小型猪肝纤维化模型制备及MSCTP研究

发布时间：2018-05-23 07:24

本文选题：动物模型 + 猪　；参考：《广西医科大学》2007年博士论文

【摘要】： 研究目的:建立小型猪肝纤维化模型,用16层螺旋CT灌注成像技术动态研究不同病理时期肝血流动力学变化的规律,获得肝纤维化小型猪门脉自由压力与肝脏灌注指数相关性的回归方程式,无创性进行肝纤维化的MSCTP诊断。材料与方法:研究对象,选择同种系巴马小型猪45头,随机分为实验组40头,对照组5头,雌雄不限,4月龄,体重10-13kg,由广西医科大学动物实验中心提供。实验组采用复合因素法造模(65%的玉米面+35%的胆固醇+四氯化碳+苯巴比妥钠的混合饲料,10%的无水乙醇作为唯一饮料),每日一次,连续16周至成模为止;对照组正常喂养(65%的玉米面+35%的米糠,清水作为唯一饮料),与实验组同期喂养。两组动物分别在0、4、8、12、16周末进行16层螺旋CT灌注扫描,抽血检测肝功能生化指标(包括T.BIL、D.BIL、I.BIL、Alb、Glo、A/G、G-GT、ALT、AST、AST/ALT测定)、开腹测量门静脉自由压力(free portal venous pressure,FPP)以及CT引导下肝穿刺病理活检。CT灌注扫描先常规轴位平扫整个肝脏后,取同时含有肝脏,脾脏,主动脉和门静脉的层面作为肝脏灌注成像扫描层面。采用仪器携带的Body-perfect multi-scans扫描程序,扫描条件:120kv,60mA,12mm/层,0.5s/次。取18~#穿刺针,穿刺耳背静脉后,经高压注射器给予碘海醇(300mgI/ml),1.5ml/kg,流率2ml/s,延迟4s后推注造影剂,连续扫描60s。所获得灌注数据采用仪器自身携带的功能软件(BodyperfCT-syngo CT2007A)分析图像。分别在主动脉,门静脉,脾脏和肝脏上的感兴趣区测定CT值(Hu),获取相对的时间密度曲线(TDC),即相对于平扫该感兴趣区的密度增加值。其中主动脉和门静脉取圆形或椭圆形的感兴趣区,肝脏和脾脏的感兴趣区沿着脏器的边缘绘制,肝脏的感兴趣区主要包括扫描层面肝叶的大部分,并尽可能的避开肝内的大血管,脾脏的感兴趣区包括该层面的整个脾脏。所取的感兴趣区离脏器的边缘1cm左右,以免产生容积效应。由软件自动生成TDC,然后根据TDC由软件采用最大斜率法进行计算,自动生成灌注参数,包括肝动脉灌注量(hepatic arteryperfusion,HAP)、门脉灌注量(portal venous perfusion,PVP)、总肝灌注量(total hepatic blood flow,THBF)以及肝动脉灌注指数(hepatic arteryperfusion index,HPI),其中THBF=HAP+PVP。每一例测量的指标均重复2次,取其平均值。采用SPSS 13.0软件对所得的各种数据进行统计及分析。对实验组和对照组以及实验组各组间的灌注参数、生化指标、门静脉自由压力进行统计学分析。所得结果用均数±标准差表示。两组均数的比较采用两独立样本的t检验,多组均数的比较采用单析因方差分析(One-Way ANOVA),方差不齐时应用非参数秩和检验(Kruskal-Wallis检验),P＜0.05有统计学差异;采用双变量Pearson线性相关分析分别对肝纤维化1～5级的HAP、PVP、THBF以及HPI四个灌注参数与同期门静脉压力的测量值进行相关性分析;采用多元线性回归的Stepwise逐步回归方式建立肝纤维化1～5级的HAP、PVP、THBF以及HPI四个灌注参数与同期门静脉压力测量值的回归方程;利用回归方程推导出肝纤维化各期的门静脉压力,所得结果与实测值进行双变量Pearson线性相关分析;判断经公式推导出的压力值落在实测值95%可信区间的例数,评价门静脉自由压的实际测量和MSCTP测量所得结果之间的符合率。结果:(1)对照组5头无1例死亡,0～16周肝脏组织病理表现正常。实验组40头猪4周末死亡14头,8周末死亡2头,造模死亡率为40%;分别获得0、1、2、3、4、5期肝纤维化病理模型,其中0级40例次,1级27例次,2级19例次,3级17例次,4级19例次,5级16例次。(2)正常组肝功能各项指标分别为ALT(42.00±19.51)u/l,AST(27.60±9.71)u/l,AST/ALT(0.69±0.10),G-GT(55.40±33.14)u/l,Alb(31.46±3.96)g/l,Glo(19.74±9.12)g/l,A/G(1.12±0.05),T.BIL(4.30±1.19)umol/l,D.BIL(1.28±0.30)umol/l,I.BIL(3.02±0.89)umol/l。实验组0-5期肝功能各项指标ALT、AST、AST/ALT、G-GT、Glo、D.BIL均表现为逐渐升高,Alb、I.BIL表现为逐渐下降,A/G比值倒置。正常组与实验组0级之间比较无统计学差异(P＞0.05);实验组各组间的ALT、AST、AST/ALT、G-GT、Alb、Glo、A/G、I.BIL均有统计学意义(P＜0.05),T.BIL无统计学意义(P＞0.05)。(3)正常组FPP为(5.00±0.61)mmHg。实验组0-5期FPP逐渐升高,分别为(5.01±0.77,6.85±1.10,10.68±1.19,14.09±4.16,17.02±3.67,18.33±3.75)mmHg。正常组与实验组0级之间比较无统计学差异(P＞0.05),实验组组间均有统计学意义(P＜0.05)。(4)小型猪肝纤维化模型CT表现,正常组及实验组0级小型猪肝脏表面光滑,密度均匀,肝实质内未见异常密度灶,肝/脾CT值的比值＞1,未见腹水形成。实验组1级和2级行椭砀卧嗤庑紊灾渍?表面尚光滑,肝脏密度减低,肝/脾CT值的比值＜1,其中6例可见少量腹水形成;3级和4级小型猪肝脏表面欠光滑,肝实质密度欠均匀,局部密度减低,肝内未见实质性肿块形成,11例出现腹水;5级小型猪肝脏体积稍缩小,表面不光滑,似可见结节状影,肝实质内密度不均匀,未见实质性肿块形成,13例出现腹水。腹主动脉旁未见肿大的淋巴结影。(5)正常组HAP为(27.74±11.77)ml.min~(-1).100ml~(-1);PVP为(94.70±9.32)ml.minl~(-1).100ml~(-1);THBF为(122.44±8.79)ml.min~(-1).100ml~(-1);HPI为(20.54±4.08)ml.min~(-1).100ml~(-1)。