转核技术建立可增殖神经细胞系的实验研究

发布时间：2018-01-04 17:42

本文关键词：转核技术建立可增殖神经细胞系的实验研究　出处：《第三军医大学》2007年博士论文　论文类型：学位论文

【摘要】： 目的:将胎鼠皮质神经元细胞核转入到骨髓间充质干细胞胞质体中构建转核细胞,以期望建立一种可增殖的神经细胞系,为脑移植的供体细胞来源探索一条新的途径。方法:(1)骨髓间充质干细胞的培养,用流式细胞仪检测骨髓间充质干细胞表面标志物CD44、CD90、CD71、CD11b进行鉴定,用免疫细胞化学染色法检测CD133的表达情况;(2)用梯度离心法制备骨髓间充质干细胞胞质体,并用HE染色、Giemsa染色、免疫荧光双标法、透射电镜检查等方法进行鉴定,用台盼蓝染色检测其活性;(3)采用胎龄17d的胎鼠进行皮质神经元的分离培养,用免疫组化法检测神经元MAP2和NeuN的表达情况;(4)用梯度离心法制备神经元细胞核,并用HE染色、免疫荧光染色、透射电镜检查等方法进行鉴定,用台盼蓝染色检测其活性;(5)用PEG介导神经元细胞核与骨髓间充质干细胞胞质体发生融合构建转核细胞;(6)用Hoechst33342和CD71免疫荧光双标法对转核细胞进行鉴定;(7)用抗BrdU和Hoechst33342免疫荧光双标法检测转核细胞是否具有增殖能力,用MTT比色法绘制生长曲线;(8)用扫描电镜和透射电镜对转核细胞进行检测;(9)用流式细胞仪检测转核细胞CD44、CD90、CD71、CD11b等抗原的表达变化;(10)用免疫荧光双标法和免疫细胞化学染色法检测转核细胞NeuN的表达情况;(11)用免疫细胞化学染色法检测转核细胞CD133、MAP2、Nestin等抗原的表达情况; 结果:(1)骨髓间充质干细胞呈长梭形、漩涡状生长,用流式细胞仪检测骨髓间充质干细胞表面标志物CD44、CD90、CD71阳性,CD11b阴性,免疫细胞化学染色显示CD133阴性。(2)HE染色和Giemsa染色显示骨髓间充质干细胞经梯度离心后在15~18%Ficoll400梯度层可见到大量胞质体,贴壁生长的胞质体呈多边形或类圆形,体积与完整细胞相比差别不大。Hoechst33342和CD71免疫荧光双标染色显示骨髓间充质干细胞经梯度离心后在15~18% Ficoll400梯度层可见到胞浆呈绿色荧光的胞质体,胞核所在部位无蓝色荧光显示。台盼蓝染色示活胞质体比例为99.5%。12.5~18% Ficoll400梯度层细胞透射电镜标本可观察到只有胞浆成分的圆形胞质体,胞浆中有内质网、线粒体等超微结构。(3)胎鼠皮质神经元呈多角形,伸出多个突起,MAP2染色显示神经元细胞浆和突起呈免疫反应阳性,NeuN染色显示神经元细胞核呈免疫反应阳性,部分核周胞质和部分近端突起也呈免疫反应阳性。在培养6h、3d、6d时MAP2和NeuN阳性细胞比例均在90%左右,培养不同时间免疫阳性细胞比例无显著性差异。(4)HE染色和Hoechst免疫荧光染色显示神经元梯度离心后在22~30% Ficoll400梯度层可见到大量圆点状细胞核,台盼蓝染色显示活细胞核比例为99.6%。在22~30% Ficoll400梯度层细胞透射电镜标本中可观察到圆形的细胞核,在细胞核的周围仅见到一层薄薄的胞浆成分。(5)用PEG可介导神经元细胞核与骨髓间充质干细胞胞质体融合构建转核细胞,转核细胞呈梭形或多边形,少数细胞长出两个或数个突起,部分转核细胞呈球状聚集生长。(6)转核细胞Hoechst33342和CD71免疫荧光双标阳性,提示细胞核来自神经元,细胞浆来自骨髓间充质干细胞。转核效率为64.8%。(7)转核细胞抗BrdU与Hoechst33342免疫荧光双标染色阳性。细胞传代次数和细胞培养天数对转核细胞MTT值变化具有显著性影响(p0.01)。二代和三代转核细胞MTT值与一代转核细胞相比明显增高(p0.05)。转核细胞培养第4天的MTT值较培养第1天的MTT值明显增高,具有显著性差异(p0.05)。(8)扫描电镜可观察到神经元细胞核与骨髓间充质干细胞胞质体发生融合的过程。透射电镜可观察转核细胞核浆比偏大,核膜有较多皱褶,内质网数量多,体积比较宽大。(9)一至四代转核细胞CD71、CD90呈阳性,CD11b呈阴性,一代转核细胞CD44为阴性,随着传代次数的增加CD44阳性细胞率逐渐增加,至第四代时CD44阳性细胞率达98.5%。(10)大多数转核细胞的胞浆NeuN均呈免疫阳性反应,部分转核细胞胞浆NeuN呈免疫阳性反应,胞核呈强阳性反应,另有少数转核细胞仅胞核部位NeuN呈强阳性。第二代转核细胞NeuN表达较第一代转核细胞明显增加(p0.05),继续传代NeuN表达逐渐下降,第四代转核细胞NeuN表达与第二代转核细胞相比明显下降(p0.05)。(11)部分转核细胞的细胞膜上出现Nestin阳性表达。一至四代转核细胞Nestin表达强弱比较无明显差异(p0.05),但阳性细胞比例逐渐增加。转核细胞胞浆中CD133表达阳性,一至四代转核细胞CD133表达水平无显著性差异(p0.05)。第一代转核细胞胞浆中MAP2呈免疫阳性反应,随传代次数增加表达水平逐渐减弱,第四代转核细胞MAP2表达呈阴性反应。结论:(1)用梯度离心法可制备骨髓间充质干细胞胞质体;(2)用梯度离心法可制备胎鼠皮质神经元细胞核;(3)采用PEG介导法可将胎鼠皮质神经元细胞核转入到骨髓间充质干细胞胞质体中,构建转核细胞;(4)本研究方法制备的转核细胞具有增殖能力,可表达NeuN和MAP2等神经元标志物,是具有增殖能力的神经元细胞;(5)转核细胞表达CD133、Nestin、CD44、CD90等神经干细胞标志物,随传代次数增加NeuN、MAP2等神经元标志物表达有所下调,CD44、Nestin阳性细胞比例增加,提示转核细胞向神经干细胞方向出现去分化。
[Abstract]:Objective: to transfer the nuclei of fetal rat cortical neurons into bone marrow mesenchymal stem cells and construct nuclear transfer cells in order to establish a proliferative neural cell line, and explore a new way for donor cells of brain transplantation.
Methods: (1) bone marrow mesenchymal stem cells cultured for detection of bone marrow mesenchymal stem cell surface marker CD44, flow cytometry, CD90, CD71, CD11b were identified, staining was used to detect CD133 expression by immunocytochemistry; (2) was prepared by gradient centrifugation of bone marrow mesenchymal stem cytoplasts, and HE staining, Giemsa staining, immunofluorescence staining, transmission electron microscopy and other methods were identified by trypan blue staining was used to detect the activity; (3) isolated by gestational age 17D of embryonic rat cortical neurons, the expression of MAP2 and NeuN neurons were detected by immunohistochemistry.; (4) preparation of neuronal nuclei by gradient centrifugation