DAPT在体外诱导人脐带间充质干细胞向内耳毛细胞样细胞分化中影响的研究

发布时间：2018-08-10 19:11

【摘要】：感音神经性耳聋(sensorineural hearing loss,SNHL)是一种常见的感觉功能障碍性疾病,在耳鼻咽喉科学临床工作及学习中尤为常见,主要是由内耳毛细胞(HCs)及螺旋神经元(SGNs)的变性、不可逆损伤所致,可伴随不同程度的言语功能障碍。鉴于哺乳动物毛细胞的有限再生能力,传统治疗方法如助听器佩戴、人工耳蜗植入(CI)等无法恢复受损内耳的生理功能,但随着对干细胞的渐进式研究,使得对内耳螺旋神经节及毛细胞受损功能的修复成为可能。人脐带间充质干细胞(hUC-MSCs)拥有较为实用的临床价值,主要是由于其具有增殖分化能力强、低免疫源性、来源广泛、无致瘤性及伦理道德争议等优点,已开始广泛应用于细胞、组织工程等相关研究,是较为理想的种子细胞。但干细胞分化的低效率性严重阻碍着其发展。故而,探讨一种促使hUC-MSCs更安全、高效的向内耳毛细胞样细胞诱导分化的实验条件变得尤为关键。Notch信号通路在进化上高度保守、普遍存在于各种生物之中,对内耳发育过程中不同听觉器官的发生、发展来说至关重要。内耳毛细胞的发育与Notch通路介导的“侧向抑制(lateral inhibition)”机制密不可分。研究发现,(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)通过作用于早老素后抑制γ-分泌酶活性使得Notch蛋白的活化形式NICD产生减少进而调控下游靶基因表达,削弱Notch通路侧抑制,使毛细胞数目有所增多。而成年豚鼠的耳毒性试验发现,当DAPT抑制Notch通路后,可产生异位的听毛细胞。但DAPT体外调控hUC-MSCs向内耳毛细胞样细胞分化的研究,目前尚未见报道。目的本研究以γ-分泌酶抑制剂DAPT为切入点,以人来源脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)为对象,探究于体外使用DAPT对hUC-MSCs定向诱导分化为内耳毛细胞样细胞的影响,并探讨可能的分子机制,以期提高干细胞向内耳毛细胞样细胞的分化效率,为感音神经性聋的治疗提供新的理论依据。方法1、使用实验室的现有方法,从脐带中分离出Wharton’s jelly组织后原代培养并获得hUC-MSCs,进一步操作,采用组织贴壁培养法分离、培养。实验分为:实验组(DFNB基础诱导培养基+EGF+bFGF)、对照组(DFNB基础诱导培养基)。实验均在琼脂糖包被的抗贴壁培养瓶中完成,分化周期为3-5天,促使hUC-MSCs向NSCs方向诱导分化。CCK-8检测各组细胞的增殖情况,免疫荧光检测神经干细胞标记物Nestin的表达情况,Western Blot、qRT-PCR检测各组细胞分化中Nestin在蛋白和mRNA水平的表达情况。2、取诱导所得的神经干细胞进一步分化,分为DAPT组(DFNB基础诱导培养基+EGF+ATRA+DAPT)、实验组(DFNB基础诱导培养基+EGF+ATRA)及对照组(DFNB基础诱导培养基)。实验均在明胶特殊包被的培养瓶中完成,分化周期为2-4周,进一步促使神经干细胞向内耳毛细胞样细胞诱导分化。CCK-8检测各组细胞的增殖情况,免疫荧光检测毛细胞标记物Math1、MyosinVIIa,Brn3c的表达情况,Western Blot、qRT-PCR检测各组细胞分化中毛细胞标记物Math1、MyosinVIIa,Brn3c及Hes1、Hes5在蛋白和mRNA水平的表达情况。结果1、分离、获取的hUC-MSCs可在体外诱导分化为神经球样结构,与对照组相比,实验组EGF、bFGF可明显促进hUC-MSCs的增殖,且神经干细胞标记物Nestin的表达明显增加。2、与对照组相比,经2-4周诱导培养基诱导分化后,DAPT组、实验组均可表达Math1、MyosinVIIa,Brn3c等毛细胞标记物,说明hUC-MSCs诱导为神经干细胞后可定向分化为类内耳毛细胞样细胞,同时DAPT组在DAPT作用下Math1、MyosinVIIa,Brn3c的蛋白和mRNA表达水平有所升高,Hes1、Hes5在蛋白和mRNA水平表达显著降低。结论1、hUC-MSCs可在体外跨胚层诱导为神经干细胞后定向分化为类内耳毛细胞样细胞,可为SNHL提供更为合理、安全的种子细胞。2、DAPT可下调Notch通路下游基因Hes1、Hes5的表达,促进hUC-MSCs向内耳毛细胞样细胞分化,为干细胞内耳移植治疗感音神经性耳聋提供新的治疗思路。
[Abstract]:Sensorineural hearing loss (SNHL) is a common sensory disorder, which is especially common in clinical work and study of otolaryngology. It is mainly caused by the degeneration and irreversible damage of inner ear hair cells (HCs) and spiral neurons (SGNs), and can be accompanied by different degrees of speech dysfunction. The limited regeneration ability of hair cells in dairy animals, traditional treatment methods such as hearing aid wearing, cochlear implantation (CI) can not restore the physiological function of damaged inner ear, but with the gradual study of stem cells, it is possible to repair the spiral ganglion of inner ear and the damaged function of hair cells. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are embracing. Because of its strong ability of proliferation and differentiation, low immunogenicity, extensive sources, non-tumorigenicity and ethical disputes, it has been widely used in cell and tissue engineering research. It is an ideal seed cell. However, the inefficiency of stem cell differentiation seriously hinders its development. Therefore, it is very important to explore a safe and efficient experimental condition for inducing differentiation of hUC-MSCs into inner ear hair cell-like cells. Studies have shown that, (3,5-difluorophenylacetyl) -L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT) reduces the production of NICD, an active form of Notch protein, by inhibiting the activity of