성2조.모형조(n=40):급여복강주사10 g/L이갑기아초알(10μL/kg),1주련속주약3 d(1차/d),지속4주;대조조(n=40):재동일시간단내주사동양제량적생리염수;분별재실험개시7,14,21,28 d처사대서,처사전좌심실취혈,혈청양품피냉동재-70℃적저온빙상중비실험용,처사후취기간조직용우광경화전경적양품제비.①용ELISA법측정재간섬유화형성과정중혈청투명질산、층점련단백、Ⅳ형효원함량적동태변화.②채용소목정-이홍염색、조직화학적천랑성홍염색관찰간섬유화형성과정중조직형태학변화급대간섬유화분급(간섬유화정도분위0～4급,0급:몰유간섬유화;1급:회관구유섬유화;2급:회관구여회관구지간형성섬유간격;3급:간소협중출현섬유간격,병차간적정상결구출현병리개변;4급:형성간경화),동시계산효원섬유적면밀도.③채용투사전경기술、면역조직화학적α-평활기기동단백염색관찰간성상세포적전화정황급분포특점.④대간섬유화형성과정중효원섬유면밀도적백분비여혈청투명질산、Ⅳ형효원、층점련단백함량적상관분석.주요관찰지표:①간섬유화형성과정중혈청투명질산、층점련단백、Ⅳ형효원함량적변화.②간섬유화형성과정중형태학변화、간섬유화적분급정황급효원섬유적면밀도.③간성상세포적전화정황급분포특점.④간섬유화형성과정중효원섬유면밀도적백분비여혈청투명질산、Ⅳ형효원、층점련단백함량적상관분석결과.결과:납입80지대서,실험조34지조모성공,여대조조40지전부진입결과분석.①모형조제7천지28천혈청투명질산、층점련단백、Ⅳ형효원함량명현고우대조조(P＜0.05),기중제28천증가정도최대.②모형조회관구재복강주사이갑기아초알후제7천출현출혈배사,제14천잉가재회관구관찰도출혈、배사,병차환가견교박적섬유간격,제21화28천가견교후적섬유간격;모형조대서복강주사이갑기아초알후제7,14,21,28천효원섬유면밀도적백분비명현고우대조조(P＜0 05),기중제28천증가적폭도최대;대조조대서섬유화병리분급여모형조대서각시간점상비차이현저(P＜0.01),모형조대서적제7천교제14,28천조유명현차이(P＜0.01).③a-평활기기동단백염색유양성세포출현,병차재저일계단재전경하관찰도정처재"전화적간성상세포";모형조제21화28천,재전경하관찰도전형적성섬유세포.④효원섬유면밀도적백분비여혈청투명질산、층점련단백、Ⅳ형효원함량정정상관(r=0.707,0.675,0.662,P＜0.01).결론:이갑기아초알유발대서간섬유화발생과정중,재불동시간단내형태학여혈청표지물투명질산、층점련단백、Ⅳ형효원정동태변화,병차재불동적시간단내효원섬유면밀도적백분비여혈청투명질산、층점련단백、Ⅳ형효원함량정정상관.
BACKGROUND: Development of liver fibrosis accompanies many morphological and functional changes. The pathological alterations of dimethylnitrosamine (DMN)-induced liver fibrosis in rats are similar to those of human liver fibrosis.OBJECTIVE: To observe the dynamic changes in morphology and serum hyaluronic acid (HA), laminin (LN), and type Ⅳ collagen in rats with liver fibrosis induced by DMN.DESIGN: Randomized controlled animal trial.SETTING: Laboratory of Teaching and Research Section of Pathology, College of Medicine, Yanbian University.MATERIALS: Eighty 3-month-old male rats of clean grade with 175-200 g body mass were selected, which were provided by the animal center of College of Medicine, Yanbian University. Agents: Dimethylnitrosamine provided by Sigma company, α smooth muscle actin by Dako company, Sirius red by Aldrich chem company, serum hyaluronic acid,laminin and type Ⅳ collagen kit by Sino-American Biotechnology Company, rabbit-anti-rat I g by Dako, Denmak company. Devices: JEM-1200EX transmission electron microscope made in Japan; enzyme linked immuno analyzer made in Japan; and CMTAS multifunction true color pathological image analysis system developed by Beijing University of Aeronautics and Astronautics.METHODS: The experiment was conducted in the College of Medicine, Yanbian University from June 2004 to December 2005. The rats were divided into 2 groups by lot: model group (n =40): The rats were intraperioneally injected with 10 g/L DMN (10 μL/kg) once daily, 3 days a week for 4 weeks; control group (n =40): The matching normal saline was injected at the same period; the blood from the left ventricle was collected and frozen in refrigerator at -70 ℃ before the rats were killed at days 7, 14, 21, and 28 and the liver tissue was removed for electron and light microscope observation. ①The dynamic changes in the content of serum HA, LN and type Ⅳ collagen were measured by enzyme-linked immunoabsorbent assay (ELISA). ②The morphological changes and liver fibrosis grading were examined by HE staining,immunohistochemical Sirius-red staining (liver fibrosis degree was classified into 5 grades: grade 0: no fibrosis; grade 1:fibrosis in portal area; grade 2: fibrotic septa between portal tracts; grade 3: fibrosis septa and structure disturbance of hepatic lobule; grade 4: cirrhosis), meanwhile, the area-density percentage of collagen fibrosis was calculated. ③The hepatic stellate cells were detected with transmission electron microscope and immunohistochemical alpha smooth muscle actin (SMA) staining. ④The correlation between area-density percentage of collagen fibrosis during liver fibrosis formation and the serum levels of HA, LN and type Ⅳ collagen was analyzed.MAIN OUTCOME MEASURES: ①The changes in the serum levels of HA, type Ⅳ collagen and LN during liver fibrosis formation; ②The morphological changes and liver fibrosis grading and area-density percentage of collagen fibrosis; ③Transformation and distribution characteristics of hepatic stellate cells; ④The correlation between area-density percentage of collagen fibrosis and the serum levels of HA, LN and type Ⅳ collagen.RESULTS: Among the 80 rats, 34 of the experimental group were modeled successfully, which were involved in the result analysis with the 40 rats in the control group. ①The levels of serum HA, type Ⅳ collagen and LN of the model group were significantly higher compared with the control group from day 7 to 28 (P ＜ 0.05), especially that on the 28th day. ②In the model group, the portal area of the rats showed hemorrhagic necrosis at day 7 after injection of DMN; at day 14,hemorrhage, necrosis and thin fibrotic septa joining central areas of liver were found; at days 21 and 28, thick septa was found; The area-density percentage of collagen fibrosis of the model group was significantly higher compared with the control group at days 7, 14, 21 and 28 (P ＜ 0.05), especially that on the 28th day. There were significant differences in the liver pathologic grading between the two groups at each time point (P ＜ 0.01); the pathologic grading of the model group at day 7 differed from those at days 14 and 28 (P ＜ 0.01). ③The α-SMA positive cells and a transitional hepatic stellate cell were found under the electron microscopy; typical myofibroblast was observed in the model group at day 21 and 28 under the electron microscopy. ④The area-density percentage of collagen fibrosis was positively correlated with the content of serum HA, LN and type Ⅳ collagen (r=0.707, 0.675, 0.662, P＜ 0.01).CONCLUSTON: There are significantly progressional changes in morphological and serum levels of HA, type Ⅳ collagen and LN in different stages of DMN-induced liver fibrosis in rats, moreover, the area-density percentage of collagen fibrosis is positively correlated with the serum levels of HA, LN and type Ⅳ collagen at different stages.
