Isolation And Culture Of BM-MSCs And The Mechanism Of Homing Ability Influenced By Culture

Objective:Bone marrow mesenchymal stem cells (BM-MSCs) are the multipotent cells with the function of undifferentiated state for long time and with proliferation ability in vitro, easy to be cultured, specific migratting to sites of injury or tumor, differentiate to the bone tissue, heart muscle, cartilage tissue, muscular tissue and so on. It has a certain therapeutic effect on acute myocardial infarction, cerebral infarction, acute and chronic liver failure, indicating its prospect of widely application. However, MSCs in bone marrow can hardly meet the needs of therapy, it often need to be cultured in vitro to meet the requirement. Previous researchs have shown that the ability of MSCs homing after transplantation will decrease along with culture and passage in vitro. And the decline of homing abilities will further influence the implantation of MSCs in the target tissue, thus seriously affect the repair effect on it. Therefore, to improve the ability of MSCs homing to the target tissue is the key to enhance therapeutic effect of stem cell transplantation. The CXCR4, CXCR6, CXCL12(SDF-1), CD44and other cytokines could be detected on the surface of MSCs, and what’s the important, the CXCR4/SDF-1axis has been confirmed to play an important role in MSCs homing.This experiment was aimed to culture and proliferate the MSCs from bone marrow of healthy human in vitro, and to observe the growth characteristics and the expression of CXCR4, CXCR6, CXCL12, CD44between different generations of the MSCs, in order to investigate the effect mechanism that how the passage in vitro affect the homing ability of MSCs.Methods:Isolating the MSCs from bone marrow by Ficoll density gradient centrifugation, then purify MSCs with the characteristics of grow adhered the wall. The MSCs were cultured into the seventh generations with the normal cultural condition, and the morphological features of the3rd,5th and7th generation MSCs were abserved. Using MTT chromatometry to detect the growth feature of the3rd,5th and7th generation MSCs, and make the growth curve. Using Real-time PCR to detect the expression of CXCR4, CXCR6, CXCL12, CD44in3rd,5th and7th generation MSCs, record the Ct value of target gene and (3-actin in each sample, then calculate correspondingΔCt(ΔCt=Ct target-Ct β-actin), andΔΔCt(ΔΔCt=ΔCta-ΔCtb),finally calculated2-ΔΔCt to get the relative value of each target gene. The results were analyzed with Paired-samples T-Test with GraphPad Prism5software package. The p<0.05was considered statistically significant.Results:The characteristics of primary MSCs were elongated spindle cell morphology, homogenization, growth fast, and have strong colony forming ability, which can be passaged about from1week to2weeks. The cell growth rate is accelerated apparently, adhered the wall completely, stretch and back to be spindle cells in24hours, growth with shorter latency (1-2), lead to complete fusion in4-7days. In vitro, the ability of growing is strong, but following the passage, the cell morphology became wider and shorter. And the proliferation rate, the overall proliferated multiple and the expression of homing related factors decreased following the passage. The expression of CXCR4, CXCR6, CXCL12(SDF-1) and CD44of MSCs will decrease following the passage. For CXCR4, the expression of the5th generation MSCs is53.18%of the3rd generation, while that the expression of the5th generation MSCs is only27.04%of the3rd generation, indicating that the expression of CXCR4in the3rd,5th and7th generation of MSCs has statistics significance compared with each other(p<0.05). For CXCL12, the expression of the5th generation MSCs is56.59%of the3rd generation, while that the expression of the5th generation MSCs is only27.41%of the3rd generation, indicating that the expression of CXCL12in the3rd,5th and7th generation of MSCs has statistics significance compared with each other(p<0.05). For CXCR6, the expression of the5th generation MSCs is76.37%of the3rd generation, while that the expression of the5th generation MSCs is only42.12%of the3rd generation, in which the expression difference of CXCR6between3rd and7th,5th and7th generation of MSCs has statistics significance (p<0.05), and the expression of CXCR6of the5th generation was decreasing when compared with the3rd generation, but with no significant, it should to be further study by increasing the sample. For CD44, the expression of the5th generation MSCs is53.99%of the3rd generation, while that the expression of the5th generation MSCs is only23.56%of the3rd generation, in which the expression difference of CD44between3rd and5th,3rd and7th generation of MSCs has statistics significance (p<0.05), and the expression of CD44of the7th generation was decreasing when compared with the5th generation, but with no significant, it should to be further study by increasing the sample.Conclusion:The mesenchymal stem cells from bone marrow were easy to culture and amplificate in vitro. The ability of multiplication is strong, but the proliferation rate, the overall proliferated multiple and the expression of homing related factors will decrease following the passage. All above results showed that the ability of MSCs homing was decreasing following the passage, and may be relevant with the lower expression of CXCR4, CXCR6, CXCL12and CD44in cultured MSCs.