本文關(guān)鍵詞：轉(zhuǎn)核技術(shù)建立可增殖神經(jīng)細(xì)胞系的實(shí)驗(yàn)研究　出處：《第三軍醫(yī)大學(xué)》2007年博士論文　論文類(lèi)型：學(xué)位論文

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【摘要】： 目的:將胎鼠皮質(zhì)神經(jīng)元細(xì)胞核轉(zhuǎn)入到骨髓間充質(zhì)干細(xì)胞胞質(zhì)體中構(gòu)建轉(zhuǎn)核細(xì)胞,以期望建立一種可增殖的神經(jīng)細(xì)胞系,為腦移植的供體細(xì)胞來(lái)源探索一條新的途徑。 方法:(1)骨髓間充質(zhì)干細(xì)胞的培養(yǎng),用流式細(xì)胞儀檢測(cè)骨髓間充質(zhì)干細(xì)胞表面標(biāo)志物CD44、CD90、CD71、CD11b進(jìn)行鑒定,用免疫細(xì)胞化學(xué)染色法檢測(cè)CD133的表達(dá)情況;(2)用梯度離心法制備骨髓間充質(zhì)干細(xì)胞胞質(zhì)體,并用HE染色、Giemsa染色、免疫熒光雙標(biāo)法、透射電鏡檢查等方法進(jìn)行鑒定,用臺(tái)盼藍(lán)染色檢測(cè)其活性;(3)采用胎齡17d的胎鼠進(jìn)行皮質(zhì)神經(jīng)元的分離培養(yǎng),用免疫組化法檢測(cè)神經(jīng)元MAP2和NeuN的表達(dá)情況;(4)用梯度離心法制備神經(jīng)元細(xì)胞核,并用HE染色、免疫熒光染色、透射電鏡檢查等方法進(jìn)行鑒定,用臺(tái)盼藍(lán)染色檢測(cè)其活性;(5)用PEG介導(dǎo)神經(jīng)元細(xì)胞核與骨髓間充質(zhì)干細(xì)胞胞質(zhì)體發(fā)生融合構(gòu)建轉(zhuǎn)核細(xì)胞;(6)用Hoechst33342和CD71免疫熒光雙標(biāo)法對(duì)轉(zhuǎn)核細(xì)胞進(jìn)行鑒定;(7)用抗BrdU和Hoechst33342免疫熒光雙標(biāo)法檢測(cè)轉(zhuǎn)核細(xì)胞是否具有增殖能力,用MTT比色法繪制生長(zhǎng)曲線;(8)用掃描電鏡和透射電鏡對(duì)轉(zhuǎn)核細(xì)胞進(jìn)行檢測(cè);(9)用流式細(xì)胞儀檢測(cè)轉(zhuǎn)核細(xì)胞CD44、CD90、CD71、CD11b等抗原的表達(dá)變化;(10)用免疫熒光雙標(biāo)法和免疫細(xì)胞化學(xué)染色法檢測(cè)轉(zhuǎn)核細(xì)胞NeuN的表達(dá)情況;(11)用免疫細(xì)胞化學(xué)染色法檢測(cè)轉(zhuǎn)核細(xì)胞CD133、MAP2、Nestin等抗原的表達(dá)情況; 結(jié)果:(1)骨髓間充質(zhì)干細(xì)胞呈長(zhǎng)梭形、漩渦狀生長(zhǎng),用流式細(xì)胞儀檢測(cè)骨髓間充質(zhì)干細(xì)胞表面標(biāo)志物CD44、CD90、CD71陽(yáng)性,CD11b陰性,免疫細(xì)胞化學(xué)染色顯示CD133陰性。(2)HE染色和Giemsa染色顯示骨髓間充質(zhì)干細(xì)胞經(jīng)梯度離心后在15~18%Ficoll400梯度層可見(jiàn)到大量胞質(zhì)體,貼壁生長(zhǎng)的胞質(zhì)體呈多邊形或類(lèi)圓形,體積與完整細(xì)胞相比差別不大。Hoechst33342和CD71免疫熒光雙標(biāo)染色顯示骨髓間充質(zhì)干細(xì)胞經(jīng)梯度離心后在15~18% Ficoll400梯度層可見(jiàn)到胞漿呈綠色熒光的胞質(zhì)體,胞核所在部位無(wú)藍(lán)色熒光顯示。臺(tái)盼藍(lán)染色示活胞質(zhì)體比例為99.5%。12.5~18% Ficoll400梯度層細(xì)胞透射電鏡標(biāo)本可觀察到只有胞漿成分的圓形胞質(zhì)體,胞漿中有內(nèi)質(zhì)網(wǎng)、線粒體等超微結(jié)構(gòu)。(3)胎鼠皮質(zhì)神經(jīng)元呈多角形,伸出多個(gè)突起,MAP2染色顯示神經(jīng)元細(xì)胞漿和突起呈免疫反應(yīng)陽(yáng)性,NeuN染色顯示神經(jīng)元細(xì)胞核呈免疫反應(yīng)陽(yáng)性,部分核周胞質(zhì)和部分近端突起也呈免疫反應(yīng)陽(yáng)性。在培養(yǎng)6h、3d、6d時(shí)MAP2和NeuN陽(yáng)性細(xì)胞比例均在90%左右,培養(yǎng)不同時(shí)間免疫陽(yáng)性細(xì)胞比例無(wú)顯著性差異。(4)HE染色和Hoechst免疫熒光染色顯示神經(jīng)元梯度離心后在22~30% Ficoll400梯度層可見(jiàn)到大量圓點(diǎn)狀細(xì)胞核,臺(tái)盼藍(lán)染色顯示活細(xì)胞核比例為99.6%。