SFRP2在调控人牙髓干细胞神经向分化中的作用研究

发布时间：2019-05-29 14:54

【摘要】：各种原因引起的视神经损伤性疾病在临床上较为常见,其中部分疾病会造成视神经进行性、不可逆性损伤,最终导致视力丧失。视神经细胞是一种终末分化细胞,再生能力差,导致损伤的视网膜神经难以修复再生,目前临床尚无有效方法治愈视神经疾病。近年来,应用干细胞治疗视网膜神经疾病这一方法极具前景,牙髓干细胞(dental pulp stem cells,DPSCs)由于其与视神经细胞同样来源于神经嵴,且较神经干细胞更易获得,而有望成为修复重建神经组织病损的种子细胞。目前研究认为,Notch及Wnt/β-catenin信号通路是脊椎动物视网膜神经发育所必需的。作为一种广泛表达于多个物种且在进化过程中高度保守的基因,Notch基因主要参与对细胞分化的抑制信号。Wnt/β-catenin信号的激活贯穿视网膜及视神经发育的整个过程,提示其在神经发育中起重要作用。分泌性卷曲蛋白2(secreted frizzled related protein 2,SFRP2)是一种Wnt信号通路抑制剂,同时也间接地参与了除Wnt信号以外的多个信号通路,如Notch及BMP信号通路等。SFRP2可与金属蛋白酶ADAM10结合,抑制Notch信号通路。已有研究表明SFRP2在神经形成过程中高表达,我们推测其可能通过抑制在干细胞增殖过程中发挥重要作用的Notch及Wnt信号通路,促进神经嵴来源的DPSCs向神经样细胞分化。为了验证上述假设,本课题借助多种细胞生物学和分子生物学手段,通过重组腺病毒载SFRP2基因体外转染人牙髓干细胞(human dental pulp stem cells,h DPSCs),同时利用h DPSCs同视网膜色素上皮(retinal pigment epithelium,RPE)细胞共培养法诱导h DPSCs的神经向分化,明确SFRP2在h DPSCs向神经样细胞分化过程中的作用,初步探讨视神经修复再生机制及临床应用前景。方法:从完整拔除的健康第三磨牙内分离牙髓组织,酶消化复合组织块法原代培养h DPSCs。取第3代h DPSCs,MTT法绘制其生长曲线,并通过成脂及成骨诱导检测其多向分化潜能。通过流式细胞术和细胞毒性实验(MTT法)筛选合适的病毒感染复数(multiplicity of infection,MOI)。采用800 particles/cell的Ad-SFRP2转染h DPSCs,RT-PCR检测SFRP2的表达;并将转染Ad-SFRP2的h DPSCs与人RPE细胞共培养诱导其神经向分化,q PCR检测神经相关基因GFAP、Nestin、MAP2、HES1、β-3tublin及ATOH7的表达。结果:倒置显微镜下见体外培养的第3代h DPSCs贴壁生长,形态规则,呈长梭形,核圆,位于中央。甲苯胺蓝染色可见细胞成集落生长,分别进行成脂和成骨诱导,35天油红O染色及21天茜素红染色可见脂滴及钙结节。生长曲线显示第4天细胞由滞留期进入对数生长期,第9天细胞进入平台期。转染Ad-EGFP的h DPSCs在转染后8 h镜下即可见绿色荧光蛋白的表达,转染Ad-SFRP2的h DPSCs在转染后72 h提取总m RNA,RT-PCR检测到SFRP2基因的表达。共培养及Ad-SFRP2转染结果表明,GFAP的表达早期表现为共培养诱导组高于转染组,晚期则表现转染组稍高表达;Nestin早期在共培养诱导组及转染组中表现为一定的上调趋势,晚期表达则呈下调趋势;β-3tublin的表达在共培养诱导组及转染组中呈持续性下调;MAP2在共培养诱导组晚期呈现较高表达;HES1在共培养诱导组早期呈现较高表达,晚期则表达降低,在转染组较非转染组呈下降趋势;ATOH7的表达在各组均无统计学意义。结论:SFRP2可以促进牙髓干细胞的神经向分化,但具体机制有待于进一步实验阐明。
[Abstract]:The optic nerve injury disease caused by various causes is more common in the clinic, in which some of the diseases can cause progressive and irreversible damage of the optic nerve and eventually lead to loss of vision. As the nerve cell is a terminal differentiation cell, the regeneration ability is poor, and the injured retinal nerve is difficult to repair and regenerate, and the present invention has no effective method to cure the optic nerve diseases. in recent year, that method of using stem cell to treat the retinal nerve disease is very promising, and the dental pulp stem cells (DPSCs) are also more accessible to the neural stem cells due to the fact that it is derived from the neural stem cells in the same way as the visual nerve cells, And is expected to be a seed cell for repairing the damaged nerve tissue. In that present study, the Notch and Wnt/ n-cata signal pathway is necessary for the development of the retinal nerve in the vertebrate. As a gene that is widely expressed in multiple species and highly conserved in the evolution process, the Notch gene is mainly involved in the inhibition of cell differentiation. The activation of the Wnt/ P-cata signal extends through the whole process of the development of the retina and the optic nerve, suggesting that it plays an important role in the development of the nerve. The secreted frizzled protein 2 (SFRP2) is a Wnt signaling pathway inhibitor, and is also indirectly involved in a plurality of signal paths other than the Wnt signal, such as a Notch and a BMP signal path, and the