实验组0-5期HAP分别为(28.24±11.74,18.02±4.70,24.24±9.62,27.45±16.40,32.07±15.77,29.48±10.98)ml.min~(-1).100ml~(-1);0-5期PVP分别为(91.69±15.44,77.73±10.52,61.824±10.52,45.08±14.98,36.18±6.22,32.39±6.15)ml.min~(-1).100ml~(-1);0-5期THBF分别为(119.93±21.87,95.76±12.09,86.06±11.65,72.54±27.00,68.26±18.91,62.54±13.65)ml.min~(-1).100ml~(-1);0-5期HPI为(22.53±6.25,28.48±5.58,30.82±6.05,37.06±5.89,40.73±10.19,48.10±13.12)%。各灌注参数正常组与实验组0级之间比较无统计学差异(P＞0.05)。实验组HAP先降后升,0级与1级之间有统计学差异(P＜0.05),0级与2、3、4、5级之间无统计学差异(P＞0.05);实验组PVP呈下降趋势,各组之间均有统计学差异(P＜0.05);实验组THBF呈下降趋势,各组之间均有统计学差异(P＜0.05);实验组HPI升高,各组之间均有统计学差异(P＜0.05)。(6)实验组FPP与HAP、HPI正相关,r值分别为+0.263、+0.656;与PVP、THBF呈负相关,r值分别为-0.633、-0.816,其中FPP与PVP的相关性最好。多元回归分析,Stepwise逐步回归将F检验统计量＜0.05的变量纳入回归方程,而F检验统计量＞0.05的变量则剔除出方程。上述4个灌注指标中,HAP和THBF被剔除,PVP和HPI两个变量则被纳入建立起最优的回归方程:Y=17.175-0.173X_1+0.135X_2(Y:FPP X_1:PVP X_2:HPI)(7)利用FPP与PVP、HPI直线回归方程Y=17.175-0.173X_1+0.135X_2计算实验组肝纤维化1～5级的门静脉压力为12.7751±4.5631mmHg,实测值为12.7867±5.4053mmHg,两者的相关系数r=0.845,P＜0.01;进行95%可信区间检验,肝纤维化1-2级诊断符合率为84.78%,3-4级诊断符合率为88.88%,5级诊断符合率为81.25%。结论:1.改良后的四氯化碳(CCL_4)、苯巴比妥、无水乙醇+高脂、低蛋白、低胆碱食物复合因素造模法可以成功建立小型猪肝纤维化模型。2.肝功能生化指标检查可以提示肝脏受损害,但对病理分期不能作出客观的评价。3.肝纤维化时肝脏血流灌注的变化与疾病的严重程度相关。4.肝纤维化MSCTP的PVP和HPI两个变量建立起最优的回归方程:Y=17.175-0.173X_1+0.135X_2(Y:FPP X_1:PVP X_2:HPI)利用回归方程可以推算不同级别肝纤维化门静脉自由压而对肝纤维化作出诊断。
[Abstract]:Objective: to establish a model of liver fibrosis in small pigs and study the dynamic changes of liver hemodynamics at different pathological stages by 16 slice spiral CT perfusion imaging technique. The regression equation of the correlation between the free pressure of the portal vein and the liver perfusion index was obtained, and the MSCTP diagnosis of liver fibrosis was noninvasive. Materials and methods: 45 Bama miniature pigs were randomly divided into 40 heads in the experimental group, 5 in the control group, 5 in the control group, 4 month old, and 10-13kg. The experimental group was provided by the Guangxi Medical University animal experiment center. The experimental group adopted the compound factor method (65% corn flour +35% cholesterol + carbon tetrachloride + phenobarbital sodium, 10% anhydrous b) Alcohol, as the only drink, was used once a day for 16 weeks. The control group was fed with normal feeding (65% corn +35% rice bran and water as the only drink) and was fed with the experimental group at the same time. The two groups of animals performed 16 layers of spiral CT perfusion scan at the weekend of 0,4,8,12,16 to detect the biochemical indexes of liver function (including T.BIL, D.BIL, I.BIL, Alb, Gl). O, A/G, G-GT, ALT, AST, AST/ALT measurement. The open abdominal measurements of the free pressure of the portal vein (free portal venous pressure, FPP) and the routine axial scan of the liver puncture biopsy were performed to scan the whole liver, and the liver, spleen, aorta and portal vein were taken as