Math1基因对新生大鼠基底膜损伤后的实验研究

发布时间：2018-05-20 12:15

本文选题：Math1基因 + 耳蜗毛细胞　；参考：《兰州大学》2010年硕士论文

【摘要】： 在哺乳动物听觉系统中,感觉神经终末细胞—耳蜗毛细胞,它能够将声刺激转换成电信号,并通过听觉神经传导至中枢,以便维持正常的听力。过度声刺激、老化、耳毒性药物(如氨基糖苷类抗生素、抗疟药、抗肿瘤药、袢利尿剂等)、感染(如风疹、流行性脑膜炎、流行性腮腺炎等)及自身免疫性疾病等多种因素均可引发耳蜗毛细胞的不可逆性损伤,从而导致永久性的感音神经性耳聋。目前对于感音神经性耳聋的治疗,主要利用放大设备(助听器)和电刺激听觉神经元的设备(耳蜗植入),但目前这两种临床治疗方法受到多种因素,如患者的耳聋程度、病程、年龄、受教育程度等多方面的影响,并且,即使使用助听器或耳蜗植入后,仍有部分患者不能改善听力,现在需要一种新的治疗方法用于临床,为更多的耳聋患者服务。运用促使非感觉上皮细胞向毛细胞分化的关键基因,是重建耳蜗Corti's结构,恢复听功能的可行方法。近20年,已有报道关于Mathl对正常体外培养基底膜使毛细胞增多的结果,但是,对于耳毒性药物损伤后的离体培养的耳蜗基底膜,导入Mathl后毛细胞的变化情况,目前还没有相关研究。本研究利用腺病毒载体将诱导毛细胞分化的关键基因---Math1导入到药物性损伤后离体培养的基底膜中,利用免疫组织化学染色、计数单位长度毛细胞等的方法,观察毛细胞数量的变化,研究分以下两个部分。第一部分新生大鼠耳蜗毛细胞损害离体动物模型的建立本部分研究的目的是建立耳毒性药物损伤离体动物模型,用于耳蜗毛细胞再生的离体实验研究。选取出生后0天(PO)的健康SD大鼠,在超净台内,在放大倍数为20-40倍的体视学显微镜下,用游丝镊完整剥取整个耳蜗基底膜,然后将其按照本身的螺旋方式平铺于六孔板中,加入含有5%胎牛血清的DMEM培养基,在含有5%CO2培养箱中进行培养,培养12小时后,可见整齐排列的一排内毛细胞和三排外毛细胞。实验可分为对照组和3个实验组,将不同浓度硫酸新霉素(1mmol/L,2mmol/L, 4mmol/L)分别加入正常培养12小时的基底膜中,继续培养24小时后,终止实验,然后进行MyosinⅦa免疫组织化学染色,在共聚焦显微镜下观察各回毛细胞缺失情况,并分别选取10个图片进行毛细胞计数(100μm),利用CHISS软件进行统计分析。加入硫酸新霉素后,耳蜗毛细胞损伤从底回外毛细胞开始,随着药物浓度增加,逐渐累及到中回,最后顶回受累,以外毛细胞为甚。以上结果说明,体外培养的耳蜗毛细胞随着硫酸新霉素药物浓度的增加损失越严重,且从底回向顶回发展,此与活体动物实验的损害规律基本相同,因此可作为耳毒性损伤后毛细胞再生研究的离体动物模型。第二部分腺病毒携带Math1基因转染损伤后新生大鼠耳蜗的实验观察本部分的研究目的是观察Math1基因对离体培养药物性损伤后耳蜗毛细胞的影响。在第一部分的基础上,对耳毒性药物损伤后耳蜗毛细胞再生做初步的研究。选取40个耳蜗基底膜,随机分为4组：正常对照组、硫酸新霉素处理组、硫酸新霉素处理+ad-Math1-EGFP组和硫酸新霉素处理+ad-EGFP组,每组10个耳蜗。Mathl基因成功转染后,利用MyosinⅦa免疫组织化学染色观察耳蜗毛细胞变化。结果显示：硫酸新霉素处理+ad-Math1-EGFP组在Math1基因成功转染后10天,除螺旋神经节部位有绿色荧光表达外,在受损毛细胞区域的支持细胞也有绿色荧光表达,而正常对照组、硫酸新霉素处理组、硫酸新霉素处理+ad-EGFP组,在毛细胞区域几乎无绿色荧光表达。说明,Math1基因可以转染受损毛细胞区域的支持细胞,提示该区域的支持细胞可以向耳蜗毛细胞转化。
[Abstract]:In the auditory system of mammals, the terminal cells of the sensory nerve, cochlear hair cells, can convert sound stimuli into electrical signals and transmit to the center through the auditory nerve to maintain normal hearing. Excessive sound stimulation, aging, ototoxic drugs (such as aminoglycoside antibiotics, antimalarial drugs, antineoplastic agents, loop diuretics, etc.), infection (like the wind) A variety of factors such as rash, epidemic meningitis, mumps, and autoimmune diseases can cause irreversible damage to the hair cells of the cochlea, resulting in permanent sensorineural deafness. Current treatment of sensorineural hearing loss mainly uses equipment (ears) to amplify (hearing aids) and electrical stimulation of auditory neurons (ears). There are many factors, such as the degree of deafness, the course of the disease, the age, the degree of education, and even the hearing aids or cochlear implants, there are still some patients who are not able to improve their hearing, and a new treatment is needed to be used in clinical and more deafness patients. Service. The use of key genes that promote the differentiation of non sensory epithelial cells to hair cells is a feasible way to reconstruct the Corti's structure of the cochlea and restore the auditory function. In the last 20 years, it has been reported that Mathl has resulted in the increase of hair cells in the normal basement membrane in vitro. However, the cochlear basement membrane in vitro culture after the ototoxic drug damage is guided. There is no related study on the change of hair cells after entering Mathl. This study uses the adenovirus vector to induce the key gene ---Math1 to induce the differentiation of hair cells into the basement membrane of the isolated culture after the drug injury. The study is divided into two parts.
