大鼠骨髓主要细胞成分的分离获取及其免疫调节作用

ISOLATING AND PURIFYING MAIN CELLS FROM RAT BONE MARROW

目的:建立分离获取大鼠骨髓主要细胞成分的方法,了解其免疫调节作用,为骨髓主要细胞成分输注诱导同种异基因移植物免疫耐受奠定基础。方法:采用密度梯度离心法、红细胞裂解法及细胞培养等方法,分离纯化供体大鼠骨髓细胞(donor bone marrow cells,DBMC)、骨髓间充质干细胞(mesenchymal stem cells,MSC)、不成熟树突细胞(immature dendritic cells,imDC),经光镜、电镜形态学、FCM测定imDC表面OX-62、MHC-Ⅱ、CD80和CD86表达、MSC表面CD80-CD86-和CD29表达等细胞表面标志的表型鉴定后,用MTT法检测DBMC、MSC和imDC对供受体混合淋巴细胞反应(Mixed lymphocytesreaction,MLR)的免疫调节作用。结果:分离获取大鼠骨髓主要细胞成分纯度可达85%-90%,活力>95%,均经光镜、电镜形态学、FCM等表型鉴定合格,其中,imDC表面OX-62、MHC-Ⅱ、CD80和CD86表达量分别是90.5%±16.4%、89.5%±6.2%、32.5%±9.2%和27.6%±8.3%;MSC表面CD80-CD86-和CD29表达量分别是86.068%±10.33%和61.77%±6.79%。MLR表明大鼠骨髓主要细胞成分对MLR均有显著抑制作用(P<0.05),DBMC对MLR具有供体特异性免疫调节作用。结论:建立的大鼠DBMC、MSC、imDC分离纯化方法简便易行,所获取的细胞活力、纯度高,可用于实验目的。DBMC、MSC、imDC对受体淋巴细胞增殖反应均有不同程度的免疫调节作用。

Objective：To isolate donor rat bone marrow cells （DBMC）, mesenchymal stem cells （MSC） and immature dendritic cells （imDC） from the rat bone marrow an analyze their immunologic regulatory effects. Methods：The rat DBMC, MSC and imDC from rat femora marrow cells were separated and purified by gradient centrifugation,cell culture,etc. The morphological character of the cultured MSC and imDC was observed under light microscope and electron microscope. Expression of CD molecules were detected by flow cytometry. The isolated DBMC, MSC and imDCs were co-cultured with allogenic spleen cells in the mixed lymphocytes reaction respectively. The spleen cells proliferation was measured by MTT colorimetry.Resolts：The isolated DBMC, MSC and imDCs had the typical morphological characterization, the expression rates of OX-62, MHC class Ⅱ molecule, CD80 and CD86 in imDCs were 90.5% ±16.4%, 89.5%±6.2%, 32.5%±9.2% and 27.6%±8.3% respectively, and the expression rates of CD80-CD86- and CD29 in MSC were 86.068%±10.33% and 61.77%± 6.79%. The isolated DBMC, MSC and imDCs could notably suppress the proliferation of allogenic spleen cells in MLR （P〈0.05）.Conclusion ：The cells obtained in this experiment possess phenotypical and functional properties of imDC and MSC respectively, and the method for isolating and culturing rat femora marrow-derived DBMC, MSCs and imDC has been established. Using these methods,we can obtain a number of highly purified DBMC, MSC and imDC, these cells have an inhibitory effect on allogeneic lymphocyte proliferation, which lay the foundation for further transplantation study.