3.0T MRI活体示踪BMSCs移植修复肾脏缺血再灌注损伤

发布时间：2018-06-24 02:51

本文选题：肾脏 + 缺血再灌注损伤　；参考：《山西医科大学》2014年硕士论文

【摘要】：1.目的 1.1研究骨髓间充质干细胞的形态及生长特征；并对其进行鉴定。 1.2建立肾脏缺血再灌注损伤模型并移植SPIO标记BMSC（s经肾动脉及肾包膜下两种移植方法）；再利用MR活体示踪BMSCs，T2及T2*map图像定量分析。 1.3综合评价BMSCs移植对肾脏缺血再灌注损伤的治疗疗效。 2.材料与方法 2.1实验对象 SD大鼠40只,雄性，5-6w,170-200g(山西医科大学实验动物中心提供)。 2.2实验方法 MRI检查方法：SD大鼠分别于造模前、造模后1d、1w、2w、3w行磁共振扫描（采用SiemensVerio3.0T磁共振成像仪，水平卧位,头先进,行轴位T2WI、T2*WI、多回波T2序列及T2*序列扫描）。细胞鉴定方法：采用流式细胞仪检测，，CellQuestPlot软件分析数据；采用常规免疫荧光染色，用激光共聚焦显微镜观察。 SD大鼠造模3w后计算体重增长率、下腔静脉采血测定肾脏功能，取材进行组织切片及HE染色。 3.结果 3.1体外实验采用全骨髓贴壁法成功分离BMSCs，免疫荧光法及流式细胞分析法鉴定细胞表型：CD34(-)、CD45（-）、CD44(+)、CD90(+)；BMSCs能够成功地被诱导分化为成骨、成脂细胞。 3.2MRI活体示踪（1）肾脏IRI后MRI表现：损伤前，T2WI图像双肾大小基本对称、信号一致，而肾脏缺血再灌注损伤后左肾体积增大、信号增高；（2）T2*WI序列在体示踪SPIO标记BMSCs：1d时，两种不同方法低信号区（铁信号）均局限在注射局部，随着时间推移，肾动脉组左肾出现斑片状低信号区，而肾包膜下组低信号区仍部分局限于包膜下。（3）损伤组与治疗组（包括肾动脉组、肾包膜下组）T2值：损伤后，三组T2值均升高，最高点位于1d；随着时间推移，T2值有下降趋势，但两组治疗组下降幅度较损伤组大；在同一时间点，治疗组T2值低于损伤组，但两组治疗组之间差异不明显。损伤组与两组治疗组（肾动脉组、肾包膜下组）T2*值：损伤组T2*值趋势比较稳定，推测肾脏缺血再灌注损伤对T2*值影响不大；两组治疗组T2*值最低点均位于2w，在同一时间点，两组治疗组T2*值低于损伤组，但两组治疗组之间变化趋势不一致。 3.3评价疗效（1）比较三组体重增长率，由高到低依次为：对照组治疗组损伤组；（2）比较三组肾脏功能，尿素氮及肌酐升高水平由高到低为：损伤组治疗组对照组，治疗组间差异有统计学意义，肾动脉组较肾包膜下组水平低；（3）比较HE染色结果：对照组肾脏组织结构基本正常，损伤组大量肾小管上皮细胞肿胀、空泡变性，治疗组肾小管上皮细胞坏死程度较损伤组轻。 4.结论 4.1采用全骨髓贴壁法可以成功分离、培养BMSCs。 4.2SPIO标记的BMSCs能够靶向迁移到肾脏缺血再灌注损伤区。 4.3MRIT2WI及T2map图像能够显示肾脏缺血再灌注损伤后改变，T2*WI及T2*map图像能够活体示踪SPIO标记的BMSCs，但是两种移植方法无明显差异。 4.4从体重增长率、肾脏功能及组织HE染色综合分析，BMSCs对肾脏缺血再灌注损伤有一定修复作用。
[Abstract]:1. purposes
1.1 to study the morphology and growth characteristics of bone marrow mesenchymal stem cells and identify them.
1.2 the model of renal ischemia-reperfusion injury was established and SPIO labeled BMSC was transplanted (two kinds of transplantation methods under renal artery and renal capsule), and the quantitative analysis of BMSCs, T2 and T2*map images was traced by MR in vivo.
1.3 to evaluate the therapeutic effect of BMSCs transplantation on renal ischemia-reperfusion injury.
2. materials and methods
2.1 experimental objects
40 SD rats, male, 5-6W, 170-200g (provided by Shanxi Medical University experimental animal center).
2.2 experimental method
MRI examination method: SD rats were performed by magnetic resonance scanning (using SiemensVerio3.0T magnetic resonance imaging instrument, horizontal position, head advanced, axis T2WI, T2*WI, multiple echo T2 sequence and T2* sequence) after model building, 1D, 1W, 2W, 3W.
Cell identification methods: flow cytometry, CellQuestPlot software analysis data, routine immunofluorescence staining, laser confocal microscopy observation.
The weight gain rate of SD rats was calculated after modeling 3W, and the renal function was measured by inferior vena cava blood collection. Tissue sections and HE staining were taken.
3. results
3.1 in vitro experiment
BMSCs was successfully separated by full bone marrow adherence, and the cell phenotype was identified by immunofluorescence and flow cytometry: CD34 (-), CD45 (-), CD44 (+), and CD90 (+); BMSCs could be successfully induced to differentiate into osteoblast and lipocyte.