method, and using HE staining, immunofluorescence staining, transmission electron microscopy and other methods were identified by trypan blue staining was used to detect the activity; (5) the nucleus neurons and bone marrow mesenchymal stem cells of cytoplasts constructed by PEG mediated fusion The nuclear transfer cells; (6) the nuclear transfer cells were identified by Hoechst33342 and CD71 double immunofluorescence method; (7) to detect whether the nuclear transfer cells have the ability of proliferation by using anti BrdU and Hoechst33342 double immunofluorescence method, MTT assay was used to draw the growth curve; (8) the nuclear transfer cells were examined by scanning electron microscopy and transmission electron microscopy; (9) to CD44 cells, flow cytometry was used to detect CD90, CD71, expression of CD11b antigen; (10) using double immunofluorescent staining and immunocytochemical staining method for detection of expression of transgenic NeuN cells; (11) using the immune cell chemical staining detection CD133, MAP2 cells, the expression of Nestin antigen;
Results: (1) bone marrow mesenchymal stem cells in the fusiform whirlpool growth, for the detection of bone marrow mesenchymal stem cell surface marker CD44, flow cytometry, CD90, CD71 positive, CD11b negative, immunocytochemical staining showed CD133 negative (2). HE staining and Giemsa staining showed that the bone marrow. Mesenchymal stem cells by gradient centrifugation in 15~18%Ficoll400 gradient layer can be seen a lot of cytoplasts, cytoplasts adherent were polygonal or round, volume difference compared with intact cells.Hoechst33342 and CD71 immunofluorescence staining showed that bone marrow mesenchymal stem cells by gradient centrifugation in 15~18% gradient layer can be seen in cytoplasm of Ficoll400 a green fluorescent cytoplasm, nucleus location no blue fluorescent display. Trypan blue stained viable cytoplasts estimated the ratio of 99.5%.12.5~18% Ficoll400 gradient layer transmission electron microscope specimens can be observed only cytoplasmic components Round cytoplast, endoplasmic reticulum in the cytoplasm, mitochondrial ultrastructure. (3) of fetal rat cortical neurons were polygonal, out of a plurality of protrusions, MAP2 staining in cytoplasm and processes of neurons were immunoreactive neurons, NeuN staining showed that the nuclei were immunoreactive, part of cytoplasm and proximal processes were also immunoreactive. In cultured 6H, 3D, 6D, MAP2 and NeuN positive cells were about 90%, the proportion of immunoreactive cells cultured for different time had no significant difference. (4) HE staining and Hoechst immunofluorescence staining showed that the neurons in the 22~30% gradient centrifugation and Ficoll400 gradient layer can be seen a large number of dot shaped nuclei and the trypan blue staining showed the nucleus of a living cell ratio of 99.6%. can be observed round nuclei in 22~30% Ficoll400 gradient layer transmission electron microscope specimens, only a thin layer of cytoplasm around the nucleus to see . (5) with PEG mediated neuron nucleus and bone marrow mesenchymal stem cells to construct the cytoplast fusion, nuclear transfer cells were spindle or polygonal, a few cells grow two or more processes, part of the nuclear transfer cells were spherical aggregation growth. (6) the nuclear transfer cells and Hoechst33342 CD71 double immunofluorescence positive, which suggested that the nucleus from