| Objectives1. To establish the method of separation, purification and identification of the rat bone marrow mesenchymal stem cells (MMSCs) in vitro. To observe the cell morphology and growth of MMSCs of different generations in vitro.2. To explore the effects of ligustrazine on the proliferation of MMSCs which culture in vitro.3. To detect the effects of Ligustrazine on the SDF-1 mRNA expression of MMSCs in vitro.4. To study and research the differentiation of MMSCs into cardiomyocyte-like cells in Ligustrazine medium in vitro.Methods1. MMSCs were isolated from adult bone marrow cells by whole bone marrow method and adherent cells separation method in vitro. Phase-contrast microscope and immumofluorescence method were used for the identification of MMSCs. Cell growth message from first to fifth generation were got by the MTT method.2. MMSCs of the 3rd generation were cultured 24h in ten different concentrations of ligustrazine. The effects of ligustrazine on the proliferation of MMSCs were reflected by MTT.3. The 3rd passage of MMSCs was chosen to undergo the experiments. MMSCs were cultured in five groups, as follows:group of control (no Ligustrazine added), three groups of Ligustrazine treatment (0.08mmol/l,1.28mmol/l, 5.12mmol/l of Ligustrazine were added, respectively), group of 5-azacytidine (5umol/l 5-azacytidine were added). Cultured MMSCs were harvested at 1d,3d, 5d and 7d. The expression of SDF-1 mRNA were analyzed with reverse transcription polymerase chain reaction.4. MMSCs were cultured in four groups, as follows:group of control (no Ligustrazine added), two groups of Ligustrazine treatment (0.08mmol/l, 1.28mmol/l of Ligustrazine were added, respectively), group of 5-AZA (5umol/l 5-AZA were added). MMSCs were treated with 5-azacytidine(5umol/l) for 1d or Ligustrazine (0.08mmol/l,1.28 mmol/1) for 10d. The cells were harvested at days 1,7and 21. The expression of CXCR-4, Nkx2-5, Gata-4 and MHC mRNA were analyzed with reverse transcription polymerase chain reaction.Results1. The cell growth of MMSCs:after primary culture for lday, some round cell adhered. The number of adherent cells increased significantly after cultured for 3 days, the cell morphology is long spindle. Cultured for 10 days, cells closed to 80-90% of integration. The passaged MMSCs were spindle-shaped and grow as the cell colony. Cell morphology is more uniform, which were whirlpool-like. CD34 was negatively expressed, CD44 was positively expressed.2. In the low concentration range (0-0.08mmol/l), the role of Ligustrazine on cell proliferation in a concentration-dependent, the higher the concentration the more obvious, but to reach 1.28mmol/l, with the further increase in the concentration of Ligustrazine, but their proliferation was inhibited. The strongest cell proliferation was 0.08mmol/l Ligustrazine on MMSCs. Compared with MMSCs, P<0.01.3. After 1d cultured, MMSCs of no induction and 5-AZA induced not obviously expressed SDF-1mRNA, but as the cultured time, SDF-1 mRNA expression increased. While MMSCs were intervened 1d by 0.08mmol/L and 1.28mmol/l concentration of Ligustrazine, the expression of SDF-1 mRNA has emerged. In the third day, different concentrations of Ligustrazine can significantly accelerate the expression of SDF-1 mRNA. In the fifth and seventh day,0.08mmol/l Ligustrazine promoted SDF-lmRNA expression, that effect was stronger than other group. In the 7d, compared with MMSCs,0.08 mmol/l Ligustrazine group was statistically significant (P<0.01).4. Increased with the incubation time, the amount of CXCR-4 mRNA expression of MMSCs group is is reduced, while the expression of CXCR-4 mRNA of 0.08 andl.28 mmol/L Ligustrazine group is the role of time-dependent enhancement. In the 21d, compared with MMSCs,0.08 mmol/l Ligustrazine group, 1.28 mmol/l Ligustrazine group were statistically significant (P<0.01). In the 1,7 and 21d, MMSCs group,0.08 mmol/l Ligustrazine group,1.28 mmol/l Ligustrazine group and 5-AZA group were expressed internal reference GAPDH, but not the expression Gata-4, Nkx2-5 and MHC mRNA.Conclusions1. The combination of whole bone marrow method and adherent cells separation method could successfully isolate MMSCs in vitro. The cell vitality and purity of MMSCs was high. This system can be used as conventional method to culture the MMSCs.2. The effect of ligustrazine on cell proliferation of MMSCs was dose-related.3. Ligustrazine may reinforce the expression of SDF-1 mRNA of MMSCs, and may contribute to homing MMSCs to the local of myocardial infarction.4. Ligustrazine can promote MMSCs expressing CXCR-4 mRNA. However, experiments in vitro have not yet found evidence of Lig differentiate MMSCs into cardiomyocyte-like cells.
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