【摘要】：目的觀察熒光染料(CM-Di I)標(biāo)記人骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BM-MSC)后,是否會(huì)脫落造成周?chē)?xì)胞被污染,標(biāo)記細(xì)胞經(jīng)靜脈移植入腦梗死大鼠后是否會(huì)造成宿主腦內(nèi)神經(jīng)元被誤染色;以及是否可以鑒定人BMMSC產(chǎn)生腦源性神經(jīng)營(yíng)養(yǎng)因子(brain derived neurotrophic factor,BDNF)。方法應(yīng)用不同濃度CM-Di I分別標(biāo)記培養(yǎng)的人BMMSC,觀察標(biāo)記效率。將轉(zhuǎn)染綠色熒光蛋白(green fluorescence protein,GFP)的人胚腎293T細(xì)胞和CM-Di I標(biāo)記的人BM-MSC細(xì)胞共培養(yǎng),7 d之后觀察CM-Di I的紅色熒光和293T細(xì)胞是否雙標(biāo)記。將CM-Di I標(biāo)記的人BM-MSC經(jīng)靜脈移植入腦梗死大鼠體內(nèi),觀察植入細(xì)胞的紅色熒光是否會(huì)沾染宿主細(xì)胞以及和BDNF免疫熒光的雙標(biāo)記。結(jié)果不同濃度CM-Di I(1 000、200、100、20 nmol/L)均可成功標(biāo)記人BM-MSC,其中1 000、200 nmol/L濃度標(biāo)記24 h后觀察標(biāo)記效率均達(dá)到98%以上,比100、20 nmol/L標(biāo)記的效率明顯升高(P0.01)。CM-Di I標(biāo)記后的人BM-MSC與GFP標(biāo)記的293T細(xì)胞共培養(yǎng),7 d之內(nèi)沒(méi)有發(fā)現(xiàn)紅色熒光沾染綠色的293T細(xì)胞。移植后第3天,靜脈植入腦梗死大鼠的標(biāo)記細(xì)胞的紅色熒光沒(méi)有沾染宿主腦內(nèi)神經(jīng)元,而且可以和BDNF的綠色熒光形成雙標(biāo)記。結(jié)論 CM-Di I可以高效地標(biāo)記人BM-MSC。采用200 nmol/L濃度標(biāo)記的人BM-MSC細(xì)胞在體外培養(yǎng)和移植入腦梗死大鼠后都未發(fā)現(xiàn)對(duì)周?chē)?xì)胞造成污染,植入細(xì)胞的CM-Di I熒光可以鑒定產(chǎn)生BDNF的人BM-MSC。
[Abstract]:Objective to observe whether fluorescent dye (CM-Di I) labeled human bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells,BM-MSC) could cause the contamination of peripheral cells. Whether the labeled cells transplanted into the cerebral infarction rats will cause the neurons in the host brain to be misstained; And whether human BMMSC can be identified to produce brain-derived neurotrophic factor (brain derived neurotrophic factor,BDNF). Methods Human BMMSC, labeled with different concentrations of CM-Di I was used to observe the labeling efficiency. Human embryonic kidney 293T cells transfected with green fluorescent protein (green fluorescence protein,GFP) and human BM-MSC cells labeled with CM-Di I were co-cultured. After 7 days, the red fluorescence of CM-Di I and whether 293T cells were double labeled were observed. Human BM-MSC labeled with CM-Di I was transplanted into cerebral infarction rats via vein to observe whether the red fluorescence of implanted cells would contaminate host cells and double labeling with BDNF immunofluorescence. Results different concentrations of CM-Di I (1 000 ~ 200 ~ 100 nmol/L) could be used to label human BM-MSC, successfully. The labeling efficiency of 1 000200 nmol/L was over 98% 24 h after labeling. The efficiency of human BM-MSC labeled with CM-Di I was significantly higher than that of 20 nmol/L (P0.01). After co-culture with 293T cells labeled with GFP, no green 293T cells were found in red fluorescent staining within 7 days. On the 3rd day after transplantation, the red fluorescence of labeled cells implanted into cerebral infarction rats was not stained with neurons in host brain and could be labeled with green fluorescence of BDNF. Conclusion CM-Di I can effectively label human BM-MSC.. Human BM-MSC cells labeled with 200 nmol/L in vitro and transplanted into cerebral infarction rats have not been found to pollute the peripheral cells. The CM-Di I fluorescence of implanted cells can identify the human BM-MSC. producing BDNF.
【作者單位】： 首都醫(yī)科大學(xué)宣武醫(yī)院細(xì)胞生物室;教育部神經(jīng)變性病重點(diǎn)實(shí)驗(yàn)室;廣州軍區(qū)廣州總醫(yī)院神經(jīng)內(nèi)科;蘇北人民醫(yī)院神經(jīng)內(nèi)科;首都醫(yī)科大學(xué)宣武醫(yī)院功能神經(jīng)外科;
【基金】：國(guó)家自然科學(xué)基金(81371377) 北京市科委健康培育項(xiàng)目(Z111107067311033) 北京市科技新星項(xiàng)目(2009B22)~~
【分類(lèi)號(hào)】：R743.3
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本文編號(hào)：2347296