neurons, cells from bone marrow mesenchymal stem cells. The nuclear transfer efficiency of 64.8%. (7) - BrdU and Hoechst33342 double immunofluorescence staining of nuclear transfer cells. The number of cells and cell culture time had significant effect on the nuclear transfer cells MTT values (P0.01). The two and three generation nuclear transfer cells MTT value and the generation of the nuclear transfer cells were significantly higher (P0.05). The nuclear transfer cells were cultured for fourth days the MTT value was cultured for first days the MTT value increased significantly, with significant difference (P0.05). (8) were detected by scanning electron microscope, neurons fine The nucleus and bone marrow mesenchymal stem cell fusion occurs cytoplast. TEM cells turn karyoplasmic ratio too large, there are many reductus endoplasmic reticulum membrane, the number, volume is relatively large. (9) to a four generation of transgenic CD71 cells, CD90 positive, CD11b negative, generation CD44 cells were negative, with the increase of passage number ratio of CD44 positive cells increased gradually, and the fourth generation of CD44 positive cell rate of 98.5%. (10) NeuN most cytoplasmic nuclear transfer cells showed positive immunoreactivity of cytoplasm of nuclear transfer cells showed NeuN immunoreactivity, the nucleus showed a strong positive reaction NeuN, a number of nuclear transfer cells only nuclear sites were strongly positive. The second generation of transgenic NeuN cells expression compared to the first generation of nuclear transfer cells increased significantly (P0.05), continue to passage of the expression of NeuN decreased gradually, the fourth generation NeuN nuclear expression and the second generation of nuclear transfer cells decreased significantly compared (p0. 05). (11) the positive expression of Nestin cell membrane of some nuclear transfer cells. One to four generation Nestin nuclear expression was no difference between the intensity (P0.05), but the proportion of positive cells increased gradually. The nuclear transfer cells in the cytoplasm of positive expression of CD133, one to four generation nuclear transfer cells CD133 expression level no significant difference (P0.05). The first generation of nuclear transfer cells in the cytoplasm showed MAP2 positive immunoreactivity expression level decreases gradually with the increase of passage, the fourth generation MAP2 nuclear expression was negative.
Conclusion: (1) preparation of bone marrow mesenchymal stem cells in plastids by gradient centrifugation; (2) by gradient centrifugation for nuclei of cortical neurons in fetal rats; (3) using PEG mediated method can be transferred to the nucleus of cortical neurons of fetal rat bone marrow mesenchymal stem cells to construct plastids. The nuclear transfer cells; (4) the preparation methods of the nuclear transfer cells have the ability of proliferation, the expression of NeuN and MAP2 neuronal markers, cells are proliferative neurons; (5) the nuclear transfer cells the expression of CD133, Nestin, CD44, CD90 and other neural stem cell marker, with the increase of passage number NeuN MAP2, neuronal marker expression decreased, CD44, the proportion of Nestin positive cells increased, suggesting that the nuclear transfer cells to neural stem cells to differentiation direction.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

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