【作者】 万云云；

【导师】 王增贤；

【作者基本信息】 泰山医学院 ， 神经生物学， 2007， 硕士

【摘要】 目的体外培养、纯化大鼠脑皮质星形胶质细胞,获得高纯度的星形胶质细胞培养物,观察其生长过程及其生物学特性,采用脂质体方法将含有目的基因SV40T的质粒pSV3neo转入纯化的星形胶质细胞内,为后续创建永生化星形胶质细胞株作准备,并为研究不同发育时期星形胶质细胞的生物学特性等奠定基础。方法胰酶消化结合机械吹打方法分离大鼠脑皮质细胞,差速粘附处理及恒温摇床振荡法获得纯化星形胶质细胞,经含SV40T基因的质粒pSV3neo脂质体转染法转入星形胶质细胞,SV40T抗原免疫荧光染色后检测转染结果。1.星形胶质细胞的纯化培养及形态学观察：取新生Wistar大鼠大脑皮质,用胰酶消化结合机械吹打方法制成初细胞悬液,差速贴壁法去除成纤维细胞,培养至细胞融合并分层生长时,以恒温摇床振荡方法去除神经元、少突胶质细胞、小胶质细胞等继续培养,获得纯化星形胶质细胞,并用GFAP免疫细胞化学方法鉴定星形胶质细胞。倒置相差显微镜下观察培养不同时间的细胞密度、突起长度及分支情况等并进行体视学测量以了解细胞的生长状态。2.质粒抽提及鉴定：质粒pSV3neo转化大肠杆菌DH5a感受态细胞,经37℃振荡培养扩增后,用质粒抽提试剂盒抽提质粒,所提质粒进行酶切后电泳分析,采集图像与质粒说明书比对,鉴定正确后大量扩增,用于后续转染实验。3.质粒转染：纯化后细胞生长1-2 d,其融合度分别达80%、90%接种密度时,以3.0、4.0、5.0、6.0μl/50μl四种脂质体浓度和0.4、0.8、1.2、1.6μg/50μl四种质粒浓度组合后,混合于培养物中进行转染,SV40T抗原免疫荧光染色标记后检测,以阳性标记细胞率最高的细胞接种密度、脂质体浓度和质粒浓度,作为最终转染条件,进行规模转染。4.转染细胞的观察：倒置相差显微镜下,观察转染过程中及转染后细胞的形态及生物学特性改变,比较转染细胞与未转染细胞的差异；免疫荧光染色方法检测SV40T抗原及GFAP在转染后细胞中的表达情况。结果1.培养细胞的生长规律：分离的大鼠脑皮质细胞在接种后1h开始贴壁,6-8 h后大部分细胞已贴壁,呈椭圆形、梭形及不规则形,部分细胞已开始铺展,并长出突起。随着培养时间的延长,细胞数量增多、体积增大,突起延长且数目增加,3-5 d时增加最明显。培养7～10d后,细胞已融合并可见分层生长,底层为星形胶质细胞,上层为神经元、少突胶质细胞、小胶质细胞等其它细胞。2.纯化鉴定：根据细胞分层生长的特性,经摇床振荡处理后,显微镜下可见贴壁细胞形态较一致,胞体轮廓清晰,一级胞突丰富,分支较多,相互交织成网状；纯化后的细胞经GFAP免疫细胞化学染色后,阳性细胞胞浆及突起均呈棕色,星形胶质细胞纯度达98%以上,可用于后续实验。3.质粒鉴定：质粒转化感受态细胞,经扩增、抽提后,酶切电泳,可清晰见到4条带,与1Kb DNA Ladder比对,分子量对应于4.6、3.1、1.4、0.64 kbp,证明所提质粒为所需质粒pSV3neo。4.转染优化试验：经脂质体、质粒浓度及细胞接种密度的转染优化试验,可得细胞融合度约90%时,以5.0μl/50μl脂质体,1.2μg/50μl质粒转染后,经免疫荧光染色可见较多阳性细胞,从而确定为转染最佳条件。5.转染细胞观察：铺板细胞达到一定融合度后,加入质粒与脂质体的混合物,作用约5h时,显微镜下观察可见细胞胞体和突起肿胀,细胞轮廓清晰,立体感增强；48 h后观察可见部分细胞裂解死亡,存活细胞形态变化不大；经免疫荧光染色后,胞浆内呈现GFAP红色荧光,清晰显现出细胞轮廓,并可见SV40T绿色荧光。结论1.原代培养的大鼠脑皮质细胞在培养3～5 d时增殖最明显,细胞数目增加,体积增大,突起增多延长并相互交织成网状；约7～10d细胞融合并分层生长时进行恒温摇床振荡传代处理,所得星形胶质细胞纯度可达98%以上。2.通过转染优化试验,可以确定转染的最佳条件为：24孔板细胞融合度约90%,脂质体5.0μl/50μl和质粒1.2μg/50μl。优化条件确定后,转染实验可以根据培养板表面积增加的比例线性放大,扩大转染量,并用于后续永生化细胞株的筛选。3.含目的基因SV40T的质粒pSV3neo转染纯化的星形胶质细胞后,免疫荧光染色检测到SV40T在星形胶质细胞内的表达产物SV40T抗原,转染成功。更多还原

