The Use Of Ganglioside GD2in The Isolation And Identification Of Rat Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs), also known as bone marrow stromal cells, a bone marrow adult stem cells, accounting for less than0.01%of the proportion of bone marrow-derived mononuclear cell populations. Had not found his ideal specific markers for the identification of BM-MSCs for a long time, usually use a variety of joint surface molecular for identification of BM-MSCs cultured in vitro. A newly discovered molecule ganglioside GD2was used to identify human bone marrow mesenchymal stem cells (BM-MSCs) and umbilical cord mesenchymal stem cells (UC-MSCs). But does rat BM-MSCs also express GD2has not been reported, This experiment studied the GD2expression in rat BM-MSCs, provide a new way to specific separation and accurate identification of rat BM-MSCs; We also used the percoll density gradient centrifugation method for optimizlation the separation of rat BM-MSCs, to obtain a high purity BM-MSCs for the follow-up study.Part1The research of rat bone mesenchymal stem cells expressing Ganglioside GD2Research methods:(1) The isolation and culture of BM-MSCs in vitro:use the whole bone marrow adherence method culture the BM-MSCs separate from8-10week-old SD rat in the a-MEM medium containing15%fetal bovine serum,1×109cells/ml were seeded and cultured at37℃,5%CO2, saturated humidity incubator.(2) Detect the mRNA of GD2synthetase:rat embryo fibroblasts (REFs) and mouse melanoma cells (B16F10) were used as positive and negative controls, using RT-PCR assay, the first, third and the fifth generation of BM-MSCs all express the mRNA of GD2synthetase.(3) BM-MSCs surface GD2molecule detection:use the cell immunohistochemical method to detect the first, third and fifth generation of BM-MSCs surface GD2expression.(4) Application of flow cytometry to detect the first, third and fifth generation of BM-MSCs surface molecular group (CD3, CD29, CD45, CD90) and GD2expression, flow-test results for comparative analysis.Results:(1) the subculture of BM-MSCs:the cycle of whole bone marrow adherence method culture the BM-MSCs from the primary to the subculture was long, need8-10d before pass-generation, after passage culture the cycle was shortened, every three days can be passaged.(2) GD2synthase mRNA expression of the BM-MSCs:GD2synthase mRNA was expressed in primary and subcultured cells, as the passage culture, GD2synthase mRNA expression level was increased, then passaged culture GD2synthase mRNA expression hold in a stable level.The REFs negative expression and B16F10positive expression.(3) BM-MSCs membrane surface GD2:Immunocytochemistry results showed that GD2was surface expressed in the first, third and fifth generation of BM-MSCs.(4) Surface molecule expression in the BM-MSCs:flow-test results of the first, third and fifth generation, CD90-positive cell ratio, respectively were59.2%,87.1%,98.2%; CD29-positive cell ratio, respectively were69%,80.9%,99.9%; CD45-positive cell ratio, respectively were13.8%,5.1%,1.1%; CD3-positive cells ratio of the first and third generations were4.4%,0.8%; GD2-positive cell ratio, the first, third, fifth, seventh generations were87.3%,48.2%,36.2%,18.9%.Conclusion:(1) The bone marrow adherent method can isolate and culture BM-MSCs in vitro successfully.Primary and subculture of rat BM-MSCs both expressing GD2synthase mRNA and membrane surface GD2, which correspondence with the human and mouse, suggesting that GD2was highly conserved between species.(2) GD2synthase mRNA expression level of the subculture cells was increased, while GD2-positive cell ratio by flow-test was decreasing, GD2synthetic enzyme is a rate-limiting enzyme in the the GD2biosynthetic process. In vitro, GD2synthase did not increase while the amount of mRNA expression levels increased. The specific regulatory mechanisms needs further study.(3) The GD2-positive cell ratio decreasing during the subculture of rat BM-MSCs, which consistent with that GD2can be used as a specific marker for identification the mouse and human MSCs in early stage.(4) With the subculture of rat BM-MSCs, CD90and CD45-positive cell ratio was increased, CD90and CD45double positive cells ratio of the fifth generation was up to98%, the high purity of cells can be used follow-up research.Part2The application of GD2for separation and identification of rat bone marrow mesenchymal stem cellsMethods:(1) Using Percoll density gradient centrifugation to separate the BM-MSCs, RT-PCR detect GD2synthase mRNA expression in the different density gradient layer cells (1.0448g/ml,1.0578g/ml,1.0708g/ml,1.0838g/ml,1.0968g/ml),which will provide a basis for quick access to acquire the BM-MSCs.(2) The BM-MSCs acquired by density gradient centrifugation was cultured in vitro.The morphology and growth characteristics of the cells was observed, using the the tetramethyl MTT analysis cell growth curve, compared with the cell acquired by the whole bone marrow adherence method.(3) Use the flow cytometry detect the expression of CD3, CD29, CD45, CD90molecules, analysis its purity.Results:(1) RT-PCR results showed that GD2synthase mRNA only expressed in the cell layer whose density is1.0708g/ml.(2) Cells obtained by density gradient centrifugation, start proliferation slow, after a period of adaptation, after about6-7d original culture can be carried out subcultured, the subculture cycle was2-3d, and similar with the subculture cycle of whole bone marrow adherent method. MTT analysis found that cell viability increased after passage15h.(3) Subcultured cells obtained by density gradient centrifugation, flow-test the generation of cell surface molecules CD90, CD29, CD45, CD3positive cell ratio were88.5%,99.9%,0.2%,0.1%, showed that cell purity was higher than the bone marrow adherent method.Conclusion:Percoll density gradient centrifugation can be quickly obtained BM-MSCs, subject to centrifugation injury the primary culture showed slow proliferation, about6-7d can be subcultured, the passaged cell viability increased; flow-test the CD90, CD29, CD45, CD3positive cell ratio was higher than the bone marrow adherent method. MTT analysis showed that the subcultured cell viability was higher than the whole bone marrow adherent method.