本文選題：成肌細(xì)胞　切入點(diǎn)：發(fā)光二極管　出處：《重慶大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：成肌細(xì)胞是指胞漿中含有肌絲的肌組織前體細(xì)胞，是一種單層貼壁式生長(zhǎng)的細(xì)胞，能自行發(fā)生分裂增殖。成肌細(xì)胞具有自我更新及損傷修復(fù)的再生能力，而且其在基因治療中較其他細(xì)胞有很明顯的優(yōu)點(diǎn)，因此成肌細(xì)胞在臨床及基礎(chǔ)研究中有重要的研究意義。成肌細(xì)胞的生長(zhǎng)狀態(tài)與環(huán)境溫度、滲透壓、PH值、光照等因素密切相關(guān)。其中光照和電流能明顯調(diào)控細(xì)胞的增殖及分化速度，所以光刺激和電刺激一直是細(xì)胞離體培養(yǎng)實(shí)驗(yàn)研究的熱點(diǎn)。目前為止，在臨床和基礎(chǔ)研究中已有大量與光刺激、電刺激有關(guān)的實(shí)驗(yàn)研究，并已取得一定的成果，一部分研究成果正逐步走向產(chǎn)業(yè)化，投入臨床使用。例如現(xiàn)今正用于臨床上治療白斑病、牛皮癬等各種皮膚病的光療法，以及用于治療坐骨神經(jīng)痛等各種疼痛性疾病的電療法。 在此背景下，本文通過(guò)自行搭建光、電輔助裝置來(lái)探討光刺激及電刺激對(duì)成肌細(xì)胞增殖生長(zhǎng)效果的影響。課題的主要研究工作如下： ①查閱文獻(xiàn)，探討實(shí)驗(yàn)可行性的理論依據(jù)。在開展本實(shí)驗(yàn)前查閱了大量相關(guān)的實(shí)驗(yàn)研究，其中包括低能量激光對(duì)細(xì)胞的光刺激實(shí)驗(yàn)研究，該研究選用LED燈作為光源，研究光對(duì)纖維原細(xì)胞的生物刺激效應(yīng)；以及微電流脈沖調(diào)控神經(jīng)干細(xì)胞增生效果的實(shí)驗(yàn)研究，該實(shí)驗(yàn)通過(guò)在自制電極對(duì)上外加微電流脈沖信號(hào)探討電刺激對(duì)神經(jīng)干細(xì)胞增生及分化效果的影響。對(duì)他們的實(shí)驗(yàn)結(jié)果進(jìn)行分析討論，總結(jié)出光刺激及電刺激的作用機(jī)理。對(duì)本課題的可行性進(jìn)行理論分析，并得出相關(guān)理論依據(jù)。 ②設(shè)計(jì)并搭建光、電刺激輔助裝置�；谥坝懻摰睦碚撘罁�(jù)，在低成本、低功耗的條件下，分別設(shè)計(jì)出光、電輔助裝置的搭建方案。選取波長(zhǎng)分別為467nm，589nm，630nm的三種LED燈作為光源，通過(guò)串聯(lián)/并聯(lián)相結(jié)合的方式搭建一個(gè)88的LED陣列燈。選定不銹鋼片作為電極材料，PDMS為澆注固化材料，搭建電輔助裝置。電極是一個(gè)7cm1cm的片狀電極，，兩電極間距離為4.5cm。 ③細(xì)胞培養(yǎng)及刺激實(shí)驗(yàn)。設(shè)計(jì)實(shí)驗(yàn)方案，確定刺激參數(shù)為刺激時(shí)機(jī)、刺激次數(shù)、刺激波長(zhǎng)及刺激時(shí)間。然后培養(yǎng)成肌細(xì)胞，當(dāng)細(xì)胞鋪滿培養(yǎng)皿（一般75%~80%）時(shí)進(jìn)行傳代培養(yǎng)，按照刺激實(shí)驗(yàn)要求確定傳代培養(yǎng)的盤數(shù)。刺激時(shí)機(jī)：接種后12h、24h，刺激次數(shù)：1次、2次、3次、4次，刺激波長(zhǎng)：467nm，589nm，630nm，刺激時(shí)間：400s、600s、1000s、1600s。分別對(duì)上述參數(shù)依次進(jìn)行刺激實(shí)驗(yàn)研究，探討各參數(shù)對(duì)成肌細(xì)胞生長(zhǎng)增殖效果的影響。 ④成肌細(xì)胞生長(zhǎng)狀態(tài)的評(píng)價(jià)及有效刺激參數(shù)的確定。完成刺激實(shí)驗(yàn)后，將細(xì)胞收集固存，24小時(shí)后用于流式細(xì)胞周期檢測(cè)。細(xì)胞收集固存過(guò)程中，胰酶消化不宜過(guò)度，否則會(huì)產(chǎn)生細(xì)胞碎片；離心時(shí)轉(zhuǎn)速應(yīng)設(shè)置為1000rpm，轉(zhuǎn)速過(guò)小，細(xì)胞不能很好的凝聚，會(huì)使細(xì)胞丟失，而轉(zhuǎn)速過(guò)大會(huì)導(dǎo)致細(xì)胞破裂。細(xì)胞碎片及細(xì)胞破裂都會(huì)影響流式檢測(cè)的結(jié)果，且流式檢測(cè)是細(xì)胞個(gè)數(shù)不少于1～2×10~6。將流式檢測(cè)結(jié)果與郭瑞雄等人的研究結(jié)果進(jìn)行對(duì)比分析，分別確定一組有效刺激參數(shù)。
[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.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R329.2

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