the scanning level of the liver perfusion imaging. Body-perfect multi-scans scanning program, scan conditions: 120kv, 60mA, 12mm/ layer, 0.5s/ times. Take the 18~# puncture needle, puncture the ear back vein, give iodide (300mgI/ml), 1.5ml/kg, flow rate 2ml/s, delay 4S backinjection contrast agent after the puncture of the auricular vein. DyperfCT-syngo CT2007A) analysis of the images. Determine the CT value (Hu) in the region of interest on the aorta, the portal vein, the spleen and the liver to obtain a relative time density curve (TDC), that is, the density increase relative to the area of the region of interest. The aorta and the portal vein take a round or oval region of interest, the liver and the spleen. Drawing along the edge of the organ, the region of interest of the liver mainly includes most of the liver leaves on the scan level, and as far as possible to avoid the large blood vessels in the liver. The region of interest of the spleen includes the whole spleen at this level. The area of interest is about 1cm around the edge of the organ so as not to produce the volume effect. The TDC is automatically generated by the software, and then according to TDC The software was calculated by the maximum slope method, and the perfusion parameters were automatically generated, including hepatic arteryperfusion (HAP), portal vein perfusion (portal venous perfusion, PVP), total liver perfusion (total hepatic blood flow, THBF), and hepatic artery perfusion index. An example of a measurement was repeated 2 times and the average value was taken. The data obtained by SPSS 13 were statistically analyzed and analyzed. The perfusion parameters, biochemical indexes and the free pressure of the portal vein were statistically analyzed in the experimental group and the control group and the experimental group. The average number of average numbers of the two groups was compared. A single factorial analysis of variance (One-Way ANOVA) was used in the t test of two independent samples. The non parametric rank sum test (Kruskal-Wallis test) was applied when the variance was inhomogeneous, and P < 0.05 had statistical difference. The four perfusion parameters of HAP, PVP, THBF and HPI of the 1~5 stage of liver fibrosis were respectively adopted by the bivariate Pearson linear correlation analysis. Correlation analysis was carried out with the measured values of portal vein pressure at the same time; Stepwise stepwise regression method of multiple linear regression was used to establish the regression equation of four perfusion parameters of liver fibrosis, PVP, THBF and HPI, and the values of portal vein pressure measured at the same time. The portal vein pressure in all stages of liver fibrosis was derived by regression equation. Results Pearson linear correlation analysis