CM-DiI标记人骨髓间充质干细胞并检测其移植后产生脑源性神经营养因子的实验研究
发布时间：2018-11-21 14:51
【摘要】：目的观察荧光染料(CM-Di I)标记人骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSC)后,是否会脱落造成周围细胞被污染,标记细胞经静脉移植入脑梗死大鼠后是否会造成宿主脑内神经元被误染色;以及是否可以鉴定人BMMSC产生脑源性神经营养因子(brain derived neurotrophic factor,BDNF)。方法应用不同浓度CM-Di I分别标记培养的人BMMSC,观察标记效率。将转染绿色荧光蛋白(green fluorescence protein,GFP)的人胚肾293T细胞和CM-Di I标记的人BM-MSC细胞共培养,7 d之后观察CM-Di I的红色荧光和293T细胞是否双标记。将CM-Di I标记的人BM-MSC经静脉移植入脑梗死大鼠体内,观察植入细胞的红色荧光是否会沾染宿主细胞以及和BDNF免疫荧光的双标记。结果不同浓度CM-Di I(1 000、200、100、20 nmol/L)均可成功标记人BM-MSC,其中1 000、200 nmol/L浓度标记24 h后观察标记效率均达到98%以上,比100、20 nmol/L标记的效率明显升高(P0.01)。CM-Di I标记后的人BM-MSC与GFP标记的293T细胞共培养,7 d之内没有发现红色荧光沾染绿色的293T细胞。移植后第3天,静脉植入脑梗死大鼠的标记细胞的红色荧光没有沾染宿主脑内神经元,而且可以和BDNF的绿色荧光形成双标记。结论 CM-Di I可以高效地标记人BM-MSC。采用200 nmol/L浓度标记的人BM-MSC细胞在体外培养和移植入脑梗死大鼠后都未发现对周围细胞造成污染,植入细胞的CM-Di I荧光可以鉴定产生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.
【作者单位】： 首都医科大学宣武医院细胞生物室;教育部神经变性病重点实验室;广州军区广州总医院神经内科;苏北人民医院神经内科;首都医科大学宣武医院功能神经外科;
【基金】：国家自然科学基金(81371377) 北京市科委健康培育项目(Z111107067311033) 北京市科技新星项目(2009B22)~~
【分类号】：R743.3
，
本文编号：2347295
[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.
【作者单位】： 首都医科大学宣武医院细胞生物室;教育部神经变性病重点实验室;广州军区广州总医院神经内科;苏北人民医院神经内科;首都医科大学宣武医院功能神经外科;
【基金】：国家自然科学基金(81371377) 北京市科委健康培育项目(Z111107067311033) 北京市科技新星项目(2009B22)~~
【分类号】：R743.3
，
本文编号：2347295
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