| BackgroudCerebrovascular disease (Cerebral vascular diseases, CVD) refers to cerebrovascular accident caused by various diseases. Cerebrovascular disease which is one of the three major deadly diseases in most country can impair human’s survival and quality of life seriously, because of high morbidity, high mortality and high morbidity. It is a hot research how to prevent and treat cerebrovascular disease and how to maximize to reduce physical disabilities caused by cerebrovascular disease, especially how to reduce the physical disability after cerebrovascular disease is vitally important. Current treatments of cerebrovascular disease in clinic contain neurotrophic factor, physical agents and motor function exercise, but there is still no a good method to cure cerebrovascular disease. Along with the advancement of stem cell engineering, more and more scholars pay attention to the potential therapeutic effects of stem cells such as the use of stem cells to repair damaged tissues, replacement therapy in tissue injury, genetic defects and degenerative diseases. Bone marrow mesenchymal stem cells (BMSCs) are the most promising seed cells among stem cells.Mesenchymal stem cells which derive from mesoderm and can differentiate to adult cells can be found in mesenchyal tissue, especially in bone marrow mesenchymal. The bone marrow mainly contains hematopoietic stem cells and progenitor cells, only few BMSCs. On the one hand, BMSCs is one of the important microenvironment members to induce hematopoietic cells proliferation and differentiation and to regulate the hematopoiesis, adhesion and homing of hematopoietic cell; On the other hand, BMSCs can differentiate into mesodermal cell and even the ectoderm or endoderm cells. BMSCs can be harvested easily and proliferate massively to ensures the plentiful source of stem cells in vitro; BMSCs can avoid immune rejection problem and restrictions from the ethics. Due to the characteristics of BMSCs, many researches in stem cells focus on BMSCs.BMSCs transplantation is applied to many animal models of clinical diseases including cardiovascular disease, blood diseases, spinal cord injuries, traumatic brain injury and cerebrovascular and so on. There are a number of studies to apply the transplantation of BMSCs treating the stroke patients with serious sequelae. It can be concluded that BMSCs transplantation can improve the motor function of stroke patients. Although the therapeutical effects of BMSCs transplantation on cerebrovascular disease are approved, there are many problems in practical use such as migration way, the number of transplanted cells, the migration time, tracking of transplanted cells etc. Another more important problem is low survival rate after BMSCs transplantation into injured tissue. The BMSCs after transplantation will distribute everywhere except for targeted transplantation. The microenvironment changes after ischemia and anoxia will lead to the death of BMSCs after transplantation into the injured tissues. All these reasons restrict the use of BMSCs.Physical agents are an important method to treat the stroke known by clinical doctors. However the clinical application of current physical agents has its advantages and disadvantages, more potential physical agents are needed to reduce the morbidity of cerebrovascular disease. Infrasound defined as mechanical vibration wave with frequency below20HZ exist in environment widely. Infrasound with sound pressure above90dB has been proved damage on body, however infrasound with sound pressure less than90dB called low sound pressure infrasound seem safe. However there are fewer researches to study the biology effects of low sound pressure infrasound. In our previous researches, we found the low sound pressure infrasound have opposite biology effects contrast to high sound pressure infrasound. Physiological studies demonstrate that organs have its fixed frequency like8～12HZ for head,4～6HZ for chest,6～9HZ for abdomen,6HZ for pelvic cavity and5HZ for heart. The fixed frequency is within the range of low sound pressure infrasound. The mechanism of biology effects is biological resonance of organs and tissues cause by low sound pressure infrasound. Researches on biological effects of infrasound are made from the organs and tissues into the cell. The current studies on biological effects of infrasound on cells focus on the proliferation, cycle and apoptosis of cells. Our previous study proved that low sound pressure infrasound could promote the proliferation and inhibit the apoptosis of BMSCs in60min. It indicates the low sound pressure infrasound can improve the survival of BMSCs and seem to be a promising method to solve the practical problem of BMSCs transplantation.Although our pilot study conclude that low sound pressure infrasound can reduce the apoptosis rate and enhance the proliferation rate of BMSCs, our design is not perfect. In order to get detailed parameters, we make further study to know the biological effects of low sound pressure infrasound on BMSCs. Next we explore the relationship between the biological effects and expression of survivin.Method1. The BMSCs harvest and culture:After rats executed by cervical dislocation, femurs and tibias were aseptically excised and the epiphyses were removed. The bone marrow was flushed from the shaft with DMEM/F12. The marrow suspension was disaggregated by pipetting several times and cells were collected by centrifugation. Then the cells were cultured in DMEM/F12with10%fetal bovine serum (FBS, Gibco/USA) containing100ul/ml penicillin-streptomycin on25cm2cell culture flasks.2. The BMSCs subculture and purification:The BMSCs were purified by anchoring culture and passaged1:3for passage one and1:2for next passages. After each subculture, the BMSCs should mark P1, P2, P3and date clearly and then culture at37℃in CO2atmosphere. The experimental BMSCs should be under logarithmic phase.3. The measurement of outcome(1) Trypan-blue staining:Cells activity was detected by Trypan-blue staining before the intervention of infrasound to guarantee the high-activity of BMSCs to decrease experimental errors.(2) The measurement of proliferation and apoptosis:Passage3BMSCs were divided into experimental group treated by infrasound for60min,90minand120min and control group exposed to air for same durations. The proliferation and apoptosis of cells were measured three times repeatly.(3) The detection of survivin:the survivin of Passage3BMSCs was detected by immunofluorescence and qRT-PCR.Result1. The passage3cell activity was both above95%accorded with experimental requirements. Cell activity=the total number of living cells/(the total number of living cells+the total number of dead cells) x100%2. OD of experiment group:60min (1.560±0.075),90min (1.160±0.096),120min (0.930±0.069); OD of control group60min (1.239±0.025),90min (1.090±0.110),120min (0.926±0.027). The OD of experiment group in60min show significantly increase compared with control group in60min, a statistic difference P<0.05. The OD of experiment groups in90min and120min show no difference compared with the corresponding control groups. The experiment group OD of60min is the most among experiment group. All results indicate60min is the ideal duration to promote the proliferation of BMSCs.3. Apoptosis rate of experiment:60min (10.70±1.47),90min (21.33±1.93),120min (22.87±1.45); apoptosis of control group:60min (18.20±1.12),90min (22.93±1.79),120min (23.47±2.50). The apoptosis rate of experiment in60min show a decrease compared with control group in60min, a statistic difference P<0.05. Both90min and120min in experiment group and control have high apoptosis rate. The experiment group apoptosis rate in60min is the least among experiment group. All results indicate that60min is the ideal duration to inhibit the apoptosis of BMSCs.4.60mim is the ideal duration base on the result that the low sound pressure infrasound can promote the proliferation and inhibit the apoptosis of BMSCs. So the survivin expression of BMSCs was measured only in experiment group and control group with60min. The immunofluorescence results indicate stronger staining in experiment group than control group; the positive rate (59.9±6.1) in experiment is higher than control group (24.3±5.8), a statistic difference P<0.05. The result of qRT-PCR show an increase in relative expression of survivin in experiment group compared (1.318±0.051) with control group (0.966±0.034). All results indicate low sound pressure infrasound can enhance the expression of survivin.Conclusion1. It is concluded that the low sound pressure infrasound can promote the proliferation and inhibit the apoptosis of BMSCs and60min is the ideal duration.2. The biologic effects of low sound pressure infrasound on BMSCs correlate with the enhancement of survivin expression. Whether the correlation is accurate need further study. |
PDF Full Text Request