| Bone mesenchymal stem cells (BMSCs) and bone marrow nucleatedcells are derived from bone marrow. Both of them play an important role inthe medicine nowadays, which have become a research hotspot in themedical field.Bone mesenchymal stem cells, as one of bone marrow-derived cells,have many advantages, such as more convenient acquisition, easier culture,quicker proliferation and other characteristics. BMSCs are considered as theideal seed cell in repairing and replacing the lesion tissues and organs, aswell as functional reconstruction. But nowadays there is insufficientnumbers of seed cell and the conventional amplification in vitro cannot meetthe requirement of clinical research and industry of tissue engineering,which greatly restricts its development. Bone marrow nucleated cells as themain ingredient in the bone marrow play an important role on humanhematopoietic and immune. When bone marrow nucleated cells reduce, thiscan cause seriously blood and immune system diseases.This means when bone marrow-derived cells are insufficient or low proliferation, this will greatly affect human tissues and organs systems. Atpresent, many scholars have found some methods and drugs to improve theproliferation of bone marrow nucleated cells, but there are someshortcomings and imperfections. Therefore the search for a safer and moreeffective technical method to obtain a sufficient and regenerate bonemesenchymal stem cell has become to resolve a vital and urgent keyquestion in the current medical realm research.In recent years, low intensity pulsed ultrasound (LIPUS) is consideredas a safe and pure "green" physical techniques with high specific, low costand other advantages. At present, LIPUS has been safely applied in healingbone and soft tissue, and achieves remarkable effect. The earlyexperiments initially explored the feasibility of LIPUS promotingproliferation of BMSCs.This project continued to deeply study the basis ofthe early experiments, and focused on bone marrow. As a result of thisstudy, LIPUS was combines respectively with Bone Marrow Stem Cells invitro and bone marrow nucleated cells in vivo rabbits to explore a new wayof identifying amplification of bone marrow-derived cells.Objectives1. Observe the influence of LIPUS of different intensity on BMSCsproliferation to investigate screen out the most suitable parameter.2. Further verify the influence of the most suitable parameter of LIPUSon BMSCs proliferation to investigate the effect and safety of bone mesenchymal stem cells proliferation by low intensity pulsed ultrasound invitro.3. Observe the influence of LIPUS on bone marrow nucleated cellsproliferation in vivo to investigate the effect of bone marrow nucleated cellsproliferation by LIPUS.Methods1. To screen out the most suitable parameter of LIPUS, the thirdgeneration of BMSCs in vitro was respectively irradiated by LIPUS ofdifferent intensity of0.03,0.1,0.2and0.3W/cm2,0.6MHz for5,10,15,20and30min，and each groups were irradiated for one time. Growth of cellswas observed under light microscope. Proliferation activity was determinedby MTT and cell cycle changes were analyzed using the flow cytometry after24h post-irradiation.2. The third generation of BMSCs which was cultivated in vitro wasrandomly divided into2groups: LIPUS groups and control groups. Thepre-experimental screening out suitable parameters0.2W/cm2,10mincontinued to irradiate for8days. Growth of cells was observed daily underlight microscope and the cell proliferation was determined by MTT; Cellcycle changing and proliferation index (PI) were detected by the flowcytometry.3.120New Zealand rabbits were randomly divided into3groups:LIPUS groups, control groups and normal groups. LIPUS irradiated for5 days. pre-irradiation and5d post-irradiation⑴Rabbit growth was observedevery day;⑵The mononuclear cells and hyperplasia degree of bone marrowwere observed6d、8d、10d、12d after5d irradiation;⑶Skin andmuscle in place of irradiation were observed under microscopepost-irradiation.Results1. Cell proliferation of different parameter groups was significantlymore active than that of the control-group and more intensive cell coloniesformed in LIPUS groups after24h post-irradiation. Both the cellproliferation activity and the cell proliferation index in different parametergroups were remarkably higher than that in the control group (P<0.05); theeffect of proliferation promotion post-irradiation with0.2W/cm2for10minis the most prominent.2. The LIPUS could promote the proliferation of BMSCs. Cellproliferation of the LIPUS group was significantly more active than that ofthe control group and more intensive cell colonies formed in LIPUS groups;MTT test showed that cell proliferation in the LIPUS group of1d、3d、5d、7d post-irradiation was significantly higher than that in the control group(P<0.05); Cell cycle changes tested by the flow cytometry showed that thecell proliferation index of LIPUS groups was significantly higher than that ofcontrol groups after irradiation (P<0.05).3. Rabbit behavior was observed in5d post-irradiation and there were no obvious difference when compared with the normal-group. The degree ofbone marrow hyperplasia, bone marrow nucleated cell was significantlyhigher than that in control group and normal group after6d,8d（P<0.05）.There was no obvious difference in statistics bone marrownucleated cells between control group and normal group (P>0.05). Skinirradiated and muscle-tissue pathological section showed muscle textureclear and staining is shallow. There were no significant abnormal andinflammatory cells in skin epidermis and dermis layer.Conclusions1. The LIPUS could prominently promote the proliferation of BMSCsin vitro.2. LIPUS can promote proliferation of BMSCs after post-irradiation forsome time and it is the optimum to promote the proliferative efficiencywhen the choice intensity0.2W/cm2for10min.3. For the bone marrow nucleated cells of living rabbit, the LIPUS maybe a safer and more effective technique method that could promote theproliferation of bone marrow nucleated cells in vitro. |
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