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
[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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