【作者】 刘鑫；

【导师】 郭启勇；

【作者基本信息】 中国医科大学 ， 影像医学与核医学， 2008， 博士

【摘要】 前言肝纤维化是肝内纤维结缔组织的异常增生与沉积,是各种慢性肝病向肝硬化发展的必经阶段。肝纤维化的早期诊断及严重程度判定在慢性肝病的早期治疗和预后判断中有重要意义。近年来各种MR功能成像方法的出现为肝纤维化的诊断带来了曙光,其中MR扩散加权成像（diffusion-weighted imaging,DWI）和磁共振灌注成像（perfusion-weighted imaging,PWI）分别通过检测组织内微观水分子的运动状态和血流灌注的变化,在肝纤维化形态学改变出现之前对肝脏分子水平的结构变化和血流动力学改变进行评价。本研究对大鼠肝纤维化模型进行DWI和PWI检查,通过与肝脏组织病理、超微结构改变、血液生化学和Ⅳ型胶原（C-Ⅳ）、层粘蛋白（LN）免疫组化进行对照分析,对DWI和PWI结果作出更深入合理的解释,评价DWI、PWI在肝纤维化中的诊断效能,以便对无创性评价肝纤维化的方法作出合理选择和应用,提高肝纤维化的诊断水平。材料和方法雄性Wistar大鼠60只,体重200-250g,随机分为6组:1、2、3、4、5组为实验组,6组为对照组,每组10只。实验组大鼠每周2次背部皮下注射60%CCl₄和40%橄榄油混合液（0.2ml/100g）,共12周。对照组大鼠背部皮下注射生理盐水（0.2ml/100g）,每周2次,共12周。在给药4、6、8、10、12周末分别取1-5实验组和对照组2只大鼠进行MR扫描。MR设备应用Philips Gyroscan Intera 1.5 T超导型磁共振仪,C3线圈。DWI采用SE-EPI序列,b值分别取333、666、1000s/mm²,TR/TE=1723ms/66ms,层厚3.0mm,层间距0mm,矩阵128×256,激励次数1。DWI原始扩散图像获得后即由计算机自动绘出ADC图。感兴趣层面定为肝门上、下1-2层,感兴趣区选在肝右叶图像质量最好的层面,在不同b值图像上测量肝脏信号强度,同一位置测量3次并取平均值,尽量避开胆管、血管和伪影等。记录DWI图像信号强度(signalintensity,SI及对应的ADC值。比较不同肝纤维化分期时DWI参数的变化,分析各参数与纤维化分期之间的相关性。灌注成像采用THRIVE（3D T1-TFE）序列,TR/TE=7.5ms/3.7ms,FA=10°,采集次数1,矩阵128×128,层厚2.0mm,层间隔0mm,块厚20mm,脂肪抑制。全肝扫描层数10层,共60个时相,扫描时间126秒。由鼠尾静脉留置针团注对比剂Gd-DTPA（0.05mmol/kg）,随后续注10ml生理盐水冲管,注射对比剂同时开始PWI扫描。选用全部PWI灌注图像,分别选择腹主动脉、门静脉和肝实质作为感兴趣区ROI,自动绘制信号强度-时间曲线（time-signal intensity curve,TIC）。观察并记录①峰值时间;②信号上升最大斜率;③信号下降最大斜率。比较不同肝纤维化分期时PWI参数的变化,分析各参数与纤维化分期之间的相关性。MR扫描后心脏穿刺取血5ml制备血清,统一进行血清学肝纤维化指标检测,包括透明质酸（HA）、Ⅲ型前胶原（PCⅢ）、Ⅳ型胶原（C-Ⅳ）和层粘连蛋白（LN）。比较不同纤维化分期时各指标的变化规律,分析血清学各指标间及与纤维化分期的相关性。MR扫描后6小时内断头处死大鼠取肝,病理取材尽量与MR中ROI设置层面相对应,10%甲醛固定,行HE、Masson和网状纤维染色,光镜下判定肝纤维化分期。采用Envision二步法进行C-Ⅳ、LN免疫组化染色,定量测量C-Ⅳ、LN的阳性面积比。对照组和实验组每组2只,处死后即刻取1×1×3mm肝组织2.5%戊二醛固定,常规电镜切片制作,透射电镜下观察肝组织超微结构改变。结果对照组死亡0只,实验组死亡12只,共48只大鼠完成实验。肝纤维化分期:对照组S0（n=10）,实验组S0（n=3）、S1（n=5）、S2（n=9）、S3（n=13）、S4（n=8）。随给药时间延长,实验组肝细胞变性、坏死及炎细胞浸润加重,胶原纤维和网状纤维数量增多、范围扩大。电镜观察随给药时间延长肝细胞损伤加重,贮脂细胞向肌成纤维细胞演变,肝内纤维沉积增多,肝窦毛细血管化早期出现并持续存在。一、血清学肝纤维化指标变化各血清学指标随纤维化分期进展逐渐升高,在S4期达到高峰。各血清学指标间及与肝纤维化分期之间显著正相关（P＜0.01）。二、免疫组化结果肝纤维化组C-Ⅳ、LN在汇管区及粗大的纤维间隔处肝窦壁阳性着色显著增强并呈连续性分布,随肝纤维化程度的增加肝窦壁C-Ⅳ、LN阳性表达逐渐增高。C-Ⅳ、LN阳性面积百分比与肝纤维化分期的相关系数分别为0.900和0.912,相关性有显著统计学意义（P＜0.01）。三、DWI结果根据肝纤维化分期进行分组,同一组内,随b值升高,SI值、ADC值逐渐降低,差异有显著统计学意义（P＜0.01）。在b取333、666和1000 s/mm²时,随肝纤维化程度加重,DWI图像信号强度SI依次升高,ADC值依次降低,差异有显著统计学意义（P＜0.01）。ADC值与纤维化分期之间有很好的相关性,以b=1000s/mm²时相关关系最密切。b=333 s/mm²和666 s/mm²时,根据SI和ADC值可以将S0～S2期与S4期进行区分,S0期与S3、S4期进行区分。b=1000s/mm²时,可将S0～S2期与S4期进行区分。ADC值与肝纤维化血清学指标之间存在不同程度的负相关（P＜0.05）。四、PWI结果获得大鼠全肝MR灌注图像和稳定的信号强度-时间曲线。随肝纤维化分期进展,门静脉灌注曲线的达峰时间逐渐递增,信号上升最大斜率和信号下降最大斜率逐渐递减。根据门静脉灌注曲线的达峰时间,S2～S4期与S0期有统计学差异（P＜0.05,＜0.001,＜0.001）。根据门静脉的最大上升斜率和最大下降斜率,S0～S2期与S3、S4期有统计学差异（P＜0.05）。肝实质灌注曲线的达峰时间逐渐递增,最大上升斜率、最大下降斜率逐渐递减。S0～S2期与S3、S4期有统计学差异（P＜0.05）。MR灌注参数与C-Ⅳ、LN相关性分析:门静脉和肝实质的达峰时间与C-Ⅳ和LN存在显著正相关关系（P＜0.01）,门静脉和肝实质的最大上升斜率和最大下降斜率与C-Ⅳ和LN存在显著负相关关系（P＜0.01）。结论1、血清学指标HA、PCⅢ、C-Ⅳ和LN随肝纤维化分期进展依次升高,血清学指标相互之间及与肝纤维化分期之间存在显著正相关关系。2、随肝纤维化分期进展,肝窦壁C-Ⅳ和LN的表达逐渐增高,C-Ⅳ、LN阳性面积百分比与肝纤维化分期之间存在显著正相关关系。3、随肝纤维化分期进展,DWI图像的SI依次升高,ADC值依次降低。DWI可检测S3、S4期中、重度肝纤维化,但对判定S0～S2期肝纤维化尚有局限性。4、ADC值与肝纤维化血液生化学各指标间存在良好的相关性,共同反映肝纤维化ECM异常增生和沉积的特征性病理改变。5、MR-PWI可能检测轻度肝纤维化（S2期）和中、重度肝纤维化/早期肝硬化（S3、S4期）,肝脏MR灌注参数与肝纤维化分期有良好的相关性,可反映肝纤维化各期的血流动力学变化趋势。6、肝纤维化大鼠肝组织C-Ⅳ、LN和PWI灌注参数间存在密切的相关关系,均可反映肝纤维化肝窦毛细血管化病理变化,可解释MR灌注成像所反映的肝脏微循环状态和血流动力学改变。更多还原