【Abstract】 Objective To culture and purify astrocytes from rat cerebral cortex to obtain an in vitro culture model of high purity astrocytes and observe their growth and the biological features, and then transfect the gene-SV40T into astrocytes to be immortalized for further study of the characteristics of astrocytes in different developing stages.Methods Cells from Wistar rat cerebral cortex disassociated by enzymatic digestion and mechanical methods were treated with differential attachment and cultured for 7 to 10 days, and then were purified in orbital shaker. The purified astrocytes were identified by GFAP-immunoreactivity, then the plasmid pSV3neo with immortalizing gene-SV40T was transfected into the astrocytes by lipofectamine, and the transfection was labelled with SV40T antigen immunofluorescence.1. Purification and morphological observation of astrocytes:The neonatal rat brain was taken and the cortex was made into suspension by aids of enzyme-digestion and mechanical dissociation. In the process of culture, fibroblasts were reduced with differential attachment and oligodentrocytes and microglial cells were eliminated with orbital shaker. The growth features of the cultured cells were observed and the astrocytes were identified with GFAP immunoreactivity. Cell density, length of processes and cell branches were tested stereologically in different stages to examine the growth of the cortical cells in the culture.2.Plasmid extraction and identification:The competent cell DH5a was transfected by plasmid-pSV3neo, cultured and shaked at 37℃for amplification. From the culture the plasmid-pSV3neo was extracted and biocatalysted to be identified and then amplified for further study.3. Plasmid transfection:In 24 well dish, for one or two days culture, when the purified astrocytes fused to 80% or 90%, the lipofectamine and the plasmid in their own gradients of 3.0,4.0,5.0,6.0μl/50μl and 0.4,0.8,1.2,1.6μg/50μl respectively were pooled into the culture, incubated for 48 hours, and then labelled with immunofluorescence and counted the SV40T positive astrocytes to screen the optimal cell density, concentration of lipofectamine and plasmid for transfection of the purified astrocytes with SV40T in large scale.4.Observation of transfected astrocytes:Under the inverted phase contrast microscope, changes in morphology and biological features of cells were examined during and after transfection, and differences of astrocytes before and after transfection were also observed. The expression of SV40T antigen and GFAP in the transfected astrocytes was tested with immunofluorescence.Results1. Morphological changes of cultured cells:Cortical cells began to adhere to the container in one hour after plantation, after 6 to 8 hours, most cortical cells attached to container, and took the ellipse, fusiform and irregular shapes, some cells had small protrusions already. As times went on, the number and volume of cells increased, there were more and longer branches. The cultured cells proliferated the fastest from the third to the fifth day, when the cell number and volume increased rapidly. Cultured for 7 to 10 days, the cultured cells fused and got demix with astrocytes underlying, on top of which were neurons, oligodendrocytes and microglial cells.2.Purification and identification of astrocytes:Treated with orbital shaker, and passaged, cells demonstrated identical shapes rich of protrusions and their processes interlaced as a network. Labelled with GFAP-immunocytochemistry, the purified cells with positive immunoreactivity were stainned brown in their endochylema and processes. The positive immunoreactivity cells, the astrocytes, were counted more than 98 percent of all cells.3.Identification of plasmid:The extracted plasmid was cut into four clips with molecular weight of 4.6,3.1,1.4,0.64 kbp matched the Marker, suggesting that the plasmid was just what we needed.4. Optimization experiment:When cells reached a confluence of 90% and incubated with lipofectamie 5.0μl/50μl and plasmid 1.2μg/50μl, the transfected cells showed more SV40T-positive immunofluorescence, indicating the optimal transfection.5. Observation of transfected astrocytes:When cells fused to about 90%, the mixture of lipofectamine and plasmid was added to the culture, and incubated for 5 hours, the cell body swelled, thus the cells became clearer in their appearance. Forty-eight hours later, some cells schizolysised into pieces, while the rest remained unchangeable. Stained with immunofluorescence, the transfected astrocytes showed red-GFAP fluorescent light in their endochylema and processes, and green-SV40T fluorescent light in their endochylema.Conclusions1.The cultured cells proliferated the fastest when they were cultured for 3 to 5 days, the cell number and volume increased rapidly and their processes interlaced as a network. When the cultured cells fused and got demix from the seventh to the tenth day, cells were treated with orbital shaker and passaged, we got astrocytes which accounted for more than 98 percent of all cells.2. The optimal transfection condition by optimization experiments was gained when cells grew by 90% fused, and incubated with lipofectamie 5.0μl/50μl and plasmid 1.2μg/50μl in 24 well flat bottom.3.Plasmid-pSV3neo was successfully transfected into purified astrocytes.更多还原