3.2MRI living body tracer
(1) MRI expression after IRI: before injury, the size of the two kidneys in the T2WI image was basically symmetrical and the signal was consistent, but the volume of the left kidney increased and the signal increased after the renal ischemia-reperfusion injury. (2) when the T2*WI sequence traced the SPIO marker BMSCs:1d, the two different methods of the low signal region (iron signal) were limited to the injection part, and the renal artery group went on with time. There was a low signal area in the left kidney, while the low signal area in the subcapsular group was still partially confined to the capsule. (3) the T2 value of the injury group and the treatment group (including the renal artery group and the renal subcapsular group): after the injury, the three groups were all increased and the highest point was in 1D; the T2 value decreased as the time went on, but the decrease of the two groups was larger than that of the injury group. At the same time point, the value of T2 in the treatment group was lower than that of the injury group, but the difference between the two groups was not obvious. The T2* value of the injured group and the two groups (renal artery group and subcapsular group): the trend of T2* value in the injured group was more stable, and the effect of renal ischemia reperfusion injury on the value of T2* was not significant; the lowest T2* value of the two groups was in the same one in the same group. At the time point, the T2* value of the two groups was lower than that of the injury group, but the trend of change between the two groups was not consistent.
3.3 evaluation of curative effect
(1) the weight growth rate of the three groups was compared from high to low to the control group, and (2) the renal function of the three groups was compared with the high to low levels of urea nitrogen and creatinine. The difference between the treatment group and the control group was statistically significant, and the renal artery group was lower than the renal subcapsular group; (3) the results of HE staining were compared: A large number of renal tubular epithelial cells were swollen and vacuolar degeneration in the injured group. The necrosis degree of renal tubular epithelial cells in the treatment group was lighter than that in the injury group.
4. conclusion
4.1 the whole bone marrow adherent method can be successfully used to isolate BMSCs..
4.2SPIO labeled BMSCs can migrate to renal ischemia-reperfusion injury area.
4.3MRIT2WI and T2map images can show changes in renal ischemia-reperfusion injury, and T2*WI and T2*map images can trace SPIO labeled BMSCs in vivo, but there is no significant difference between the two methods of transplantation.
4.4 comprehensive analysis of body weight growth rate, renal function and tissue HE staining indicated that BMSCs had certain restorative effect on renal ischemia-reperfusion injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R699.2;R445.2

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