【Abstract】 PrefaceLiver fibrosis is a pathologic state with abnormal synthesis and deposition of extracellular matrix.It is a procedure of cirrhosis resulted from all kinds of chronic liver diseases.Make early diagnosis and assess the severity of liver fibrosis accurately is very important for therapy and prognosis.In recent years the functional imaging techniques of MR including difffusion weighted imaging（DWI）and perfusion-weighted imaging（PWI）have developed fast. DWI sensitizes the MR signal related to the incoherent microscopic motion of water molecules,and PWI evaluates the hemodynamic characteristics of the hepatic lesions. Both of them can assess ultrastructural changes and hemodynamic alterations before morphological alteration in liver fibrosis.In our experimental study,DWI and PWI examinations were underwent in rat model of liver fibrosis.The changes of them in different stages of liver fibrosis were analyzed.DWI and PWI results were compared with histopathological, ultramicrostructural,blood biochemiology,typeⅣcollagen（C-Ⅳ）and laminin（LN） immunohistochemistry expressions to make further interpretations.The diagnosis efficacies of DWI and PWI parameters for liver fibrosis as well as the correlations among them were analyzed,so as to choose noninvasive method for assessing liver fibrosis reasonably.Materials and MethodsSixty Wistar rats were divided into six groups:1 to 5 as experimental group,and 6 as control group with 10 rats each.Mixed resolution（0.2ml/100g）with 60%carbon tetrachloride（CCl₄）and 40%olive oil were hypodermic injected twice a week on rats of experimental group,while in control group were injected 0.2ml/100g saline twice a week.At 4,6,8,10,12 weekends after injection,rats of 1 to 5 experimental groups and two rats of control group were underwent MR examination respectively.All MR examinations were performed on a 1.5 Tesla unit（Philips Intera）with the C3 coil.Three different b values were tested:333,666 and 1000s/mm².Spin echo echo-planar DWI was performed using the following acquisition parameters: TR/TE=1723ms/66ms,slice thickness/slice gap=3mm/0mm,matrix=128×256, NSA=1.The software may provide ADC images automatically after DWI original diffusion images were acquired.Then all images were transferred to Philips workstation.Slice of interesting were selected at hepatic right lobes.Three ROI avoiding large vessels and artifacts were placed over 1-2 representative sections involving hepatic hilar.Signal intensity（SI）and ADC value were recorded and compared among different stages of liver fibrosis.The relationships among DWI parameters,liver fibrosis stage were analyzed.First-pass contrast enhanced perfusion MR imaging was performed on rats in experimental and control groups.A THRIVE（T1-TFE）three dimensional sequence was undertaken with the following acquisition