不同分化状态的间充质干细胞向肝细胞生长因子的趋化性迁移研究

发布时间：2018-06-24 04:10

  本文选题：骨髓间充质干细胞 + 细胞分化　； 参考：《苏州大学》2010年硕士论文

【摘要】： 神经胶质瘤是颅内最常见的恶性肿瘤,具有扩散和浸润的特性,即使经过外科手术切除,并辅以放疗或化疗,也难以彻底治愈。因此,需要新的治疗方法来追踪逃离常规治疗的散在瘤细胞。间充质干细胞(mesenchymal stem cells, MSCs)是具有多向分化潜能的成体干细胞,而且能够趋向胶质瘤及多种趋化因子迁移,从而成为治疗胶质瘤的理想载体细胞。然而MSCs定向迁移的机制还不是完全清楚,尤其是MSCs分化状态与迁移之间关系的研究还未见报道。本实验旨在研究不同分化状态的骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)向肝细胞生长因子(hepatocyte growth factor, HGF)的趋化性迁移。 本研究首先采用Percoll密度梯度离心法从大鼠骨髓中分离出BMSCs,体外扩增培养,通过观察细胞形态、生长特性,免疫荧光染色,成骨、成脂诱导分化,对BMSCs进行鉴定。结果显示,分离培养的BMSCs为长梭形,增殖速度快,表面抗原CD29、CD90、CD106呈阳性,CD34、CD45为阴性,并且体外能够诱导分化为成骨细胞及脂肪细胞,证明分离培养的细胞是具有多向分化潜能的BMSCs。 随后通过丁羟基茴香醚(butyl hydroxy anisol, BHA)联合碱性成纤维生长因子(basic fibroblast growth factor, bFGF)诱导BMSCs成神经分化,即先用10 ng/ml bFGF预诱导24 h,再用200μM BHA和2% DMSO诱导5 h,最后用含有N2的H-DMEM维持培养。观察该过程中细胞的形态变化,免疫细胞化学染色检测神经细胞特异性标志物nestin、β-III-tubulin及NSE的表达变化。结果显示,未分化BMSCs呈成纤维细胞样;预诱导24 h后,胞体稍有收缩,边缘变得不齐整;诱导5 h,胞体收缩成圆形或椭圆形,折光性强,有突起伸出;维持培养18 h后,细胞出现3个或更多突起;维持培养48 h,细胞突起更为复杂,出现二级分叉,突起长度增加,与其他细胞的突起相互接触。免疫荧光染色显示,nestin与β-III-tubulin的表达都呈现出先增高后降低的趋势,均在诱导5 h时达到最高值,之后显著下降;而NSE阳性细胞比率在诱导期以及维持前期都很低,到维持48 h显著升高;GFAP在整个诱导分化过程中始终为阴性。 接着运用Dunn chamber装置研究了BMSCs及成神经分化不同状态BMSCs向HGF的趋化性迁移,计算细胞迁移速率和迁移效率。结果显示,在趋化性迁移实验中,即外槽加入不同浓度HGF、内槽只加L-DMEM时,细胞的迁移速率没有变化,迁移效率随着HGF浓度的增加而增高;与对照组(内外槽均只加入L-DMEM)相比,50 ng/ml及100 ng/ml HGF均显著提高了细胞的迁移效率,但两者之间没有差异。当内外槽均加入50 ng/ml HGF,细胞的迁移速率及迁移效率与对照相比无差异,表明HGF能够诱导BMSCs定向迁移。接着我们研究了成神经分化的BMSCs向HGF的趋化性迁移,结果显示外槽加入50 ng/ml HGF未能改变分化各点细胞的迁移速率,但显著提高了未分化、预诱导24 h、诱导5 h及维持48 h细胞的迁移效率,表明成神经分化的BMSCs向HGF的趋化迁移能力与其分化状态密切相关。用PI3K抑制剂LY294002(30μM)预处理1 h的BMSCs进行趋化迁移实验,外槽加入HGF后,细胞迁移效率与未处理组细胞相比显著降低,即LY294002阻断了HGF诱导BMSCs的定向迁移,证明PI3K信号通路参与介导HGF诱导BMSCs的定向迁移过程。 最后,通过改变Rac1的表达水平,观察BMSCs随机迁移行为及HGF诱导的定向迁移行为的变化。发现Rac1过表达后,BMSCs迁移速率及趋向HGF的迁移效率显著提高;干扰Rac1表达后,迁移速率及趋向HGF的迁移效率显著降低。结果表明Rac1调节BMSCs的迁移速率,并且参与介导HGF诱导BMSCs的定向迁移过程。 以上结果表明,HGF能够趋化BMSCs的定向迁移,PI3K信号通路参与介导这一过程;成神经分化的BMSCs趋向HGF的迁移能力与其分化状态密切相关。Rac1调节BMSCs迁移速率,并且介导HGF诱导BMSCs的定向迁移。本研究为进一步揭示BMSCs的定向迁移机制及临床应用BMSCs进行移植提供了理论基础。
[Abstract]:Glioma, the most common malignant tumor of the brain, has the characteristics of diffusion and infiltration. It is difficult to be cured by surgical excision, combined with radiotherapy or chemotherapy. Therefore, new treatments are needed to trace the scattered tumor cells that escape conventional treatment. Mesenchymal stem cells (MSCs) is multidirectional. The adult stem cells of differentiated potential, and can tend to glioma and a variety of chemokine migration, become ideal carrier cells for the treatment of glioma. However, the mechanism of MSCs directed migration is not completely clear, especially the study of the relationship between MSCs differentiation and migration. The experiment aims to study the different state of differentiation. Bone marrow mesenchymal stem cells (BMSCs) migrates to chemotaxis of hepatocyte growth factor (hepatocyte growth factor, HGF).
In this study, Percoll density gradient centrifugation was used to isolate BMSCs from rat bone marrow and expand culture in vitro. By observing cell morphology, growth characteristics, immunofluorescence staining, osteogenesis, and lipid induced differentiation, BMSCs was identified. The results showed that the isolated BMSCs was long spindle shape, rapid proliferation, surface antigen CD29, CD90, CD106 presented. Positive, CD34, CD45 are negative, and can be induced to differentiate into osteoblasts and adipocytes in vitro. It is proved that the isolated cells are BMSCs. with multiple differentiation potential.
