【作者】 杨欢；

【导师】 许文荣；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的建立PKH26、DAPI标记骨髓间质干细胞(mesenchymalstem cell，MSC)的方法，并探讨标记MSC的基本生物学活性；SD大鼠经50％的甘油肌内注射建立大鼠急性肾功能衰竭(acute renalfailure，ARF)模型，评价经尾静脉注射的MSC对大鼠ARF的治疗效果及体内外初步探讨其可能机制。方法MSC按PKH26、DAPI标记程序进行标记后培养，采用激光共聚焦显微镜(confocol)、流式细胞分析仪(FCM)等观察细胞生长状态和荧光标记活性变化；应用RT-PCR检测标记后MSC的nucleostemin及Bmi-1基因的表达；选用碱性磷酸酶(ALP)染色、Von kossa染色及骨形成蛋白3(BMP3)基因表达分析等技术，观察标记后MSC体外分化成骨细胞的特性；SD大鼠经50％的甘油肌内注射建立大鼠ARF模型，尾静脉注射MSC，综合运用临床生物化学、分子生物学、免疫组织化学、病理学、荧光原位杂交(FISH)等技术，评价MSC的治疗效果并找寻修复肾损伤的机制。体外利用Transwell培养板将大鼠MSC与受损肾组织共培养，运用confocol、RT-PCR、巢式PCR、荧光定量PCR鉴定细胞是否能分化为肾小管样上皮细胞，进一步佐证MSC的治疗修复机制。结果PKH26标记后细胞呈红色荧光，DAPI标记的细胞核呈蓝色荧光，荧光的强度随着荧光染料浓度的增加而递增，并与传代时间和代数相关；标记后细胞生长状态良好，基本生长特性如传代培养和生长曲线无明显改变；细胞nucleostemin及Bmi-1基因表达未见改变；标记MSC诱导后ALP、Von kossa染色阳性，呈现典型的成骨细胞形态和生物学特征，并表达BMP3基因；成功建立大鼠ARF模型，ARF大鼠肾组织内具有较多的外源MSC，其他组织如心、肝、肺、脑也可见少量的外源MSC；利用胎儿MSC进行的定位实验中，在大鼠的心、肝及不同肾脏部位扩增出人17α卫星DNA序列。FISH实验显示大鼠冰冻肾脏切片中可见人源性细胞。MSC实验组大鼠血清肌酐、尿素氮下降水平较对照组具有明显的差异(P＜0.05)。实验组大鼠肾脏恢复情况较对照组有明显改善。体外共培养后，大鼠MSC形态由长梭形变为圆形，并且高表达上皮细胞特异性基因CK18和肾小管上皮细胞特异性基因AQP1。结论PKH26、DAPI可稳定标记大鼠骨髓MSC并能传代培养，标记后细胞形态、生长活力及多向分化潜能等无明显影响，该技术可用于追踪MSC转归、可塑性及干细胞移植方面的实验研究；外源MSC体内能够归巢到受损肾脏，并能有效地治疗大鼠ARF。体外受损肾组织能诱导大鼠MSC分化为肾小管样上皮细胞。本研究为ARF的有效治疗和肾损伤的修复等提供新的实验模型和依据，为临床治疗肾脏疾病开辟一条新途径。更多还原

【Abstract】 Objective To establish a method of labeling bone marrow mesenchymal stem cell with PKH26 and DAPI in vitro and explore the biological activity of labeled MSC. To induce rat acute renal failure model by intra-muscle injection of 50% glycerol in SD rats and evaluate the therapeutic efficacy by injecting MSC intravenously and explore the possible mechanisms in vivo and in vitro.Methods Mesenchymal stem cell was stained with red fluorescent PKH26 and blue fluorescent DAPI. The growth and fluorescent intensity was observed by confocol laser microscopy and flow cytometer. The expression of nucleostemin, Bmi-1 genes in the cells was detected by RT-PCR. The characteristics of labeled MSC differentiating into osteoblasts in vitro were identified by ALP stain, Von kossa stain and bone morphogenetic protein3(BMP3) expression. Rat acute renal failure model was induced by intra-muscle injection of 50% glycerol in SD rats. MSC was injected intravenously into them. The therapeutic efficacy of MSC as well as the possible mechanisms about it were investigated by clinical biochemistry, molecular biology, immunohistochemical and pathological methods, fluorescence in situ hybridization(FISH) and so on. Rat MSC was cocultured with injured kidney by transwell in vitro, and whether the cells could differentiate into renal tubular-like epithelial cells was identified by confocol, RT-PCR, Nested PCR, real-time quantitative PCR.Results The labeled cells appeared red fluorescence. The intensity of fluorescence was related to the concentration of fluorochrome and the passages. The growth characteristic between the labeled and unlabeled cells was not different significantly. The expression of nucleostemin, Bmi-1 genes between them was not observed. After being induced, the cells appeared typical cell shape and biological characteristics of osteoblasts, positive for ALP stain, Von kossa stain and expressed BMP3 gene. Rat ARF model was established successfully. There were more exogenous MSC in injured kidney than in other organs such as heart, liver, lung and brain. The human 17αsatellite DNA sequence was amplified in rat heart, liver and different parts of rat kidney in the experiment for fetal MSC location. The exogenous cells from human MSC could be seen in rat kidney section by FISH analysis. The levels of serum creatinine and nitrogen decreased significantly in experimental groups than in control groups(P＜0.05). The kidney recovery situation in experimental groups was better than in control groups. After cocultured with injured kidney in vitro, the shape of rat MSC turned round, and they highly expressed cytokeratin 18(CK18) and aquaporin-1(AQP1).Conclusions MSC could be labeled with PKH26 and DAPI stably. The labeled cells still had the abilities of self-renewal and multi-differentiation. It was suggested that the labeling method could be applied to study the homing, plasticity and transplantation of MSC. Exogenous MSC could home to the injured kidney and cure rat ARF efficiently. The injured kidney tissue could induce rat MSC trans-differentiate into renal tubular-like epithelial ceils. These investigations could offer original experimental model and evidence about efficient cure of MSC to kidney disorders such as ARF, acute glomerulonephritis, and also open up a new path to them.更多还原