parameters:TR/TE=7.Sms/3.Sms,slice thickness/slice gap=2mm/0mm,block thickness=20mm,matrix=128×128,NSA=1. Sixty phases of dynamic images for ten slices of the whole liver were obtained during 126 seconds after intravenous bolus administration of Gd-DTPA（0.05mmol/kg）via caudal vein.The scan began as soon as bolus administration started.Region-of-interest analyses over the aorta,the portal vein,and liver parenchyma enabled generation of concentration-time curves.Perfusion parameters were recorded as follows:①Time to peak（TP）;②Wash-in rate;③Wash-out rate.Three parameters of portal vein and liver parenchyma were compared among different stages of liver fibrosis.The relationships among PWI parameters,fibrosis stage were analyzed. Blood samples were obtained in all animals after MR examination to observe serum markers of liver fibrosis including hyaluronic acid（HA）,procollagen typeⅢ（PCⅢ）,typeⅣcollagen（C-Ⅳ）and laminin（LN）.The differences of results at various stages of liver fibrosis were compared.Analysis of relationships among serum markers and stage of liver fibrosis were performed.Liver was resected within six hours after MR scanning and fixed in 10%formalin. HE,Masson and reticular fiber staining stained the samples separately.The stage of liver fibrosis was decided by light microscopy.C-Ⅳand LN immunohistochemistry staining were undertaken by Envision two-step method.A sample with 1×1×3mm volume was obtained and fixed in 2.5%glutaral for making electron microscopic section in 2 rats of control group and of each experimental group.Hepatic ultrastructure was observed by transmission electron microscope.Correlation between ADC and serum marker,perfusion parameter and C-Ⅳcollagen and LN expression were analyzed separately.ResultsTwelve rats of experimental group were eliminated from experiment due to death in the process of breeding.With the prolongation of injection,the severity of hepatic degeneration,necrosis and inflammatory cell infiltration progressed,the amount of collagen and reticular fiber increased.The injury of hepatic cell progressed and the deposition of fibrous tissue increased under electron microscope as the injection time prolonged.Morphology of fat-storing cell changed and hepatic sinusoidal capillarization developed in the process of liver fibrosis.1.Results of serum indices in liver fibrosisAll serum fibrosis indices increased as the stage of liver fibrosis increased,and reached the peak on S4 stage.There was a significantly positive correlation among all serum fibrosis indices and between serum indices and stage of liver fibrosis（P＜0.01）.2.Results of C-Ⅳand LN immunohistochemistry staining We found C-Ⅳand LN dramatically increased and successionally deposited along the wall of sinusoids and thick fibrous septa.The positive expression of C-Ⅳand LN increased as the stage of liver fibrosis progressed.There was a significantly positive correlation between C-Ⅳor LN and stage of liver fibrosis（P＜0.01）.The r values amount to 0.900 