| Bone marrow mesenchymal stem cells (BMSCs)also known as bone marrow stromal-derived stem cells, mesoderm development of the early cells exist in the bone marrow, in addition to hematopoietic stem cells other than another type of cells are multipotency,selfrenewal and easily to be harvested. In addition, they have the unique ability to escape immune recognition and to inhibit different functions of the immune system.Under appropriate circumstances, such cells can be directly induced to differentiate into osteoblasts, the bone cells into chondrocytes, fat cells, tendon cells, muscle cells, hepatocyte-like cells, nerve cells and other tissue cells. BMSCs multilineage differentiation potential characteristics coupled with the growth of adherent cells in vitro easily separated and amplified, and also easily transferred and expression of exogenous gene, so BMSCs by the medical profession’s favour, it becomes a kind of ideal cells to treat tools and gene therapy to target cells. It heralds the regenerative medicine and tissue engineering precedent.But the content of mesenchymal stem cells in the bone marrow is very low, Given that dramatically lower quantity, it has been reasonable to investigate how to get efficient generation, purification, and expansion of BMSCs.Objective To study the effects of different micro-environments on the growth of rat BMSCs and improve the methods of bone marrow stromal stem cells in vitro. Using Hoechst33342to labelbone marrow mesenchymal stem cells, observing the fluorescence duration and cell markers of cell proliferation,lay a foundation for MSCs transplantation in vivo.MethodsSince6-8W age SD male rats femoral, tibia and fibula extraction in bone marrow stromal stem cells. Adopted the bone marrow stick wall of training. The cells were cultured under the following serum microenvironment:The primary cells of autoserum group were cultured with autoserum, changing the medium with fetal bovine serum after passage. The primary cells of homogeneity foreign serum group were cultured with homogeneity foreign serum, changing the medium with fetal bovine serum after passage. The primary cells of fetal bovine serum group were cultured with fetal bovine serum, and cultured with fetal bovine serum after passage. The primary cells of Dulbecco’s modified Eagle’s medium (DMEM) group were cultured with serum-free DMEM, changing the medium with f ovine serum after passage. Analysis of biological characteristics of mesenchymal stem cells in the bone marrow and preliminary identification it from the following aspects:The morphologic changes in BMSCs were detected under an inverted phase contrast microscope in different serum microenvironment, growth curve were measured and flow cytometry. Surface markerCD34and CD44expression was analyzed by flow cytometry. Observed the labeling rate of bone marrow mesenchymal stem cells Hoechst33342.The result of the application of the above data SPSS17.0software for statistical analysis of data.ResultsCultured rat serum and allogeneic serum microenvironment cells were highly homogeneous, spindle-shaped colony growth, the degree of integration than other groups,the days of the first passages was less than other groups. The cells were cultured under serum-free microenvironment scarce, only slightly colony type growth.Doubling rate was fastest in the growth curve of autoserum group, followed by homogeneity foreign serum group and fetal bovine serum group. However, no doubling phenomenon was found in the DMEM group.Flow cytometric detection contain serum medium under the culture condition of the third generation of the cells CD44positive expression rate of90%or more, CD34positive expression rate under2%. And in serum-free culture conditions CD44positive expression rate at65.79%.This shows that under the environment of serum-free micro cultured cells purity was significantly lower than the serum micro environment with the training of cells. rMSCs labeled by Hoechst33342nucleus are displayed blue under UV excitation light and Hoechst33342labeling rate of almost100%. rMSCs marked on the Hoechst33342can still continue to value-added growth.With the the division algebra increase, the fluorescence intensity gradually weakened, faint fluorescence is still visible after4weeks.ConclusionsThe morphology of the cells cultured in the rats of the volume fraction of10%autologous serum microenvironment culture conditions good uniformity, integration and high, the first passage time is short, rapid multiplication rate. The above results suggest that the rat serum microenvironment culture conditions more conducive to the mesenchymal stem cells in the bone marrow adherent, value-added and purification. Labeled BMSCs effect Hoechst33342vitro labeling high and no significant effects on cell growth of value-added, prompted Hoechst vitro BMSCs marker research as lay the foundation for MSCs vivo transplantation mark vivo tracking. |
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