胚胎后肾组织体外诱导骨髓间充质干细胞向肾干细胞分化实验研究

发布时间：2018-05-12 23:09

本文选题：骨髓间充质干细胞 + 后肾　；参考：《第三军医大学》2009年博士论文

【摘要】： 背景和目的: 慢性肾脏疾病(Chronic kidney disease,CKD)发病率不断增加,在成人中的发生率已达10%,其主要特征是细胞外基质(Extracellular matrix,ECM)过度沉积和纤维化,引起肾功能进行性下降,发展的最终结局是终末期肾衰(end stage renal disease,ESRD),目前只能依赖血透、腹透和肾移植来治疗。血透、腹透只能部分替代肾脏的滤过功能。肾移植供体短缺、抗排异药物副作用大,这些治疗方法均存在严重缺陷。寻找更完美的新治疗方法和途径是肾脏病工作者迫在眉睫的任务。近来研究发现骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)具有高度可塑性(plasticity),在体的胚胎组织可诱导骨髓间充质干细胞向肾干细胞(renal stem cells)或肾脏固有细胞分化,异种胚胎组织也可诱导人骨髓间充质干细胞向肾干细胞或肾脏固有细胞分化,但在胚胎内诱导骨髓间充质干细胞向肾干细胞分化无临床应用前景,因为难以从胚胎组织中分离纯化由骨髓间充质干细胞分化而来的肾干细胞。如果能在体外诱导骨髓间充质干细胞分化为肾干细胞,则诱导的细胞可被轻而易举地分离和纯化,具有广阔的临床应用前景。大量资料表明,干细胞的分化发育及其发育方向,取决于其所处的微环境(microenvironment)。在后肾发育过程中,后肾间充质细胞(metanephric mesenchyme cells,MMCs)即胚肾干细胞,具有自我更新及多向分化的能力,除了集合管起源于输尿管芽(ureteric bud,UB)外,后肾间充质细胞分化形成所有的肾单位和基质细胞,在离开胚肾组织仍能发育成肾固有细胞,这提示胚肾组织具有干细胞微环境特征。鉴于以上科学研究背景,本课题拟在体外,用Transwell小室共培养体系,将骨髓间充质干细胞与后肾间充质细胞和输尿管芽分室共培养,利用胚肾组织按内在规律释放的可溶性因子,诱导骨髓间充质干细胞向肾干细胞分化,观察诱导后的干细胞在体外诱导输尿管芽分枝的能力,为以后探讨诱导骨髓间充质干细胞向肾干细胞分化所需的调控因子及建立单纯用诱导因子在体外诱导骨髓间充质干细胞向肾干细胞分化的方法提供实验依据,为肾脏组织工程研究以及将来采用肾干细胞在纤维化的肾组织形成有功能的新肾单位的实验研究提供充足的无免疫原性的肾脏种子细胞。方法: 1.用密度梯度离心和贴壁培养相结合的方法分离培养大鼠骨髓间充质干细胞,倒置相差显微镜观察细胞形态及生长特点,四唑盐比色法(Methylthio tetrazole, MTT)观察细胞增殖及更新能力,透射电镜观察细胞内部结构,流式细胞仪检测细胞周期及细胞表面CD44、CD45、CD90分子的表达,免疫细胞技术检测细胞波形蛋白(Vimentin)、纤维连接蛋白(Fibronectin,FN)、角蛋白(Keratin)、E-钙粘蛋白(E-cadherins)、CD34等的表达,同时对培养细胞进行成脂、成骨诱导分化,采用油红(Oil red)O染色及骨钙素(osteocalcim,OCN)观察大鼠骨髓间充质干细胞诱导分化情况。