光损伤视网膜片培养上清液诱导大鼠骨髓间充质干细胞分化为视网膜样细胞的研究

发布时间：2018-06-14 01:08

  本文选题：骨髓间充质干细胞 + 视网膜样细胞　； 参考：《福建医科大学》2011年硕士论文

【摘要】：目的:研究应用SD(Sprague-Dawley)大鼠视网膜片光损伤后的培养上清液,诱导SD大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在体外跨胚层诱导为视网膜样细胞的可能性。 方法:1、SD大鼠MSCs的分离和培养:采用贴壁筛选法分离、培养SD大鼠MSCs,并用流式细胞仪对细胞纯度进行鉴定。2、SD大鼠视网膜片的取材:在显微镜下,彻底取下SD大鼠视网膜神经上皮层作为视网膜片。并对其常规石蜡切片HE染色鉴定各层组织结构的完整性。3、视网膜片光损伤:在超净台内,将取下的SD大鼠视网膜片放入盛有Neurobasal培养基A(其中加入1:50的B27和0.5M L-glutamine)中,用(1950±200) Lux的光照强度照射45min,用电镜观察其光损伤程度。4、大鼠MSCs诱导分化的条件培养液制备:条件培养液Ⅰ:将SD大鼠视网膜片光损伤后的培养上清液与10%FBS的LG-DMEM以2:3混合而成。条件培养液Ⅱ:未光损伤SD大鼠视网膜片培养的上清液与10%FBS的LG-DMEM以2:3混合而成。条件培养液Ⅲ:直接用Neurobasal培养基A与10%FBS的LG-DMEM以2:3混合制成。5、大鼠MSCs体外诱导成视网膜样细胞:3种条件培养液均培养诱导至第3代的SD大鼠骨髓间充质干细胞7-8d。用倒置相差显微镜观察诱导后细胞形态学改变。6、用免疫细胞化学染色和RT-PCR检测视紫红质(Rhodopsin)、神经元特异性烯醇化酶(neuron-specific enolase,NES)、胶质纤维酸性蛋白(glial fibrillary acidic protein ,GFAP)等特征性标记在诱导后细胞中的表达情况。 结果:1、流式细胞仪鉴定经贴壁筛选法可获得大量高纯度的大鼠MSCs。2、HE染色显示所取的SD大鼠神经上皮层结构完整。3、电镜结果显示光损伤后的SD大鼠视网膜片结构损伤严重。4、诱导7-8d后的SD大鼠MSCs置倒置相差显微镜下观察:条件培养液Ⅰ诱导组的细胞有较多突起,发生迁移并建立突触联系,而条件培养液Ⅱ及条件培养液Ⅲ只有少数细胞发生上述改变。5、免疫细胞化学染色显示:3组不同检测指标阳性率分别为:条件培养液Ⅰ组Rhodopsin(37.97±7.84)%、NSE(21.59±2.98)%、GFAP(20.73±5.25)%,条件培养液Ⅱ组Rhodopsin(10.23±2.05)%、NSE(16.24±1.10)%、GFAP(15.92±0.96)%,条件培养液Ⅲ组Rhodopsin(0)、NSE(6.24±4.58)%、GFAP(4.96±1.79)%。将三种培养条件下所表达的阳性细胞分别进行统计学分析,组间差异有统计学意义。RT-PCR鉴定也显示同样结果:条件培养液Ⅰ组Rhodopsin(0.3915±0.00644)、NSE(0.2019±0.00682)、GFAP(0.1972±0.00211),条件培养液Ⅱ组Rhodopsin(0.0983±0.00319)、NSE(0.1048±0.00323)、GFAP(0.1040±0.00254),条件培养液Ⅲ组Rhodopsin(0.0044±0.00126)、NSE(0.0498±0.00149)、GFAP(0.0467±0.00333)。 结论:光损伤SD大鼠视网膜片培养上清液可诱导SD大鼠骨髓间充质干细胞分化为视网膜样细胞,研究结果为干细胞治疗视网膜变性疾病提供新思路。
[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，

本文编号：2016349