在22~30% Ficoll400梯度層細(xì)胞透射電鏡標(biāo)本中可觀察到圓形的細(xì)胞核,在細(xì)胞核的周?chē)鷥H見(jiàn)到一層薄薄的胞漿成分。(5)用PEG可介導(dǎo)神經(jīng)元細(xì)胞核與骨髓間充質(zhì)干細(xì)胞胞質(zhì)體融合構(gòu)建轉(zhuǎn)核細(xì)胞,轉(zhuǎn)核細(xì)胞呈梭形或多邊形,少數(shù)細(xì)胞長(zhǎng)出兩個(gè)或數(shù)個(gè)突起,部分轉(zhuǎn)核細(xì)胞呈球狀聚集生長(zhǎng)。(6)轉(zhuǎn)核細(xì)胞Hoechst33342和CD71免疫熒光雙標(biāo)陽(yáng)性,提示細(xì)胞核來(lái)自神經(jīng)元,細(xì)胞漿來(lái)自骨髓間充質(zhì)干細(xì)胞。轉(zhuǎn)核效率為64.8%。(7)轉(zhuǎn)核細(xì)胞抗BrdU與Hoechst33342免疫熒光雙標(biāo)染色陽(yáng)性。細(xì)胞傳代次數(shù)和細(xì)胞培養(yǎng)天數(shù)對(duì)轉(zhuǎn)核細(xì)胞MTT值變化具有顯著性影響(p0.01)。二代和三代轉(zhuǎn)核細(xì)胞MTT值與一代轉(zhuǎn)核細(xì)胞相比明顯增高(p0.05)。轉(zhuǎn)核細(xì)胞培養(yǎng)第4天的MTT值較培養(yǎng)第1天的MTT值明顯增高,具有顯著性差異(p0.05)。(8)掃描電鏡可觀察到神經(jīng)元細(xì)胞核與骨髓間充質(zhì)干細(xì)胞胞質(zhì)體發(fā)生融合的過(guò)程。透射電鏡可觀察轉(zhuǎn)核細(xì)胞核漿比偏大,核膜有較多皺褶,內(nèi)質(zhì)網(wǎng)數(shù)量多,體積比較寬大。(9)一至四代轉(zhuǎn)核細(xì)胞CD71、CD90呈陽(yáng)性,CD11b呈陰性,一代轉(zhuǎn)核細(xì)胞CD44為陰性,隨著傳代次數(shù)的增加CD44陽(yáng)性細(xì)胞率逐漸增加,至第四代時(shí)CD44陽(yáng)性細(xì)胞率達(dá)98.5%。(10)大多數(shù)轉(zhuǎn)核細(xì)胞的胞漿NeuN均呈免疫陽(yáng)性反應(yīng),部分轉(zhuǎn)核細(xì)胞胞漿NeuN呈免疫陽(yáng)性反應(yīng),胞核呈強(qiáng)陽(yáng)性反應(yīng),另有少數(shù)轉(zhuǎn)核細(xì)胞僅胞核部位NeuN呈強(qiáng)陽(yáng)性。第二代轉(zhuǎn)核細(xì)胞NeuN表達(dá)較第一代轉(zhuǎn)核細(xì)胞明顯增加(p0.05),繼續(xù)傳代NeuN表達(dá)逐漸下降,第四代轉(zhuǎn)核細(xì)胞NeuN表達(dá)與第二代轉(zhuǎn)核細(xì)胞相比明顯下降(p0.05)。(11)部分轉(zhuǎn)核細(xì)胞的細(xì)胞膜上出現(xiàn)Nestin陽(yáng)性表達(dá)。一至四代轉(zhuǎn)核細(xì)胞Nestin表達(dá)強(qiáng)弱比較無(wú)明顯差異(p0.05),但陽(yáng)性細(xì)胞比例逐漸增加。轉(zhuǎn)核細(xì)胞胞漿中CD133表達(dá)陽(yáng)性,一至四代轉(zhuǎn)核細(xì)胞CD133表達(dá)水平無(wú)顯著性差異(p0.05)。第一代轉(zhuǎn)核細(xì)胞胞漿中MAP2呈免疫陽(yáng)性反應(yīng),隨傳代次數(shù)增加表達(dá)水平逐漸減弱,第四代轉(zhuǎn)核細(xì)胞MAP2表達(dá)呈陰性反應(yīng)。 結(jié)論:(1)用梯度離心法可制備骨髓間充質(zhì)干細(xì)胞胞質(zhì)體;(2)用梯度離心法可制備胎鼠皮質(zhì)神經(jīng)元細(xì)胞核;(3)采用PEG介導(dǎo)法可將胎鼠皮質(zhì)神經(jīng)元細(xì)胞核轉(zhuǎn)入到骨髓間充質(zhì)干細(xì)胞胞質(zhì)體中,構(gòu)建轉(zhuǎn)核細(xì)胞;(4)本研究方法制備的轉(zhuǎn)核細(xì)胞具有增殖能力,可表達(dá)NeuN和MAP2等神經(jīng)元標(biāo)志物,是具有增殖能力的神經(jīng)元細(xì)胞;(5)轉(zhuǎn)核細(xì)胞表達(dá)CD133、Nestin、CD44、CD90等神經(jīng)干細(xì)胞標(biāo)志物,隨傳代次數(shù)增加NeuN、MAP2等神經(jīng)元標(biāo)志物表達(dá)有所下調(diào),CD44、Nestin陽(yáng)性細(xì)胞比例增加,提示轉(zhuǎn)核細(xì)胞向神經(jīng)干細(xì)胞方向出現(xiàn)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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R329

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