细胞鉴定后,将其接种于细胞外基质胶中进行三维立体(three-dimensional,3D)培养,倒置相差显微镜观察细胞在细胞外基质胶中的形态变化,激光扫描共聚焦显微镜观察细胞外基质胶不同层面的细胞形态。 2.在解剖显微镜下分离孕13.5d(E13.5d)大鼠胚胎,获取胚胎后肾并去除输尿管芽,将胚胎后肾间充质细胞以组织块法进行原代培养,用倒置相差显微镜观察细胞形态演变,MTT法观察细胞增殖及更新能力,透射电镜和扫描电镜观察细胞内部结构和表面结构,流式细胞仪检测细胞周期及细胞表面CD44、CD45、CD90分子的表达,免疫细胞技术检测细胞Vimentin、Fibronectin、Keratin、E-cadherins及CD34的表达,同时进行细胞多向诱导分化(成脂肪细胞、成骨细胞)实验,分别采用油红O及OCN染色观察诱导分化情况。细胞鉴定后,将其接种于细胞外基质胶中进行三维立体培养,倒置相差显微镜观察细胞在细胞外基质胶中的形态变化,激光扫描共聚焦显微镜观察细胞外基质胶不同层面的细胞形态变化。 3.在解剖显微镜下分离孕13.5d(E13.5d)大鼠胚胎,获取胚胎后肾,消化法得到完整的输尿管芽,将其种植于Transwell小室上室中的细胞外基质胶中,后肾间充质细胞种植于Transwell小室下室中,二者进行分室共培养,倒置相差显微镜下观察输尿管芽的形态,免疫细胞技术对其进行鉴定,激光扫描共聚焦显微镜下观察输尿管芽的形态变化。 4.建立大鼠骨髓间充质干细胞向肾干细胞分化的体外诱导共培养模型,并分为5组,A:骨髓间充质干细胞+后肾间充质细胞;B:骨髓间充质干细胞+输尿管芽;C:骨髓间充质干细胞+后肾间充质细胞+输尿管芽;D:骨髓间充质干细胞;E:空白对照组。用倒置相差显微镜观察诱导后的大鼠骨髓间充质干细胞形态变化,流式细胞仪检测诱导后细胞周期及细胞表面CD44、CD45、CD90分子的表达,免疫荧光化学法检测诱导后细胞的细胞标志物Vimentin、Fibronectin、Keratin、E-cadherins蛋白的表达。诱导后细胞与输尿管芽共培养,用激光扫描共聚焦显微镜观察诱导后细胞对输尿管芽的诱导能力。结果: 1.大鼠原代BMSCs呈集落样生长,细胞形态呈梭形,传代后细胞生长迅速,形态比较均一,大部分细胞为成纤维样或多角形,细胞增殖能力强,具有自我更新能力;透射电镜结果显示细胞核较大,核呈椭圆形或不规则形,有核突,含有1-2个核仁,胞浆内含有粗面内质网和线粒体,其他细胞器较少,胞浆内可见分泌小泡,表现出未分化细胞的特征;细胞周期结果显示,只有少数细胞进入增殖活跃期,大部分细胞处于静止期;流式细胞仪检测细胞表面分子标志物的结果显示,细胞强表达CD44、CD90,不表达CD45;免疫细胞化学染色结果显示BMSCs明显表达Vimentin、Fibronectin,不表达Keratin、E-cadherin及CD34;BMSCs在特定诱导条件下可以向脂肪细胞、成骨细胞分化,加入成脂肪诱导培养基3w后,细胞内可见多个折光性强的圆形脂肪滴,油红O染色呈红色;在加入成骨诱导培养基诱导后,可见圆形或卵圆形矿化结节,OCN表达阳性。