本文選題：骨髓間充質(zhì)干細(xì)胞 + 視網(wǎng)膜樣細(xì)胞��； 參考：《福建醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:研究應(yīng)用SD(Sprague-Dawley)大鼠視網(wǎng)膜片光損傷后的培養(yǎng)上清液,誘導(dǎo)SD大鼠骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)在體外跨胚層誘導(dǎo)為視網(wǎng)膜樣細(xì)胞的可能性。 方法:1、SD大鼠MSCs的分離和培養(yǎng):采用貼壁篩選法分離、培養(yǎng)SD大鼠MSCs,并用流式細(xì)胞儀對(duì)細(xì)胞純度進(jìn)行鑒定。2、SD大鼠視網(wǎng)膜片的取材:在顯微鏡下,徹底取下SD大鼠視網(wǎng)膜神經(jīng)上皮層作為視網(wǎng)膜片。并對(duì)其常規(guī)石蠟切片HE染色鑒定各層組織結(jié)構(gòu)的完整性。3、視網(wǎng)膜片光損傷:在超凈臺(tái)內(nèi),將取下的SD大鼠視網(wǎng)膜片放入盛有Neurobasal培養(yǎng)基A(其中加入1:50的B27和0.5M L-glutamine)中,用(1950±200) Lux的光照強(qiáng)度照射45min,用電鏡觀察其光損傷程度。4、大鼠MSCs誘導(dǎo)分化的條件培養(yǎng)液制備:條件培養(yǎng)液Ⅰ:將SD大鼠視網(wǎng)膜片光損傷后的培養(yǎng)上清液與10%FBS的LG-DMEM以2:3混合而成。條件培養(yǎng)液Ⅱ:未光損傷SD大鼠視網(wǎng)膜片培養(yǎng)的上清液與10%FBS的LG-DMEM以2:3混合而成。條件培養(yǎng)液Ⅲ:直接用Neurobasal培養(yǎng)基A與10%FBS的LG-DMEM以2:3混合制成。5、大鼠MSCs體外誘導(dǎo)成視網(wǎng)膜樣細(xì)胞:3種條件培養(yǎng)液均培養(yǎng)誘導(dǎo)至第3代的SD大鼠骨髓間充質(zhì)干細(xì)胞7-8d。用倒置相差顯微鏡觀察誘導(dǎo)后細(xì)胞形態(tài)學(xué)改變。6、用免疫細(xì)胞化學(xué)染色和RT-PCR檢測(cè)視紫紅質(zhì)(Rhodopsin)、神經(jīng)元特異性烯醇化酶(neuron-specific enolase,NES)、膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein ,GFAP)等特征性標(biāo)記在誘導(dǎo)后細(xì)胞中的表達(dá)情況。 結(jié)果:1、流式細(xì)胞儀鑒定經(jīng)貼壁篩選法可獲得大量高純度的大鼠MSCs。2、HE染色顯示所取的SD大鼠神經(jīng)上皮層結(jié)構(gòu)完整。3、電鏡結(jié)果顯示光損傷后的SD大鼠視網(wǎng)膜片結(jié)構(gòu)損傷嚴(yán)重。4、誘導(dǎo)7-8d后的SD大鼠MSCs置倒置相差顯微鏡下觀察:條件培養(yǎng)液Ⅰ誘導(dǎo)組的細(xì)胞有較多突起,發(fā)生遷移并建立突觸聯(lián)系,而條件培養(yǎng)液Ⅱ及條件培養(yǎng)液Ⅲ只有少數(shù)細(xì)胞發(fā)生上述改變。5、免疫細(xì)胞化學(xué)染色顯示:3組不同檢測(cè)指標(biāo)陽(yáng)性率分別為:條件培養(yǎng)液Ⅰ組Rhodopsin(37.97±7.84)%、NSE(21.59±2.98)%、GFAP(20.73±5.25)%,條件培養(yǎng)液Ⅱ組Rhodopsin(10.23±2.05)%、NSE(16.24±1.10)%、GFAP(15.92±0.96)%,條件培養(yǎng)液Ⅲ組Rhodopsin(0)、NSE(6.24±4.58)%、GFAP(4.96±1.79)%。將三種培養(yǎng)條件下所表達(dá)的陽(yáng)性細(xì)胞分別進(jìn)行統(tǒng)計(jì)學(xué)分析,組間差異有統(tǒng)計(jì)學(xué)意義。RT-PCR鑒定也顯示同樣結(jié)果:條件培養(yǎng)液Ⅰ組Rhodopsin(0.3915±0.00644)、NSE(0.2019±0.00682)、GFAP(0.1972±0.00211),條件培養(yǎng)液Ⅱ組Rhodopsin(0.0983±0.00319)、NSE(0.1048±0.00323)、GFAP(0.1040±0.00254),條件培養(yǎng)液Ⅲ組Rhodopsin(0.0044±0.00126)、NSE(0.0498±0.00149)、GFAP(0.0467±0.00333)。 結(jié)論:光損傷SD大鼠視網(wǎng)膜片培養(yǎng)上清液可誘導(dǎo)SD大鼠骨髓間充質(zhì)干細(xì)胞分化為視網(wǎng)膜樣細(xì)胞,研究結(jié)果為干細(xì)胞治療視網(wǎng)膜變性疾病提供新思路。
[Abstract]:Aim: to study the possibility of inducing mesenchymal stem cells (MSCs) from SD rat bone marrow mesenchymal stem cells (MSCs) into retinoid cells in vitro by using culture supernatant of SD Sprague-Dawley (SD) rat retina. Methods: MSCs from SD rats were isolated and cultured by adherent screening method. The cell purity of SD rats was identified by flow cytometry. The retinal slices of SD rats were obtained by flow cytometry. The retinal nerve epithelium of SD rats was removed as retina slice. The HE staining of the paraffin sections was used to identify the integrity of the tissue structure of each layer, and the light damage of the retina: in the ultra-clean platform, the removed retina slices of SD rats were added to the neurobasal medium A (including 1:50 B27 and 0.5M L-glutamine), and the retinal slices of SD rats were added to the neurobasal culture medium A (B27 and 0.5M L-glutamine at 1:50). The light damage was observed by electron microscope for 45 min. The conditioned medium for differentiation of rat MSCs was prepared: conditioned medium 鈪，

本文編號(hào)：2016349