like. The SFRP2 can be combined with the metal protease ADAM10 to inhibit the Notch signaling pathway. It has been shown that SFRP2 is highly expressed in the process of neural formation, and we speculate that it is possible to promote the differentiation of DPSCs from the source of neural stem cells to neural-like cells by inhibiting the Notch and Wnt signaling pathways that play an important role in stem cell proliferation. In order to verify the above-mentioned hypothesis, human dental pulp stem cells (h DPSCs) were transfected with the recombinant adenovirus-loaded SFRP2 gene in vitro by means of a variety of cell biology and molecular biology methods. The nerve-to-differentiation of h-DPSCs was induced by the co-culture of RPE cells, and the role of SFRP2 in the differentiation of h-DPSCs into the neural-like cells was determined. Methods: The pulp tissue was separated from the intact third molar, and the primary cultured h-DPSCs were cultured by enzyme-digested composite tissue-block method. The growth curves of the third generation h DPSCs and the MTT method were obtained, and their multi-directional differentiation potential was detected by lipogenesis and osteogenesis. The appropriate number of viral infections (MOI) was selected by flow cytometry and cytotoxicity assay (MTT method). The expression of SFRP2 was detected by using an Ad-SFRP2 of 800 parts/ cell, and the expression of SFRP2 was detected by RT-PCR. The expression of GFAP, Nestin, MAP2, HES1, HCO3-3 and ATOH7 was detected by co-culture of h-DPSCs transfected with Ad-SFRP2 and human RPE cells. Results: The growth and morphology of the third generation h DPSCs cultured in vitro under the inverted microscope were in the center. In the staining of toluidine blue, the growth of the cells was observed, and the lipid and calcium nodules were observed by the staining and osteogenesis induction, the 35-day oil-red O-staining and the 21-day fluorescein-red staining. The growth curve shows that the 4-day cell is in the logarithmic growth phase from the retention period, and the 9-day cell enters the plateau phase. The expression of the green fluorescent protein was observed at 8 h after the transfection of Ad-EGFP, and the expression of the gene of SFRP2 was detected by RT-PCR at 72 h after the transfection of Ad-SFRP2. The results of co-culture and the transfection of Ad-SFRP2 showed that the expression of GFAP was higher than that of the transfected group in the early stage. The expression of 1-3 tubuin was down-regulated in the co-culture induction group and the transfected group; MAP2 showed a high expression in the late stage of the co-culture induction group; the expression of HES1 in the early stage of the co-culture induction group was higher, and the expression of the later stage decreased, and the expression of the non-transfected group in the transfection group was decreased. The expression of ATOH7 was not statistically significant in each group. Conclusion: SFRP2 can promote the differentiation of the nerve of the dental pulp stem cells, but the specific mechanism is to be further clarified.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782

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