骨髓间充质干细胞与纳米再生蜘蛛丝生物相容性及向脂肪细胞诱导分化关键点研究

发布时间：2018-03-14 00:04

  本文选题：骨髓间充质干细胞　切入点：纳米再生蜘蛛丝　出处：《苏州大学》2009年硕士论文　论文类型：学位论文

【摘要】： 骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是骨髓中的非造血干细胞,能够自我更新和多向分化。在不同的诱导条件下可以分化为多种细胞,包括成骨细胞、成纤维细胞、软骨细胞、脂肪细胞、肌肉细胞、内皮细胞及神经细胞等。骨髓间充质干细胞还具有取材方便,对机体损伤小,体外培养增殖能力强等优点,选用自体骨髓间充质干细胞进行组织再生或移植,不仅没有移植排异反应,还避免了伦理纷争。因此,对骨髓间充质干细胞的研究成为当今生物医学的热点,骨髓间充质干细胞也被认为是最有前途的组织工程的种子细胞。 1骨髓间充质干细胞的分离培养 为了建立一个有效的分离培养,体外扩增的方法,我们采用Percoll淋巴细胞分离液进行不连续密度梯度离心法分离培养BMSCs,实现在较短的时间内体外扩增和纯化骨髓间充质干细胞。免疫组织细胞化学检测骨髓间充质干细胞表面抗原,结果显示细胞表达CD29、CD44、CD90、CD106,不表达CD34、CD45。 2纳米再生蜘蛛丝与骨髓间充质干细胞生物相容性研究 为了研究纳米再生蜘蛛丝(SHSH)与骨髓间充质干细胞(BMSCs)之间的生物相容性,我们将BMSCs培养在SHSH和多聚赖氨酸(PLL)上。通过死活试剂染色我们对培养在SHSH上24 h、48 h、72 h后的BMSCs进行存活分析;同时以BrdU作为标记物,观察这些细胞在0-24 h、24-48 h和48-72 h的增殖状况;利用油红O染色检测细胞的定向成脂分化能力。结果显示SHSH和常用的细胞培养基质PLL一样能够很好地支持BMSCs的生长。BMSCs在SHSH上表现出正常的粘附、铺展和生长行为。接种后的48 h内,与PLL相比较,细胞呈现较低的增殖速度;之后恢复为与PLL相同的增殖。经过定向成脂诱导,BMSCs在SHSH上能够分化为脂肪细胞。这些数据说明SHSH与BMSCs之间具有良好的生物相容性,同时考虑到其自身优秀的力学性能,SHSH将成为组织工程与再生医学中极具应用潜力的生物支架。 3骨髓间充质干细胞向脂肪细胞分化过程中关键点的研究 为了研究BMSCs向脂肪细胞分化过程中间关键点,我们加入脂肪诱导剂对BMSCs进行成脂诱导,分别诱导12 h、18 h、1 d、2 d、3 d后撤去诱导剂,换入生长培养(L-DMEM + 10%FCS),在第6 d,第9 d时油红O染色。结果显示BMSCs在加入脂肪诱导剂诱导12 h、18 h后,换入生长培养基,第6 d,第9 d油红O染色成阴性;诱导1 d、2 d、3 d后,换入生长培养基,第6 d,第9 d油红O染色成阳性。这些数据说明BMSCs在向脂肪细胞分化过程中非常可能存在一个关键点,这个关键点是细胞的分化决定点,关键点之前的细胞仍然未分化,关键点之后的细胞已经被决定了命运,即使不存在分化诱导剂,仍然能够向脂肪细胞分化。在BMSCs向脂肪细胞分化过程中它的关键作用时间点是BMSCs成脂肪诱导24 h。 同时我们检测了关键点前后时间点Rac1及相关蛋白的表达变化:通过western blotting检测BMSCs向脂肪细胞分化早期12 h、18 h、24 h、48 h的Rac1及相关蛋白的表达变化;Pull-down assay分析Rac1-GTP及相关蛋白活性变化。结果显示Rac1及其相关蛋白总蛋白的表达量发生了变化;并且Rac1-GTP蛋白随着诱导时间的延长,在诱导24 h内蛋白表达不断升高,诱导24 h时达到最大,随后的24 h蛋白表达下降,低于诱导12 h时蛋白表达。RhoA-GTP蛋白表达则与Rac1-GTP相反,随着诱导时间的延长,RhoA-GTP蛋白表达逐渐降低,诱导24 h时降到最低,在随后的24 h中,RhoA-GTP蛋白表达则明显升高,高于诱导12 h时RhoA的蛋白表达水平。Cdc42-GTP蛋白表达在诱导24 h内保持稳定,诱导24 h后显示出与Rac1-GTP蛋白相同的趋势,表达下降且低于诱导12 h时的水平。 总的来说,BMSCs在向脂肪细胞分化的过程中存在一个关键点,这个关键点的作用时间为BMSCs成脂肪诱导24 h时,在这个关键点前后细胞内Rac1及其相关蛋白的表达量发生了变化。
[Abstract]:Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) in the bone marrow non hematopoietic stem cells capable of self-renewal and multilineage differentiation. Differentiation into a variety of cells in different conditions can be induced, including osteoblasts, fibroblasts, cartilage cells, fat cells, muscle cells, and endothelial cells nerve cells. Bone marrow mesenchymal stem cells also has convenient, small injury to the body, has the advantages of strong proliferation ability and in vitro selection of autologous bone marrow mesenchymal stem cells for tissue regeneration or transplantation, not only did the graft rejection, but also avoid the ethical disputes. Therefore, the study of bone marrow mesenchymal stem the cell has become the hotspot of biomedical, bone marrow mesenchymal stem cells are also considered to be the most promising seed cells for tissue engineering.
Isolation and culture of 1 bone marrow mesenchymal stem cells
In order to establish an effective method of isolation and culture, in vitro, we used Percoll lymphocyte separation medium by discontinuous density gradient centrifugation method BMSCs, achieved in a relatively short time in vitro amplification and purification of bone marrow mesenchymal stem cells. Immunohistochemical detection of bone marrow mesenchymal stem cell surface antigen results show CD29 expression, CD44, CD90, CD106, the expression of CD34, CD45.
Biocompatibility of 2 nanoscale regenerated spider silk and bone marrow mesenchymal stem cells
In order to study the nano regenerated spider silk (SHSH) and bone marrow mesenchymal stem cells (BMSCs) compatibility between organisms, we will BMSCs cultured in SHSH and poly-L-lysine (PLL). The death of our reagent staining cultured in SHSH 24 h, 48 h, 72 h after BMSCs survival analysis; at the same time using BrdU as a marker to observe these cells in 0-24 h, 24-48 h and 48-72 h proliferation; directed by oil red O staining was used to detect the cell adipogenic differentiation ability. The results showed that SHSH and PLL as the common cell culture matrix can support the growth of BMSCs.BMSCs show normal adhesion on SHSH, spreading and growth behavior. After inoculation of 48 h, compared with PLL cells showed a lower proliferation rate; after restoration with the same PLL proliferation. After adipogenesis induction, BMSCs SHSH can differentiate into fat cells. These data suggest that SHSH and BM SCs has good biocompatibility and takes account of its excellent mechanical properties. SHSH will become a potential scaffold for tissue engineering and regenerative medicine.
3 key points in the differentiation of bone marrow mesenchymal stem cells into adipocytes
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