【作者】 尚全良；

【导师】 周启昌； 肖恩华；

【作者基本信息】 中南大学 ， 影像医学与核医学， 2009， 博士

【摘要】 第一章同种异体骨髓单个核细胞肝内移植治疗急性肝损伤的实验研究目的：探讨骨髓单个核细胞肝内移植治疗D-氨基半乳糖诱导的兔急性肝损伤模型的疗效。材料与方法：由雄性健康大白兔四肢长骨提取骨髓单个核细胞,用D-氨基半乳糖腹腔注射诱导建立兔急性肝损伤模型,模型兔(n=66)随机分为2组：移植组(n=32)：直接肝内多点穿刺注射5 m1骨髓单个核细胞悬液(细胞约2×107个)；对照组(n=34)：直接肝内多点注射5 ml D-hanks液。定期观察各组实验兔的行为变化、生存率、肝功能(ALT、AST、ALB)的改变；移植术后,相当于制模后第7天、14天、28天分别处死两组实验兔,每次处死7只,观察肝脏组织病理学变化,计算病理评分。所有数据资料均借助SPSS 11.0统计软件分析。结果：移植组在骨髓单个核细胞移植后的第1周内生存率高于对照组(p<0.05),第2周内及第3—4周内移植组的生存率与对照组比较差异无显著性意义(p>0.05)。肝功能统计分析显示：移植前,两组肝功能无明显差别；移植后,随时间推移,肝功能逐渐好转,在细胞移植后各观察时点移植组转氨酶ALT和AST均较对照组降低明显(p<0.05)；除制模后24h及制模后第28天外,在其余细胞移植后的各观察时点,移植组血清白蛋白均较对照组明显升高(p<0.05)。组织病理学评分的结果显示：随时间推移,病理评分逐渐好转,在各观察时点移植组的病理评分明显好于对照组(p<0.05)。结论：骨髓单个核细胞直接肝内多点注射移植可有效改善D-氨基半乳糖诱导的兔急性肝损伤模型的肝功能。直接肝内多点注射是一种简单有效的骨髓单个核细胞肝脏移植途径。第二章同种异体骨髓单个核细胞肝内移植治疗急性肝损伤肝组织的MVD改变及VEGF的表达研究目的：探讨骨髓单个核细胞移植后急性肝损伤模型肝组织的VEGF(血管内皮生长因子)表达及平均MVD(微血管密度)值的动态变化；探讨平均MVD值与肝功能的相关性。材料和方法：以论文第一章随机选择用以病理评分的42只急性肝损伤模型兔为研究对象,分为2组,移植组(n=21)：直接肝内注射5 ml骨髓单个核细胞(约2×107个)；对照组(n=21)：直接肝内注射5 ml D-hanks液。在行移植手术后相当于制模后的第7天、第14天、第28天,移植组及对照组分别处死实验兔7只,行肝脏病理切片及免疫组化,进行VEGF表达阳性细胞数计数及平均MVD值测定,而后进行对比分析。并将各组平均MVD值与各时点样本兔处死前所测得的各组肝功能指标进行相关性分析。结果：各观察时点移植组的VEGF表达阳性细胞数及平均MVD值均高于对照组(p<0.05)。移植组内部各时点相互比较,VEGF表达阳性细胞数逐渐降低,第7天、第14天较第28天高,差异有显著意义(p<0.05),第7天较第14天高,差异无显著意义(p>0.05)；平均MVD值逐渐增高,第14天及第28天平均MVD值较第7天高,差异有显著意义(p<0.05),第28天较第14天平均MVD值高,差异无显著意义(p>0.05)。对照组内部各时点相互比较,VEGF表达阳性细胞数逐渐降低,第7天较第14天及第28天高,差异有显著意义(p<0.05),第14天较第28天高,差异无显著意义(p>0.05)；平均MVD值逐渐增高,各时点两两比较差异均有显著性意义(p<0.05)。各组平均MVD值与肝功能指标ALT(谷丙转氨酶)、AST(谷草转氨酶)呈负相关(移植组平均MVD值与ALT、AST相关系数为-0.913、-0.918,对照组为-0.776、-0.678)(p<0.05),与ALB(白蛋白)呈正相关(移植组平均MVD值与ALB的相关系数为0.924,对照组为0.729)(p<0.05)。结论：骨髓单个核细胞移植可以促进急性肝损伤模型的VEGF表达及MVD值增加,有利于改善急性损伤肝脏的血供,促进受损肝脏功能的恢复。第三章同种异体骨髓单个核细胞肝内移植治疗急性肝损伤的磁共振扩散成像研究目的：通过分析骨髓单个核细胞移植后急性肝损伤模型的肝脏平均ADC(表观扩散系数)值的动态变化以及其平均ADC值与病理评分、平均MVD值的相关性,探讨利用磁共振扩散成像评价急性肝损伤模型肝功能恢复的可行性；探讨随b值的改变,受损肝脏的平均ADC值受病理评分及血流灌注的影响程度。材料和方法：以10只健康同龄兔及论文第一章随机选择用以病理评分的42只急性肝损伤模型兔为研究对象。42只急性肝损伤模型兔分为细胞移植组及对照组,移植组(n=21)：直接肝内注射5 ml骨髓单个核细胞(约2×107个)；对照组(n=21)：直接肝内注射5 ml D-hanks液。在行移植手术后,相当于制模后的第7天、第14天、第28天,移植组及对照组分别选择实验兔7只进行肝脏磁共振扩散成像及肝脏平均ADC值测定后处死,行肝脏病理切片及免疫组化,进行病理评分及平均MVD值测定,而后进行对比分析。10只健康同龄兔作为正常对照组,用于正常兔肝脏平均ADC值的测定。结果：移植术后,相当于制模后第7d,移植组与对照组的平均ADC值明显低于正常对照组(p<0.05),且随时间的推移,移植组与对照组的平均ADC值逐渐增高向正常肝组织的平均ADC值接近；同一观察时点、相同b值时,移植组肝脏的平均ADC值明显高于对照组(p<0.05)；无论是b取100 s／mm2还是400 s／mm2,移植组与对照组的病理评分值均与平均ADC值呈负相关(移植组b为100 s／mm2及400s／mm2病理评分值与平均ADC值的相关系数分别为-0.805、-0.883,对照组分别为-0.628、-0.762)(p<0.05),其平均MVD值均与平均ADC值呈正相关(移植组b为100 s／mm2及400 s／mm2平均MVD值与平均ADC值的相关系数分别为0.946、0.840；对照组分别为0.884、0.654)(p<0.05)；b取100 s／mm2时,移植组与对照组病理评分与平均ADC值的相关系数均小于平均MVD值与平均ADC值的相关系数,且其病理评分与平均ADC值的相关系数均小于b取400 s／mm2时病理评分与平均ADC值的相关系数,其平均MVD值与平均ADC值的相关系数均大于b取400 s／mm2时平均MVD值与平均ADC值的相关系数。b取400 s／mm2时,移植组及对照组的病理评分与平均ADC值的相关系数均大于平均MVD值与平均ADC值的相关系数。结论：利用损伤肝脏的平均ADC值动态变化可以了解其肝脏修复情况；b值及b值差越小,受损肝脏的平均ADC值与平均MVD值的相关性越强,平均ADC值受血流灌注影响就越大,平均ADC值的动态变化就越能反映受损肝组织血流灌注恢复情况；b值及b值差越大,受损肝脏的平均ADC值与病理评分的相关性越强,平均ADC值的变化越能真实地反映肝组织内水分子自身的扩散运动的变化。更多还原