and 0.912,respectively.3.Results of DWIGrouping according as different stage of fibrosis,we found that SI and ADC values decreased along with b values increasing（P＜0.01）.When b values equal to 333, 666 and 1000 s/mm² respectively,SI increased and ADC decreased gradually as the stage of fibrosis progressed（P＜0.01）.There was a good relationship between ADC and fibrosis stage,especially when b value was 1000 s/mm².When b values equal to 333 and 666s/mm²,S0-S2 can be distinguished from S3 and S4 stage,and S3 can be distinguished from S4 stage according to SI and ADC values.Moreover b value equal to 1000 s/mm²,S0-S2 can be distinguished from S4 stage.There was excellent negtive correlation between ADC and serum indices（P＜0.05）.4.Appearances and results of PWIOn perfusion-weighted image,inferior vena cava,abdominal aorta and portal vein visualized in order.As the stage of fibrosis progressed,TP of portal vein increased progressively,and the wash-in rate and wash-out rate decreased progressively. According to TP of portal vein,S2-S4 stage can be distinguished from S0（P＜0.05,＜0.001,＜0.001）.According to wash-in rate and wash-out rate of portal vein,S0-S2 stage can be distinguished from S3 and S4 stage（P＜0.05）.As the stage of fibrosis progressed,TP of liver parenchyma increased progressively,and the wash-in rate and wash-out rate decreased progressively.S0-S2 stage can be distinguished from S3 and S4 stage（P＜0.05）.Of all parameters,TP of portal vein and liver parenchyma had a positive correlation with the expressions of C-Ⅳand LN（P＜0.01）.The wash-in and wash-out rate of portal vein and liver parenchyma had a negative correlation with the expressions of C-Ⅳand LN（P＜0.01）.Conclusions1.All serum fibrosis indices increased as the stage of liver fibrosis progressed. There was a significantly positive correlation among all indices and between serum indices and stage of liver fibrosis.2.The positive expression of C-Ⅳand LN increased as the stage of liver fibrosis progressed.There was a significantly positive correlation between C-Ⅳor LN and stage of liver fibrosis.3.SI increased and ADC value decreased gradually as the stage of fibrosis progressed.There was a good relationship between ADC and stage of fibrosis, especially when b value equal to 1000 s/mm².DWI was an effective method to detect liver fibrosis on S3 and S4 stage.4.There was excellent correlation between ADC and serum indices.It reflected the characteristic pathologic state of abnormal synthesis and deposition of extracellular matrix.5.PWI should be an effective method to quantify severity of liver fibrosis as well as to detect early cirrhosis（S2 stage）and severe fibrosis（S3-S4 stage）.MR perfusion parameters were better correlated with fibrosis staging,and were associated with hemodynamic alternations.6.There were excellent correlations between the perfusion parameters and the expressions of C-Ⅳand LN.These correlations would better interpreted sinusoidal capillarization in liver fibrosis.更多还原