| Objective: To seek effect of weeks on rat BMSCs cultured in vitro, toobserve cell morphology, and to assess surface marker. Provide adequate sourceof seed cells for tissue engineering. The optimal week old SD rat’s BMSCs wasinduced apoptosis by dexamethasone, conduct research on detection of apoptoticcells, to explore the feasibility of Raman spectroscopy for the detection ofapoptosis.Methods: Take1weeks,4weeks,8weeks SD rats, each5only, male orfemale, Aseptic conditions take limbs long bone of rats, use L-DMEM whichcontain fetal bovine serum(FBS) rush out of the bone marrow. BMSCs from1week,4weeks and8weeks old rats were isolated, cultured and purified by thewhole bone marrow adherence method, for morphology observations, the growth curve was drawn, cell surface markers were assessed by flow cytometry.Making use of single cell laser tweezer Raman spectroscopy and flow cytometrytechnology, it conducts the apoptosis of bone mesenchymal stem cells underdexamethasone-induced concentrations of10-3,10-4,10-5mol/L in24h and10-3mol/L in0h,6h,12h,24h,36h, and48h.For each group fo theconcentration-time random testing60BMSCs Raman spectra, turn each BMSCsspectrum’s Raman spectral data into ASCII data. After background subtraction,smoothing, baseline correction on the data, to obtain the real average Ramanspectrum.The spectrum of BMSCs treated with Micro-Origin8.1softwareprocessing, the interception of600~1800cm-1fingerprint region, access to thereal spectrum of BMSCs for finding and analyzing BMSCs constitute the mainpeaks of Raman spectroscopy. The data conducts the principal componentanalysis (PCA) to compare similarities and differences of the analysis results ofRaman spectroscopy and flow cytometry.Results: BMSCs can be effectively harvested by the whole bone marrowadherence separation method. Cells were spindle cell-based, showing radialcolony arrangement. Passage to the third generation to be able to harvest higherpurity of BMSCs, BMSCs were positive for CD44and CD90, but negative forCD34and CD45. Compare cell culture time of reaching confluency betweenprimary and passaged cells of different weeks rats BMSCs, the time of smallweeks rat BMSCs is lesser than old weeks rat BMSCs’s.Cell culture time up to 80%confluency: primary cells were (5±0.7) days,(7.8±0.8) days,(10.2±1.1), the third passage cells were (3.8±0.4) days,(5.2±0.5) days,(6±0.7) days, the results were significant differences (P <0.05). The PCAanalysis showed that:(1) after24h treatment with different concentrations ofdexamethasone, the10-3mol/L dexamethasone showed the strongest apoptosis,which is similar to the flow cytometry results.(2) after36h,10-3mol/Ldexamethasone induced totally apoptosis of bone mesenchymal stem cells, whileafter48h, induction of apoptosis of flow cytometry was only72±2.6%; thePCA load spectra showed that, in apoptosis cells and normal cells, there are tensignificant differences of Raman peaks:714,785,939,1002,1097,1233,1255,1343,1446and1662cm-1, which are the signal peaks of nucleic acids, proteins,carbohydrates and lipids.Conclusion:1week,4weeks and8weeks old rats BMSCs after multiplepassages,1week rats BMSCs can maintain a better cell viability and a higherproliferation rate than4weeks and8weeks old rats BMSCs. To use1week oldrats to isolate, culture and purify BMSCs by the whole bone marrow adherencemethod can be more economical, faster and more efficiently to provide adequatesource of seed cells for tissue engineering.The results of Raman spectroscopyand flow cytometry showed that, compared with the traditional techniquemeasure, Raman spectroscopy can achieve more sensitive and more accuratedetection of apoptosis through the detection of intracellular macromolecules structure or content changes, such as intracellular nucleic acids, proteins,carbohydrates, lipids. These features of Raman spectroscopy detection techniquemakes it possible to be a new and effective method to detect early apoptotic cells,research the structure and content of the material change in apoptotic cells. |
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