光、电刺激对成肌细胞生长增殖影响的实验研究

发布时间：2018-03-16 06:12

本文选题：成肌细胞　切入点：发光二极管　出处：《重庆大学》2013年硕士论文　论文类型：学位论文

【摘要】：成肌细胞是指胞浆中含有肌丝的肌组织前体细胞，是一种单层贴壁式生长的细胞，能自行发生分裂增殖。成肌细胞具有自我更新及损伤修复的再生能力，而且其在基因治疗中较其他细胞有很明显的优点，因此成肌细胞在临床及基础研究中有重要的研究意义。成肌细胞的生长状态与环境温度、渗透压、PH值、光照等因素密切相关。其中光照和电流能明显调控细胞的增殖及分化速度，所以光刺激和电刺激一直是细胞离体培养实验研究的热点。目前为止，在临床和基础研究中已有大量与光刺激、电刺激有关的实验研究，并已取得一定的成果，一部分研究成果正逐步走向产业化，投入临床使用。例如现今正用于临床上治疗白斑病、牛皮癣等各种皮肤病的光疗法，以及用于治疗坐骨神经痛等各种疼痛性疾病的电疗法。在此背景下，本文通过自行搭建光、电辅助装置来探讨光刺激及电刺激对成肌细胞增殖生长效果的影响。课题的主要研究工作如下： ①查阅文献，探讨实验可行性的理论依据。在开展本实验前查阅了大量相关的实验研究，其中包括低能量激光对细胞的光刺激实验研究，该研究选用LED灯作为光源，研究光对纤维原细胞的生物刺激效应；以及微电流脉冲调控神经干细胞增生效果的实验研究，该实验通过在自制电极对上外加微电流脉冲信号探讨电刺激对神经干细胞增生及分化效果的影响。对他们的实验结果进行分析讨论，总结出光刺激及电刺激的作用机理。对本课题的可行性进行理论分析，并得出相关理论依据。 ②设计并搭建光、电刺激辅助装置。基于之前讨论的理论依据，在低成本、低功耗的条件下，分别设计出光、电辅助装置的搭建方案。选取波长分别为467nm，589nm，630nm的三种LED灯作为光源，通过串联/并联相结合的方式搭建一个88的LED阵列灯。选定不锈钢片作为电极材料，PDMS为浇注固化材料，搭建电辅助装置。电极是一个7cm1cm的片状电极，，两电极间距离为4.5cm。 ③细胞培养及刺激实验。设计实验方案，确定刺激参数为刺激时机、刺激次数、刺激波长及刺激时间。然后培养成肌细胞，当细胞铺满培养皿（一般75%~80%）时进行传代培养，按照刺激实验要求确定传代培养的盘数。刺激时机：接种后12h、24h，刺激次数：1次、2次、3次、4次，刺激波长：467nm，589nm，630nm，刺激时间：400s、600s、1000s、1600s。分别对上述参数依次进行刺激实验研究，探讨各参数对成肌细胞生长增殖效果的影响。 ④成肌细胞生长状态的评价及有效刺激参数的确定。完成刺激实验后，将细胞收集固存，24小时后用于流式细胞周期检测。细胞收集固存过程中，胰酶消化不宜过度，否则会产生细胞碎片；离心时转速应设置为1000rpm，转速过小，细胞不能很好的凝聚，会使细胞丢失，而转速过大会导致细胞破裂。细胞碎片及细胞破裂都会影响流式检测的结果，且流式检测是细胞个数不少于1～2×10~6。将流式检测结果与郭瑞雄等人的研究结果进行对比分析，分别确定一组有效刺激参数。
[Abstract]:Myoblast refers to the cytoplasm containing filament muscle precursor cells is a monolayer adherent growth of cells, can be split into muscle cells. The proliferation of self-renewal and damage repair ability of regeneration, and in gene therapy than other cells have obvious advantages, so muscle cells have important significance in clinical and basic research. Growth condition and environmental temperature, muscle cell osmotic pressure, pH, illumination and other factors are closely related. The light and electricity can regulate cell proliferation and differentiation rate obviously, so the light and electrical stimulation of cells in vitro has been the research hot spot the in vitro culture. So far, in the clinical and basic research has been a large number of stimulation and light, the experimental study on the electrical stimulation, and has achieved certain results, part of the research is gradually moving towards industrialization, put into clinical use. Such as now being used in clinical treatment of vitiligo, psoriasis and other light therapy of various skin diseases, as well as for the treatment of sciatica and other pain disorders.
Under this background, this article through the self built light electric auxiliary device to investigate light stimulation and electrical stimulation of the myoblast proliferation effect. The main research topics are as follows:
In the literature, explore the theoretical basis of the experimental feasibility. Before carrying out the experiment of access to a large number of studies, including experimental study on stimulation of low energy laser on cell light, this research used the LED lamp as light source, light on the study of biological fibroblasts stimulated effect; and experimental research of micro current pulse control neural stem cell proliferation effect, with the self-made electrode on putting micro current pulse signal to investigate the effect of electrical stimulation on neural stem cell proliferation and differentiation effect. The results of their experiment were analyzed and discussed, summed up the light stimulation and electrical stimulation of the mechanism. To analyze the feasibility of this project, and the relevant theoretical basis.
How to design and build the optical and electrical stimulation device. Based on the theory discussed before, at a low cost, low power consumption under the condition of light were designed, built scheme of electric auxiliary device. The wavelength selection were 467nm, 589nm, three LED 630nm lamp as the light source, through the serial / parallel combination the way to build a 88 LED array light. The selected stainless steel sheet as electrode material for PDMS, pouring and curing materials, building the electric auxiliary device. The electrode is a 7cm1cm electrode, the two electrode distance 4.5cm.
Cell culture and stimulation experiments. The experimental program was designed to determine the stimulation parameters for the stimulation time, stimulation frequency, stimulation and stimulation time. Then the wavelength of myoblasts in vitro, when the cells covered with a Petri dish (usually 75%~80%) when they were cultured, in accordance with the stimulation requirements to determine subcultured plate number. 12h after inoculation, stimulation time 24h stimulation: 1 times, 2 times, 3 times, 4 times, stimulation wavelength: 467nm, 589nm, 630nm, 600s, stimulation time: 400s, 1000s, 1600s. respectively for the above parameters in order to stimulate research, explore the influence of parameters on myoblast proliferation effect.
To determine the evaluation of muscle cell growth state and effective stimulation parameters. Complete stimulation after the experiment, the cells were collected after 24 hours for sequestration, FCM. Cell collection sequestration process, trypsin digestion should not be excessive, otherwise it will produce cell debris; centrifugal speed should be set to 1000rpm, the speed is too small cell aggregation is not good, will cause the loss of cells, and lead to excessive speed cell rupture and cell debris. Rupture will affect the flow cytometry results and flow cytometry on cell number less than 1~2 * 10~6. will flow cytometry results and Guo Ruixiong et al. The results are compared and analyzed. A set of effective stimulus parameters were determined.

【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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