在本实验中,大鼠BMSCs在由80%Ⅰ型胶原与20%的微量生长因子基质胶组成的细胞外基质胶中生长状态较好。 2.培养的大鼠MMSCs贴壁生长,梭形,成纤维细胞样,单个核,核仁大,,形态均一,细胞增殖能力强,具有自我更新能力;透射电镜结果显示,细胞为长梭形或不规则形状,核不规则,核仁大,胞浆中滑面内质网发达,可见分泌颗粒;扫描电镜结果示,细胞呈梭形或多角形,核仁明显,细胞连接紧密,表面有微绒毛突起,梭形细胞胞浆向两极伸出长突起,多角形细胞突起呈树枝状,紧紧贴于盖玻片上;细胞周期结果显示,90%以上的细胞处于静止期,只有少部分细胞处于增殖期;流式细胞仪检测结果显示,几乎所有细胞都明显表达CD44、CD90,几乎所有细胞不表达CD45;免疫细胞化学染色法结果显示,大鼠MMSCs明显表达间充质细胞标志蛋白Vimentin、Fibronectin,不表达Keratin、E-cadherin及CD34;细胞在特定诱导条件下具有多向分化能力,可以向脂肪细胞、成骨细胞分化,诱导分化后的脂肪细胞油红O染色呈特异性红色,OCN表达呈阳性,并可形成钙结节。倒置相差显微镜下观察三维立体培养的大鼠MMSCs结果显示,不同层面细胞都呈长梭形,培养2-3d可见细胞连接,细胞增殖能力强;激光扫描共聚焦显微镜下观察可见三维立体培养的细胞明显表达Vimentin、Fibronectin,不表达Keratin及E-cadherin。三维立体培养体系中,大鼠MMSCs形状立体感强,生长状态好。 3.倒置相差显微镜下观察E13.5d大鼠的UB呈T形,接种于细胞外基质中不断分枝发芽。激光扫描共聚焦显微镜下,用FITC-DB标记UB,结果显示大鼠UB以二叉分枝方式进行分枝、生长,UB顶端的细胞生长迅速,其颈部细胞较顶端(壶腹)部细胞的生长速度慢。随着生长时间的延长,大鼠UB不断以二叉分枝方式扩展延伸,逐渐形成树状结构。 4.倒置相差显微镜下观察诱导后的大鼠BMSCs,结果显示细胞生长密集,界限不清,呈梭形;流式细胞仪检测诱导后的细胞周期结果显示,90%以上的细胞处于静止期,只有少部分细胞处于增殖期;流式细胞仪检测诱导后的细胞表面标记物,结果显示几乎所有细胞都明显表达CD44、CD90,几乎所有细胞不表达CD45;免疫荧光检测诱导后的细胞结果显示,大鼠后肾间充质干细胞明显表达间充质细胞标志蛋白Vimentin、Fibronectin,不表达上皮细胞标志蛋白Keratin及E-cadherin;激光扫描共聚焦显微镜下观察到C组诱导后的细胞对大鼠UB有诱导作用,C组的UB以二叉分枝的方式分枝发芽。结论: 1.我们成功分离培养了大鼠BMSCs和MMSCs,这些细胞具有典型的间充质细胞形态及干细胞表型,具有多向分化能力。 2.我们成功分离、培养、鉴定了大鼠UB。 3.我们建立了大鼠BMSCs在体外向肾干细胞分化模型,实现了BMSCs向肾干细胞的转型。在体外诱导后的大鼠BMSCs能促进大鼠UB分枝发芽。
[Abstract]:Background and Purpose :

