| ObjectiveDiscuss the changes of the biological characteristics of mesenchymalstem cells(BMSCs) in the microenvironment of C6glioma cells and build awell model for study of BMSCs in nude mouse bearing C6glioma, in orderto lay the experimental foundation of the clinical security application ofBMSCs.Material and Methods1. Experimental animalsThe weight range of SPF SD rats was between forty gram and sixtygram, and the age range was between three weeks and four weeks; Theweight range of the female nude mouse was between eighteen gram andtwenty gram, and the age range was between five weeks and six weeks, thistwo kinds of animal were purchased from the Animal Experimental Centerof Chongqing Medical University.2. Sources of Cells Isolated and cultured BMSCs by whole bone marrow adherentcultivation methods; C6glioma cells and293cells were given by theInstitute of Oncology of the Key Laboratory of Child Development andDisorders founded by Ministry of Education, Children’s Hospital ofChongqing Medical University.3. Experimental GroupsThis study had two parts. The first part had three groups:(1) Theexperimental group was CM-Dil labeled BMSCs(CM-Dil+BMSCs) beingdirect-co-cultured with C6glioma cells;(2) The positive control group wasC6glioma cells being single cultured;(3) The negative control group wasBMSCs being single cultured. The second part had two groups:(1) Theexperimental group was the nude mouse bearing C6glioma that had beeninjected Ad-GFP+BMSCs;(2) The positive control group was the nudemouse bearing C6glioma.4. Methods4.1Adopt whole bone marrow adherent cultivation to isolate andculture SD rat BMSCs; Observed the shape and the states of cells byinverted microscope.4.2Detected the expression of GFAP, CD105, CD45, GFAP proteinusing IF assay.4.3FCM was used to detect the labeling efficiency and to sort BMSCsafter being direct-co-cultured with C6glioma cells. 4.4RT-PCR was applied to detect the expressions of GFAP, PTEN,Bcl-xl, CyclinD1, CD90, CD105genes in each group.4.5HE assay was used to observe the tissue and cell structure oftumor.Result1. It was showed by IF that more than ninety percent of the thirdgeneration of BMSCs express CD90, CD105, and don’t express CD45. Theexpression of GFAP of BMSCs in experimental group was increasedcompared with negative control group, and the expression of CD90, CD105were decreased.2. It was showed by FCM that the labeling efficiency of CM-Dil was98.3%. And the corresponding infection efficiency of different volumes(0ul,5ul,6ul,8ul,10ul,15ul) of Ad-GFP were0.22%,8.58%,29.03%,36.54%,42.27%,51.43%.3. Gene expressions of GFAP, PTEN, Bcl-xl, CyclinD1genes ofBMSCs in experimental group were significantly increased compared withnegative group(P＜0.05)，while the gene expressions of CD90and CD105genes were significantly decreased(P＜0.05).4. BMSCs were still alive after being injected in the nude mousebearing C6glioma, and BMSCs could be found by the green fluorescentsignals of Ad-GFP. Conclusion1. In the microenvironment of C6glioma cells, the biologicalcharacteristics of BMSCs were changed, and the tumor associated geneshad a high level expression in BMSCs. It was showed that BMSCs had thepotential malignant transformation tendency in the microenvironment ofC6glioma cells.2. BMSCs, labeled by Ad-GFP, could alive in nude mouse bearing C6glioma and could be detected by the fluorescent signals after beingorthotropic implantation. It will be a basic model for the study of malignanttransformation of BMSCs in C6glioma growing in nude mouse. |
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