was carried out with the measured value, and the number of pressure values deduced by the formula fell on the 95% confidence interval of the measured value, and the coincidence rate between the actual measurement of the free pressure of the portal vein and the results obtained by the MSCTP measurement was evaluated. Results: (1) there were no 1 cases of death in the 5 head of the control group, and the pathological changes of the liver tissue were normal in 0~16 weeks. In group 40, 14 pigs died at the end of 4 weeks, 2 died at the end of 8 weeks, and the death rate was 40%. The pathological model of liver fibrosis in 0,1,2,3,4,5 stage was obtained respectively, including 0 grade 40 times, 1 level 27 times, 2 grade 19 times, 3 level 17, u/l, AST, u/l, AST/ALT (AST/ALT), respectively. 0.69 + 0.10), G-GT (55.40 + 33.14) u/l, Alb (31.46 + 3.96) g/l, Glo (19.74 + 9.12) g/l, A/G (1.12 + 0.05), T.BIL (4.30 + 1.19) umol/l, D.BIL (1.28 + 55.40) umol/l. There was no statistical difference between the 0 levels of the normal group and the experimental group (P > 0.05), and the ALT, AST, AST/ALT, G-GT, Alb, Glo, A/G, I.BIL in the experimental group were statistically significant (P < 0.05), T.BIL was not statistically significant (P > 0.05). (3) the normal group was (5 + 0.61) 0-5 of the experimental group increased gradually, respectively (5.01 + + + + 1.). 19,14.09 + 4.16,17.02 + 3.67,18.33 + 3.75) there was no statistical difference between the normal group and the experimental group (P > 0.05), and there were statistical significance (P < 0.05) between the experimental group (P < 0.05). (4) the liver fibrosis model of the small pig was CT, the liver surface of the normal and experimental group 0 small pigs was smooth, the density was uniform, and the liver parenchyma did not have abnormal density, liver The ratio of the CT value of the spleen was more than 1, and no ascites was found. The 1 and 2 levels of the experimental group were smooth, the liver density decreased and the ratio of the liver / spleen CT value was less than 1, of which 6 cases showed a small amount of ascites; the liver surface of the 3 and 4 small pigs was less smooth, the liver parenchyma density was less uniform, the local density was reduced, and no substantial mass was found in the liver. Formation of ascites in 11 cases; the liver volume of the 5 grade miniature pig was slightly reduced, the surface was not smooth, the nodular shadow was visible, the density of the liver was not uniform, no substantial mass was formed, and the ascites was found in 13 cases. (5) the normal group was (27.74 + 11.77) ml.min~ (-1).100ml~ (-1) in the normal group; and PVP was (94.70 + 9.32) ml.minl~ (-). (1).100ml~ (-1), THBF (122.44 + 8.79) ml.min~ (-1).100ml~ (-1), HPI (20.54 + 4.08) ml.min~ (-1).100ml~ (-1). The 0-5 period of the experimental group is (28.24 +, + + + + 10.98), and 0-5 (91.69 + + + + + 14.98, respectively). 