Then butyl hydroxy ANISOL (BHA) combined with basic fibroblast growth factor (basic fibroblast growth factor, bFGF) to induce neuronal differentiation of BMSCs, that is, first inducement of 10 ng/ml bFGF to induce 24, and then 200 micron and 2% induction to induce 5. Changes, immunocytochemical staining was used to detect the expression of neuron specific markers nestin, beta -III-tubulin and NSE. The results showed that the undifferentiated BMSCs was fibroblast like, after 24 h preinduction, the cell body was slightly contracted and the edge became unintegrated; the induced 5 h, the cell body contracted into a circular or oval shape, and the light was protruded and protruded. After 18 h, 3 or more protrusions were found in the cells; the culture of 48 h was maintained, the cell protruding was more complex, the two branching, the increase of the protuberance and the contact with the protuberances of other cells. Immunofluorescence staining showed that the expression of nestin and beta -III-tubulin showed a tendency to increase first and then decrease, and then reached the highest value at the time of inducing 5 h. The ratio of NSE positive cells was very low in the induction period and in the prophase, and to maintain 48 h significantly, and GFAP was always negative during the whole induction of differentiation.
Then the Dunn chamber device was used to study the chemotactic migration of BMSCs and BMSCs to HGF, and the migration rate and migration efficiency of the cell were calculated. The results showed that in the chemotactic migration experiment, the migration rate of the cells was not changed when the external slot added different concentrations of HGF and the inner slot was only L-DMEM, and the migration efficiency was with the HGF concentration. Compared with the control group (both internal and external slot only added L-DMEM), 50 ng/ml and 100 ng/ml HGF significantly increased the cell migration efficiency, but there was no difference between them. When both internal and external slots were added to 50 ng/ml HGF, the migration rate and migration efficiency of the cells were not different from those of the photographic ratio, indicating that HGF could induce the directional migration of BMSCs. We studied the chemotactic migration of BMSCs into HGF. The results showed that the addition of 50 ng/ml HGF in the outer slot failed to change the migration rate of the cells at different points, but significantly increased the undifferentiation, preinduced 24 h, induced 5 h and maintained the migration efficiency of 48 h cells, indicating the chemotactic migration ability of the deity BMSCs to HGF and its differentiation. The state of the PI3K inhibitor LY294002 (30 mu M) was pretreated with 1 h BMSCs for chemotactic migration experiment. After HGF, the cell migration efficiency was significantly lower than that of the untreated group. That is, LY294002 blocked the directional migration of BMSCs induced by HGF, and demonstrated that PI3K signaling pathway involved the directed migration process of HGF inducible BMSCs.
Finally, by changing the expression level of Rac1, the changes of BMSCs random migration and HGF induced migration were observed. It was found that after Rac1 overexpression, the migration rate of BMSCs and the migration efficiency of HGF were significantly improved, and the migration rate and the migration efficiency of HGF were significantly reduced after the interference of Rac1 expression. The results showed that Rac1 regulated the migration of BMSCs. Rate and participate in the directional migration of BMSCs induced by HGF.
The above results suggest that HGF can chemotaxis directed migration of BMSCs, and PI3K signaling pathway participates in this process. The migration ability of BMSCs toward HGF is closely related to the differentiation state of HGF, and.Rac1 regulates the migration rate of BMSCs and mediates the directional migration of HGF to induce BMSCs. This study further reveals the directional migration mechanism of BMSCs. And the clinical application of BMSCs provides a theoretical basis for transplantation.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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