Chronic kidney disease ( CKD ) is increasing and the incidence of chronic kidney disease ( CKD ) is up to 10 % . It is characterized by excessive deposition and fibrosis of extracellular matrix ( ECM ) , which causes progressive decline in renal function .

It has been found that bone marrow mesenchymal stem cells can induce differentiation of bone marrow mesenchymal stem cells into renal stem cells or kidney cells .

In the process of post - renal development , the differentiation of mesenchymal cells ( MMCs ) , i.e . , embryonic kidney stem cells , has the ability to self - renew and multi - directional differentiation .

In view of the above scientific research background , in vitro and in vitro , the bone marrow mesenchymal stem cells are co - cultured with the posterior renal mesenchymal stem cells and the ureter bud subchambers , and the differentiation of the bone marrow mesenchymal stem cells into the renal stem cells is induced by utilizing the soluble factors released by the embryo and kidney tissues in vitro , and the experimental research on the induction factor for inducing the differentiation of the bone marrow mesenchymal stem cells into the renal stem cells in vitro provides an experimental basis for further exploring the experimental research of inducing bone marrow mesenchymal stem cells to differentiate into renal stem cells in vitro , and provides sufficient non - immunogenic renal seed cells for experimental research on the renal tissue engineering research and the future use of renal stem cells to form a functional new kidney unit .

Method :

1 . The rat bone marrow mesenchymal stem cells were isolated and cultured by means of density gradient centrifugation and adherent culture . The cell morphology and growth characteristics were observed by reversed phase contrast microscope . The expression of CD44 , CD90 , E - cadherins , CD34 , etc . were observed .

2 . The rat embryo of 13.5 days ( E13.5 days ) was isolated under an anatomical microscope . After the embryo was obtained , the kidney was removed and the ureter bud was removed . The cell morphology and surface structure were observed by using an inverted phase contrast microscope . The cell cycle and the expression of CD44 , CD90 and E - cadherins and CD34 were observed by MTT assay . The morphological changes of the cells in extracellular matrix glue were observed by MTT assay . The morphological changes of the cells in extracellular matrix glue were observed by reversed phase contrast microscope .

3 . The rat embryo was isolated from 13.5 days ( E13.5 days ) under an anatomical microscope . After the embryo was obtained , the complete ureter bud was obtained by digestion method , and then it was planted in the extracellular matrix glue in the upper chamber of Transwell chamber . The posterior renal mesenchymal cells were cultured in the lower chamber of Transwell chamber . The morphology of the ureter bud was observed under the reversed phase contrast microscope . The morphological changes of the ureter bud were observed under the laser scanning confocal microscope .

4 . In vitro induction and co - culture of bone marrow mesenchymal stem cells into renal stem cells was established and divided into five groups : A : bone marrow mesenchymal stem cells + post - renal mesenchymal cells ; B : bone marrow mesenchymal stem cells + ureter buds ; C : bone marrow mesenchymal stem cells + post - renal mesenchymal stem cells + ureteral buds ; D : bone marrow mesenchymal stem cells ; E : blank control group .

Results :

The results of cell cycle showed that only a small number of cells entered the proliferative activity period , and the cells were in quiescent period . The results showed that only a small number of cells entered the proliferative activity period , and the cells were in the quiescent period . The results of the cell cycle showed that the cells were more likely to express vimentin , Fibronectin , and the cells were not expressed by Keratin , E - cadherin and CD34 .

The results showed that the cells were spindle - shaped or irregular . The results showed that the cells were spindle - shaped or irregular . The results showed that the cells were spindle - shaped or irregular . The results showed that the cells were spindle - shaped or polygonal . Fibronectin did not express Keratin and E - cadherin . In the three - dimensional three - dimensional culture system , the shape of MMSCs was strong and the growth state was good .

3 . The UB was T - shaped in E13.5 rats under reversed phase contrast microscope . UB was labeled with FITC - DB labeled UB . The results showed that the proliferation of UB at apical ( ampulla ) was slower than that of apical ( ampulla ) .

The results showed that almost all the cells expressed CD44 , CD90 and almost all the cells did not express CD90 . The results showed that almost all the cells expressed CD44 , CD90 , almost all the cells did not express CD90 . The results showed that almost all the cells expressed CD44 and CD90 . The results showed that almost all the cells did not express CD44 and CD90 . The results showed that almost all the cells did not express CD44 and CD90 .

Conclusion :

1 . We have successfully isolated the cultured and MMSCs , which have typical mesenchymal cell morphology and stem cell phenotype , and have multi - directional differentiation ability .

2 . We successfully isolated and cultured rat UB .

3 . We established a rat model of differentiation into renal stem cells in vitro , and the transformation of bone marrow cells into renal stem cells was achieved . In vitro , the rats were able to promote the sprouting of UB branches in rats .

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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