36.18 + 6.22,32.39 + 6.15) ml.min~ (-1).100ml~ (-1), 0-5 phase THBF (119.93 + 21.87,95.76 + 11.65,72.54 + 27.00,68.26 + 13.65) ml.min~, respectively. The 0-5 period was (22.53 + + + + + 13.12)%. The normal group of the perfusion parameters and the experimental group 0 There was no statistical difference between the experimental group (P > 0.05). There was a statistical difference (P < 0.05) between the 0 and the 1 levels in the experimental group (P < 0.05). There was no statistical difference between the grade 0 and the 2,3,4,5 level (P > 0.05); the PVP in the experimental group had a downward trend (P < 0.05), and the THBF in the experimental group had a downward trend, and there was a statistical difference between the groups. ( P < 0.05); the experimental group HPI increased, and there was a statistical difference between each group (P < 0.05). (6) the experimental group FPP was related to HAP and HPI, R value was +0.263, +0.656, and PVP, THBF was negatively correlated, and the R values were the best. In the regression equation, the F test statistic > 0.05 of the variables eliminated the equation. Among the above 4 perfusion indexes, HAP and THBF were eliminated, and the two variables of PVP and HPI were incorporated into the optimal regression equation: Y=17.175-0.173X_1+0.135X_2 (Y:FPP X_1:PVP X_2:HPI) (7) using FPP and PVP, HPI linear regression equation The portal vein pressure of the 1~5 grade liver fibrosis in the experimental group was 12.7751 + 4.5631mmHg, the measured value was 12.7867 + 5.4053mmHg, the correlation coefficient was r=0.845, P < 0.01, the 95% confidence interval test, the 1-2 diagnosis coincidence rate of liver fibrosis was 84.78%, the 3-4 diagnosis coincidence rate was 88.88%, and the 5 diagnosis coincidence rate was 81.25%. conclusion: 1. improved four Chlorinated carbon (CCL_4), phenobarbital, anhydrous ethanol + high fat, low protein, low choline food compound factor modeling method can successfully establish the liver fibrosis model of small pig.2., the liver function biochemical indexes can be detected, but the pathological staging can not make an objective evaluation of the changes of liver blood perfusion and disease in.3. liver fibrosis and disease. The optimal regression equation for the severity of.4. liver fibrosis MSCTP PVP and HPI is established: Y=17.175-0.173X_1+0.135X_2 (Y:FPP X_1:PVP X_2:HPI) can be used to calculate the free pressure of portal vein in different levels of hepatic fibrosis and to diagnose liver fibrosis by using Y=17.175-0.173X_1+0.135X_2 (Y:FPP X_1:PVP X_2:HPI).
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R575.2;R-332

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