| Background:Mesenchymal stem cells(MSCs) is a kind of stem cells derived from early embryonic mesoderm and ectoderm pluripotent. They are widely distributed in the body. Bone marrow mesenchymal stem cells (BMSCs) have many abilities like hematopoiesis support, self-renewal, multiple differentiation, immune regulation and secretion. BMSCs have great potential in the treatment of tissue regeneration, wound repair, and a wide variety of diseases. BMSCs is a group of heterogeneous cells. The cell surface antigens of subgroups differ. Thus, the specific surface marker of BMSCs is controversial. BMSCs have a strong ability of self-renewal. BMSCs have an S-shaped growth curve. BMSCs have good growth characteristics before the 7th generation. BMSCs have a multiple differentiation potential. BMSCs can differentiate into a variety of non-hematopoietic tissue cells under different induced conditions, such as the osteoblasts, chondrocytes, adipocytes, myoblasts, astrocytes and so on. In addition, BMSCs can also support hematopoiesis, regulate immune and secrete some factors. They include transforming growth factor-β1(TGF-β1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), matrix metalloprotein-9 (MMP-9), interleukin-10 (IL-10) and so on.In addition to these excellent properties, BMSCs are easy to be extracted, easy to be proliferated in vitro and easy for the introduction and expression of genes. Therefore, BMSCs are expected to be a powerful tool for cell therapy and gene therapy. BMSCs will play an important role in the treatment of non-hematopoietic deficiency diseases, degenerative diseases and genetic diseases. At present, autologous stem cell transplantation is an important method of clinical application of BMSCs. It has the following advantages.1. The cells come from each patient’s own, and the donor source is adequate.2. No immunological rejection will be provoked.3. The cost of treatment is relatively low.4. No ethical problems will be produced. Hematopoiesis support is one of the main physiological roles of BMSCs in vivo. They say, the co-transplantation of autologous BMSCs and peripheral blood hematopoietic stem cells can be used for the treatment of malignant hematological diseases. A large number of reports confirm the good performance of autologous BMSCs transplantation in the trauma repair of bones, cartilages and joints. For instance, the transplantation of autologous BMSCs compound with allogeneic bones can be used for the treatment of canine segmental mandibular defect; the transplantation of autologous BMSCs compound with alginate carrier can be used for the treatment of cartilage defect in rabbits; the injection of autologous BMSCs associated with fibrin glue is good for the postoperative recovery of the reconstruction of anterior cruciate ligament in rabbits; the infusion of autologous BMSCs via medial femoral circumflex artery can be used for the treatment of ischemic necrosis of femoral head. Some studies found that autologous BMSCs can also be used for the repair of tissue traumas or tissue defects. For instance, autologous BMSCs can be used for the treatment of skin wounds in nude mice; autologous BMSCs can help to rebuild the damaged ocular surface, alleviate edema and inhibit angiogenesis; autologous BMSCs can be used for the repair of canine prtial cystectomy. In addition, autologous BMSCs can also play an important role in the treatment of hypoxic ischemic injuries. Although the mechanism is not very clear. Local injections of autologous BMSCs can be used for the treatment of acute myocardial infarction (AMI) in miniature pigs; the infusion of autologous BMSCs via coronary artery during the percutaneous coronary intervention (PCI) can inhibit the ventricular reconstruction and improve the heart function in AMI patients; local injection of autologous BMSCs can improve the blood infusion of lower limb ischemia; autologous BMSCs can alleviate the local symptoms of diabetes foot. Autologous BMSCs can also be used for the repair of the nervous system. For instance, the Injection of autologous of BMSCs via veins can alleviate the nerve dysfunction cased by central nervous system injury; the Injection of autologous of BMSCs via veins can be used for the treatment of cerebral infarction; the infusion of autologous BMSCs into ventricle haematoma intracavity contributes to the postoperative recovery of cerebral hemorrhage; autologous BMSCs can be used for the treatment of Alzheimer’s disease (AD); autologous BMSCs can be used for the repair of peripheral nerve injury; the transplantation of autologous BMSCs combined with peripheral nerves can be used for the treatment of spinal cord injury. However, functional neurons should have not only the typical morphologies and surface markers of typical neuronal cells but also excitability, the ability to form synapses and the ability to produce synaptic potentials. Thus, the neural differentiation of BMSCs is still controversial. Autologous BMSCs can also play a role in the treatment of different organ-dysfunction caused by various reasons. For instance, autologous BMSCs can be used in the treatment of acute hepatic failure in pigs; autologous BMSCs transplantation can promote the liver regeneration in SD rat with hepatocirrhosis; autologous BMSCs can alleviate the acute rejection of liver transplantation in allogeneic miniature pigs; autologous BMSCs transplantation can be used for the treatment of end-stage liver disease; autologous BMSCs transplantation can promote the recovery of renal structure and function after a renal ischemia reperfusion injury in rabbit; autologous BMSCs transplantation can protect the rats from damage in the early stage of severe acute pancreatitis; autologous BMSCs transplantation combined with insulin or anti-diabetic drug therapy can effectively decrease the patients’blood glucose and alleviate their clinical symptoms.Autologous BMSCs transplantation is an effective way to give full play to the strong functions of BMSCs, such as tissue repair, immune regulation and hematopoiesis support. Therefore, many scholars are devoted to extent BMSCs to more treatment fields. However, autologous BMSCs transplantation is affected by the donor age. BMSCs are experiencing a