gamma-secreting enzymes after acting on presenilin. In adult guinea pigs, when DAPT inhibited Notch pathway, heterotopic auditory hair cells could be produced. However, the study of DAPT regulating the differentiation of hUC-MSCs into inner ear hair cell-like cells in vitro has not been reported. The aim of this study was to investigate the effect of DAPT on the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into inner ear hair cell-like cells in vitro, and to explore the possible molecular mechanism in order to improve the differentiation efficiency of stem cells into inner ear hair cell-like cells. Methods 1. Wharton's jelly tissues were isolated from the umbilical cord and cultured in vitro to obtain hUC-MSCs. The hUC-MSCs were isolated and cultured by tissue adherence culture. The experiment was divided into experimental group (DFNB basic induction medium + EGF + bFGF) and control group (DFNB basic induction medium + EGF + bFGF). The experiment was carried out in agarose-coated anti-adherence culture flask. The differentiation cycle was 3-5 days, which promoted the differentiation of hUC-MSCs toward NSCs. CCK-8 was used to detect the proliferation of cells in each group, immunofluorescence was used to detect the expression of neural stem cell marker Nestin, Western Blot and qRT-PCR were used to detect the differentiation of cells in each group. At protein and mRNA levels, neural stem cells were further differentiated into DAPT group (DFNB-based induction medium + EGF + ATRA + DAPT), experimental group (DFNB-based induction medium + EGF + ATRA) and control group (DFNB-based induction medium). The experiment was completed in a special gelatin-coated culture flask with a differentiation cycle of 2-4. CCK-8 was used to detect cell proliferation, immunofluorescence to detect the expression of hair cell markers Math1, MyosinVIIa, Brn3c, Western Blot and qRT-PCR to detect the expression of hair cell markers Math1, MyosinVIIa, Brn3c and Hes1, Hes5 in protein and m. Results 1. The isolated hUC-MSCs could be induced to differentiate into neurosphere-like structures in vitro. Compared with the control group, EGF and bFGF could significantly promote the proliferation of hUC-MSCs, and the expression of neural stem cell marker Nestin was significantly increased. 2. Compared with the control group, the DAPT group was induced to differentiate after 2-4 weeks. All the hair cell markers such as Math1, Myosin VIIa and Brn 3C could be expressed in the test group, indicating that the hair cell-like cells could be differentiated into inner ear hair cell-like cells after induction of neural stem cells by hUC-MSCs. The protein and mRNA expression levels of Math1, Myosin VIIa and Brn 3C were increased in DAPT group, while the protein and mRNA expression levels of Hes 1 and Hes 5 were significantly decreased in DAPT group. HUC-MSCs can differentiate into hair cell-like cells of inner ear after transembryonic induction in vitro, which can provide more reasonable and safe seed cells for SNHL. 2. DAPT can down-regulate the expression of Hes1 and Hes5 genes downstream Notch pathway, promote the differentiation of hUC-MSCs into hair cell-like cells of inner ear, and provide stem cell transplantation for the treatment of sensory nerve. New treatment ideas for deafness.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.43

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