The first part is the establishment of neonatal rat cochlear hair cell damage model in vitro.
The aim of this study is to establish an animal model of ototoxic drug injury in vitro and to study the regeneration of cochlear hair cells in vitro.
In a healthy SD rat 0 days after birth (PO), the whole cochlear basement membrane was completely stripped with a swimming tweezers under a stereological microscope with a magnification of 20-40 times. Then a DMEM medium containing 5% fetal bovine serum was added to the six hole plate according to its own spiral mode, and was cultured in a 5%CO2 incubator. After 12 hours of culture, a row of neatly arranged inner hair cells and three xenophobic hair cells can be seen. The experiment can be divided into control group and 3 experimental groups, adding different concentrations of neomycin sulfate (1mmol/L, 2mmol/L, 4mmol/L) to normal culture for 12 hours of basal membrane, continuing to culture for 24 hours, terminating the experiment, and then conducting Myosin VII a immunization The loss of hair cells was observed under the confocal microscope, and 10 pictures were selected to count the hair cells (100 m), and the CHISS software was used to analyze the hair cells. After adding neomycin sulfate, the damage of the cochlear hair cells started from the bottom of the hair cells. With the increase of the drug concentration, the hair cells were gradually involved in the middle gyrus, and the final apex was involved. The above results indicate that the loss of cochlear hair cells in vitro is more serious with the increase of neomycin sulfate concentration, and it develops from the bottom back to the top, which is basically the same as in vivo animal experiments. Therefore, it can be used as an isolated animal model for the study of hair cell regeneration after ototoxicity.
The second part of adenovirus carrying Math1 gene transfected into neonatal rat cochlea after injury.
The purpose of this part is to observe the effect of Math1 gene on the cochlear hair cells after the drug induced injury in vitro. On the basis of the first part, a preliminary study of the cochlear hair cell regeneration after the ototoxic drug damage is made.
40 cochlear basement membranes were selected and randomly divided into 4 groups: normal control group, neomycin sulphate treatment group, neomycin sulphate treatment group +ad-Math1-EGFP and neomycin sulfate treatment group +ad-EGFP. 10 cochlear.Mathl genes in each group were transfected successfully. The changes of cochlear hair cells were observed by Myosin VII a immuno histochemical staining. The results showed: new sulfuric acid 10 days after the successful transfection of Math1 gene, 10 days after the successful transfection of mycophenolate gene, there was green fluorescent expression in the damaged hair cell area except for the green fluorescence expression in the part of the spiral ganglion, while the normal control group, neomycin sulfate treatment group, neomycin sulphate treatment + ad-EGFP group had almost no green fluorescence expression in the hair cell region. It indicates that Math1 gene can be transfected into Sertoli cells in damaged hair cells, suggesting that Sertoli cells in the region can be transformed into cochlear hair cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

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