physiological aging, along with the human age. According to the oxidative stress theory, the body’s antioxidant system becomes imbalance, with the growth of age. With a large amount of active oxygen released and accumulated, cells gradually become aging and even apoptotic, and organ decline or organ failure occurs accordingly. BMSCs could not escape this rule as well. A study finds that embryonic BMSCs have strong proliferation abilities and no contact inhibition. But its secretion function is poor. In adult BMSCs, the biological characteristics of which extracted from people aged 0 to 20 years old are optimal. Therefore, people aged 0 to 20 years old will be a good source of BMSCs. Some people think that the oxidative stress theory cannot perfectly explain the phenomenon of cell aging. Maybe we can find a method to improve the biological characteristics of BMSCs extracted from aging individuals.The function of hypoxia treatments on BMSCs is an interesting subject. It seems to give people some hopes. Some people speculate that as the oxygen concentration of bone marrow is lower than that of other tissues. Hypoxic environment should be more conducive to the growth of BMSCs. Some experiments confirm that:sustained hypoxia of 3%O2 could promote the proliferation of human BMSCs; sustained hypoxia of 1%O2 can promote the proliferation of human BMSCs, the colony formation, the expression of hypoxia-inducible factor-1, (HIF-1α), the secretion of stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF), and inhibite the osteogenic differentiation of BMSCs. But the opposite conclusions are:hypoxia can promote the proliferation of BMSCs cultured in the serum medium, but showed an inhibition of proliferation in serum-free medium; the concentration of 1%O2 is a non-lethal condition, but can temporarily suppress the proliferation of rat BMSCs. Therefore, people think that effects of hypoxia treatment on BMSCs depend on the degree of hypoxia, the duration of hypoxia, the concentration of serum in the medium, the species of the donor and so on.Objective:To repeat the hypoxia treatment on the BMSCs of C57 mice, and exert this treatment on the poorly-proliferated BMSCs extracted from aging C57 mice, in order to promote its proliferation. For moderate hypoxia may promote the proliferation of BMSCs.Method:The MIC101-type hypoxia cells-culture system of Billups-rothenberg’s company was consulted and transformed. A novel self-made hypoxia culture device was manufactured. The design principle of this device is as follows:The mixed gas containing 5% CO2 was introduced from the ordinary culture box which was used as a gas source. The O2 of gas was consumed by the "activated-iron vermiculite activated-carbon battery" before transferred to BMSCs. In comparison with the MIC101-type hypoxia cells-culture system, this device doesn’t have to connect a steel cylinder. It is flexible, convenient, safe and reliable. This device can ensure the adequate exchange of gases and ensure the accuracy of results displayed by the oxygen analyzer. This device can keep the concentration of CO2 stable, when O2 was consumed at the same time. According to the data provided by The Jackson Laboratory, C57 mice aged 3 to 6 weeks were equivalent to human aged 20 to 30 years old, C57 mice aged 18 to 24 months were equivalent to human aged 56 to 69 years old. C57 mice aged 3 to 6 weeks were chosen as the young group, while C57 mice aged 18 to 24 months were chosen as the aging group. BMSCs were extracted from the young C57 mice aged 3 to 6 weeks by whole bone marrow adherence method. The BMSCs was expanded to the 6th generation. And some of the cells were frozen. The 6th-generation BMSCs of young C57 mice were cultured. The morphology and the number of cells were observed under the microscope. The growth curve was drawn in 5 consecutive days, which can confirm the strong proliferation ability of BMSCs extracted from young C57 mice. The osteogenic differentiation and adipogenic differentiation of 6th-generation BMSCs of young C57 mice were induced, which can confirm the good ability of multilineage differentiation kept by the BMSCs of young C57 mice, which can confirm the feasibility of whole bone marrow adherence method we use. The 6th-generation BMSCs of young C57 mice were treated under hypoxia of 5%O2 for 30 minutes and then routinely cultured for 8 hours. The cells were stained by crystal violet, and the morphology and the number of cells were observed under the microscope. BMSCs were extracted from the aging C57 mice aged 18 to 24 months by whole bone marrow adherence method. The cells were stained by crystal violet, and the morphology and the number of cells were observed under the microscope. The growth curve was drawn in 12 consecutive days, which can confirm the poor proliferation ability of bone marrow adherent cells of aging C57 mice. It is hard to extract BMSCs from aging C57 mice. The bone marrow adherent cells of aging C57 mice, routinely cultured for 72 hours, were treated under hypoxia of 5%O2 for 30 minutes and then routinely cultured for 24 hours. The cells were stained by crystal violet, and the morphology and the number of cells were observed under the microscope.Results:The BMSCs extracted from C57 mice aged 3 to 6 weeks was expanded to the 6th generation. The multilineage differentiation of BMSCs extracted from the young C57 mice was kept. The cells flaked off under hypoxia of 5%O2 for 30 minutes. The proliferation ability of bone marrow adherent cells of aging C57 mice was poor. Only one colony formed in 12 days. The number of adherent cells decreased obviously under a hypoxia of 5%O2 for 30 minutes and a routinely culture for 24 hours. And no novel colony formed.Conclusion:BMSCs can be successfully extracted from the young C57 mice by whole bone marrow adherence method. The 6th-generation BMSCs of young C57 mice grew well, and the multilineage differentiation ability was kept. But hypoxia treatment can damage the proliferation of BMSCs extracted from the young C57 mice. It is hard to extract BMSCs from aging C57 mice. Hypoxia treatment can damage the proliferation of bone marrow adherent cells extracted from the aging C57 mice. |
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