【Abstract】 PartⅠ:The experimental study on therapeutic effect of allogeneic mononuclear bone marrow cells transplantation to rabbit liver with acute hepatic injuryObjective:To investigate the therapeutic effect of mononuclear bone marrow cells (MBMCs) transplantation to rabbit liver with acute hepatic injury (AHI) induced by D-galactosamine (D-GaIN)Materials and Methods:The prepared D-GaIN solution was injected sterilely into the hypogastrium of each rabbit at a dosage of 10g/kg to establish 66 AHI rabbit models. AHI rabbits (n=66)were randomly divided into 2 groups, a transplanted group (n=32) and a control group(n=34). MBMCs were derived from bone marrow obtained from all limb long bones of male albino rabbits.Each rabbit of the transplanted group was directly infused 5 ml MBMCs suspension (about containing 2×107 MBMCs) into its liver at multiple sites’. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension. The survival rates were observed and liver functions (ALT, AST, ALB) were estimated for the two groups.7 model rabbits from each group were killed on day 7, day 14 and day 28 of AHI establishment after transplantation of MBMCs, respectively, to be carried out pathological examination of liver and pathological score. All data were analyzed by SPSS 11.0 software package.Results:Survival rate of the transplanted group during the first week after MBMCs transplantation was higher than that of the control group(p<0.05). However, there was not significant difference of the survival rate between the two groups during the second week and the fourth week after MBMCs transplantation (p> 0.05). With regard to liver function, the liver functions of the two groups both got improvement gradually, but the level of liver enzyme ALT and AST significantly decreased in the transplanted group compared to the control group at any same time point observed after MBMCs transplantation (p<0.05).There was also a significant improvement of serum albumin (ALB) in the transplanted group compared to the control group (p<0.05)except the observed time point of the 24th hour and 28th day after AHI establishment and MBMCs transplantation. Results of the histopathological scores showed that the histopathological scores of the two groups both gradually decreased over time, but the histopathological score of the transplanted group was better than that of the control group at any time point observed(p<0.05).Conclusions:Direct injection of MBMCs suspension into liver at multiple sites can effectively improved the liver function of the acute liver injury in D-GalN-induced rabbits. Direct injection of MBMCs suspension into liver at multiple sites is a simple and effective route of MBMCs transplantation in experimental model.PartⅡ:Evaluation of MVD and VEGF protein expression of liver tissue in the treatment of acute hepatic injury with allogeneic MBMCs transplantation into liverObjective:To investigate the dynamical changes of VEGF (Vascular endothelial cell growth factor) protein expression and the average value of average MVD(micro-vessel density) in acute hepatic injury model after transplantation of MBMCs (mononuclear bone marrow cells). To investigate the correlation between the average value of MVD and liver function.Materials and Methods:In this part,there were 42 model rabbits of acute liver injury,which were chosen to carry out pathological examination of liver and pathological score in the first part of the thesis. So, in this part there were two groups too,the transplanted group and the control group.There were 21 model rabbits in each group. Each rabbit of the transplanted group was directly infused 5 ml MBMCs suspension (about containing 2×107 MBMCs) into its liver at multiple sites. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension.7 model rabbits from each group were killed on day 7, day 14 and day 28 of AHI establishment after transplantation of MBMCs, respectively. A series of comparative assays performed were including:functional assay of liver, immunohistochemical pathological examination of liver sections, VEGF-expressing cells count and quantification of the average MVD value. At the same time,we investigated the the correlation between the average MVD value and liver function.Results:Before MBMCs transplantation, the difference of liver function was not significant between the transplanted group and the control group. But after MBMCs transplantation,the liver functions of model rabbits in the transplanted group were significantly better than those of the control group at all time points observed (p<0.05). More VEGF-expressing cells and higher average MVD value were observed in the transplanted group than the control group (p<0.05). In the transplanted group, the count of VEGF-expressing cells gradually reduced, the count on day 7 was higher than that on day 14 (p>0.05), while both were significantly higher than the count on day 28 (p<0.05). In contrast, the average value of MVD increased gradually in the transplanted group, the value on day 28 was higher than the one on day 14 (p>0.05), while both were significantly higher than the one on day 7 (p<0.05). In the control group, the count of VEGF-expressing cells also gradually reduced, the VEGF-expressing cell number on day 7 was higher than the one on day 14 and day 28 (p<0.05), and the number on day 14 was higher than the one on day 28 (p>0.05). The average MVD value increased with significance between each time points observed over time (p<0.05). The correlation between the average MVD value and the ALT or AST was negative (the transplanted group:-0.913,-0.918, the control group:-0.776,-0.678) (p<0.05), at the same time, the correlation between the average MVD value and the ALB was positive (the transplanted group:0.924, the control group:0.729) (p<0.05).Conclusions:Transplantation of MBMCs promotes the expression of VEGF and also up-regulates the average MVD value in the acute injury livers. This facilitates the recovery of liver function.PartⅢ:The study of magnetic resonance diffusion-weighted imaging(MR-DWI) to the treatment of acute hepatic injury with allogeneic MBMCs transplantation into liverObjective:By analyzing the dynamic changes of the mean ADC values of the injury livers after MBMCs transplantation and the correlation between the mean ADC values and the injury liver histopathological score or MVD values,to investigate the feasibility that evaluated the functional recovery of acute hepatic injury model with MR-DWI and the degree of the influence of the histopathological score and blood perfusion to the mean ADC value of the injury liver. Materials and Methods:In this part,there were 10 healthy rabbits and 42 model rabbits with acute liver injury,which were chosen to carry out pathological examination of liver and pathological score in the first part of the thesis. So, in this part there were three groups:the normal control group(n=10), the transplanted group(n=21) and the control group(n=21). Each rabbit of the transplanted group was directly infused 5 ml MBMCs suspension (about containing 2×107 MBMCs) into its liver at multiple sites. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension.7 model rabbits from the transplanted group and the control group were killed on day 7, day 14 and day 28 of AHI establishment after transplantation of MBMCs, respectively. A series of comparative assays performed were including:immunohistochemical pathological examination of liver sections, quantification of pathological score and the average MVD value. Before the rabbits killed,each model rabbit was carried out MR-DWI scan and calculated the mean ADC value of the injury liver. Each rabbit of the normal control group was only carried out MR-DWI scan and calculated the mean ADC value of the normal liver.Results:On day 7 of AHI establishment after MBMCs transplantation, the mean ADC values of the injury livers of the transplanted group and the control group were significant lower than those of the normal control group (p<0.05). The mean ADC values of the injury livers of the transplanted group and the control group increased to the mean ADC values of the normal control group over time. At the same time point observed, The mean ADC values of the injury livers of the transplanted group were significant higher than those of the control group when the b value was same.No matter the b value was 100 s/mm2 or 400 s/mm2, there was negative correlation between the histopathological scores and the mean ADC values in the transplanted group and the control group (the transplanted group:-0.805,-0.883; the control group:-0.628,-0.762) (p<0.05), but there was positive correlation between the average MVD values and the mean ADC values in the transplanted group and the control group (the transplanted group:0.946,0.840; the control group:0.884,0.654) (p<0.05). When the b value was 100 s/mm2, the correlation coefficient between the histopathological scores and the mean ADC values was lower than that between the average MVD values and the mean ADC values,at the same time, it also was lower than that between the histopathological scores and the mean ADC values when the b value was 400 s/mm2. When the b value was 100 s/mm2, the correlation coefficient between the average MVD values and the mean ADC values was higher than that when the b value was 400 s/mm2. When the b value was 400 s/mm2, the correlation coefficient between the histopathological scores and the mean ADC values was higher than that between the average MVD values and the mean ADC valuesConclusions:The recovery of the inury liver can be detected with observing dynamic change of its mean ADC value. The lower the b value, the higher the correlation coefficient between the average MVD values and the mean ADC values, which meant the influence of blood perfusion became more obvious to the mean ADC values of the injury livers with the decrease of b value. The higher the b value, the higher the correlation coefficient between the histopathological scores and the mean ADC values, which meant the mean ADC values became more truly reflect the diffusion of water molecules themselves in the injury liver with the increase of the b value.更多还原