BMSCs多分化潜能鉴定、向肝细胞分化、体外标记和MR活体示踪研究

发布时间：2018-05-08 12:34

本文选题：骨髓间充质干细胞 + 分化　；参考：《吉林大学》2008年硕士论文

【摘要】： 目前,关于肝脏组织损伤后,如何使肝细胞再生和肝功能恢复是人们一直非常关注的课题,也是临床治疗中遇到的难点。利用干细胞移植和干细胞工程肝细胞来治疗肝组织损伤,促使肝细胞再生以及改善肝功能等日益受到人们重视。本研究主要采用具有多分化潜能的骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)向肝细胞进行定向诱导分化,获得肝样细胞,为干细胞移植提供可靠的种子细胞来源;同时,通过细胞标记,研究移植后的细胞迁移及MRI活体示踪,为MRI动态观察建立一个良好的标记方法和技术平台。目的:用本实验室建立的大鼠BMSCs培养方法,分离、培养和纯化大鼠BMSCs,对BMSCs进行多分化潜能鉴定,检测BMSCs表面抗原;体外诱导分化为肝样细胞后进行免疫细胞化学和RT-PCR方法检测,鉴定肝细胞特异性标志物;将标记的肝样细胞经门静脉移植、肝内移植入CCl_4肝损伤大鼠肝脏,利用MR影像系统进行细胞移植后的动态观察,探讨细胞移植前后信号变化及变化规律,活体显示标记肝样细胞的特异性及敏感性,为细胞移植临床治疗提供可靠的理论依据。方法:采用全血培养法和密度梯度离心法,经传代和控制消化时间得到纯度较高的BMSCs;传代后用免疫化学法检测其表面抗原;用成脂诱导和成骨诱导的方法鉴定多分化潜能,用损伤肝组织和血清作为诱导剂定向诱导分化BMSCs,利用免疫细胞化学和RT-PCR等方法对MSCs和诱导后肝样细胞进行细胞特异性标志物的检测;利用CCl_4肝损伤模型鼠,经门静脉、肝实质移植标记的肝样细胞,采用MRI技术对大鼠肝脏进行活体扫描,然后进行统计学分析。结果:利用本实验室的方法获得了高纯度贴壁生长的BMSCs呈均匀分布的集落样生长,呈梭形或纺锤形,细胞传代稳定,随传代次数增加纯度提高。传代培养第3代BMSCs经免疫细胞化学检测CD105呈阳性;传代培养BMSCs诱导分化后,油红O结合苏木素染色结果显示细胞核呈浅兰色,胞浆内有橘红色脂肪滴出现,显示为脂肪细胞;BMSCs诱导分化后,ALP染色显示细胞内有致密颗粒形成,矿化结节染色结果显示BMSCs经诱导后有很好的成骨能力。诱导BMSCs向肝细胞分化实验显示:白蛋白呈阳性,糖原染色阳性,可表达肝脏特有的基因AFP、ALB。标记的肝样细胞体外MRI成像显示,在T1W,T2W序列均可见信号改变;标记的肝样细胞移植入大鼠肝脏后,采用MRI技术对大鼠肝脏进行扫描:对照组在移植前肝脏均未见明显的异常低信号区;门静脉组大鼠移植后24h内在肝右叶可见明显信号改变;肝内注射组大鼠移植后24h内同样出现信号改变。1W后MRI仍能检测到信号改变。结论: (1).利用本实验室建立的方法,可获得高纯度贴壁生长的BMSCs。在体外可以实现大量扩增; (2).培养的BMSCs可表达表面抗原CD105,具有分化为脂肪细胞,成骨细胞的能力,证明BMSCs具有多分化潜能; (3).利用本实验室建立的方法,可诱导BMSCs分化为肝样细胞; (4).标记的肝样细胞在体内、体外均可引起MRI信号的明显改变,可进行较长时间的MRI动态观察,有较好的特异性和灵敏度。
[Abstract]:At present , after liver tissue injury , how to regenerate liver cells and recover liver function is a problem that people have been very concerned about , and it is a difficult problem in clinical treatment . The research mainly adopts stem cell transplantation and stem cell engineering liver cells to induce differentiation of liver tissues , and to obtain liver - like cells , which can provide reliable source of seed cells for stem cell transplantation .

Objective : To isolate , culture and purify the bone marrow cells of rats by the method of culture and culture and to purify the bone marrow cells of the rats , and to detect the surface antigen of bone marrow cells . After the cells were transplanted into the liver , the specific markers of liver cells were identified . The changes of signal before and after the transplantation of the cells were investigated . The changes and the changes of the signal were observed before and after the transplantation of the cells . The specific and sensitivity of the cells were examined in vivo .

Methods : Using whole blood culture method and density gradient centrifugation method , the cells with high purity were obtained by means of immuno - chemical method .

Results : The results showed that the cells with high purity adherent growth had a uniform distribution of colony - like growth , spindle - shaped or spindle - shaped , and increased purity with the number of passages .

Conclusion :

( 1 ) By using the method established in this lab , the high - purity adherent growth can be obtained , and a large amount of amplification can be realized in vitro ;

( 2 ) The expressed surface antigen CD105 can be expressed by cultured bone marrow cells , which has the capability of differentiating into adipocytes and osteoblasts , and proves that the bone marrow cells have the multi - differentiation potential ;

( 3 ) By using the method established in this lab , the cells can be induced to differentiate into liver - like cells ;

( 4 ) In vivo and in vitro , the marked liver - like cells can cause obvious changes of MRI signal , and can be used for MRI dynamic observation for a long time , and has good specificity and sensitivity .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【参考文献】