The Effects Of Bone Marrow Mesenchymal Stem Cells Differentiated Into Endothelium And The Underlying Mechanism On Condition Of Modeled Microgravity Under The Guidance Of "Theory Of Body And Spirit"

BackgroudIn recent two decades of development, regenerative medicine is the multidisciplinaryfields related to biology, materlals science, engineering and clinical medicine, theapplication of which has been able to reengineering a variety of tissues and organs. Inparticular, stem cell research brings a new opportunity in nearly decade for thebreakthrough progress of regenerative medicine. Ischemic injury of tissues and organs is aproblem which plagued clinicians for centuries. However, the traditional method oftreatment is difficult to restore the number and function of endothelial cells and improveblood perfusion of tissues and organs. With the rapid development of tissue engineering, regenerative medicine and gene therapy, bone marrow mesenchymal stem cells are playingan enormous application potential in treatment of various ischemic diseases. For theapplication of stem cells, the main method is employed by gene modification, drug effectsor cytokines joint use. Although these methods can successfully induce, the excessivedifferentiation problems cannot ignore. At present, there is no report on above way.With the development of the areospace industry, people in the field of space scienceresearch has made a number of innovations, especially in microgravity, people have beenaware of the changes of microgravity on cell proliferation and apoptosis, and each systemfunction. Studies have shown that morphology and function is closely related to itsinternal environment under microgravity. However, our study found that change ofBMSCs morphology under microgravity stimulation from spindle to round is obviously.Shape with the quality of God, God with the purposes.The changes of morphologicaland its function are unified. Theory of Body and Spirit indicates that the morphologicalchanges of external influence intercourse movements and affect its function. We think thecells with round change are in a primitive state. Could these changes make mesenchymalstem cells with higher differentiation potential? Therefore, we carried out the experimentalresearch as follow.Aims:1. To explore the feasibility of the clutivation, identification and marking of rat bonemarrow mesenchymal stem cells (BMSCs) in vitro.2. To observe the differentiation of bone marrow mesenchymal stem cells (BMSCs)under modeled microgravity intervention in vitro.3. To observe transplantation of modeled microgravity intervention with bone marrowmesenchymal stem cells (BMSCs) may give better way in a mouse hindlimb ischemiaangiogenesis.4. From the level of RhoA expression, to demonstrate the mechanisms involved in effectof cell morphology, cytoskeleton, and cytoskeletal protein molecules RhoA undermodeled microgravity intervention on the angiogenesis of BMSCs.5. By means of modern medical knowledge to prove the theory of traditional Chinese medicine theory, to explore the meeting point involved in the unity of morphologyand function, and Theory of Body and Spirit of Circumference Philosophicalparadigm.Methods:1. We utilized bone marrow adherence to isolate and culture mouse bone marrowmesenchymal stem cells; and utilized MTT assay BMSCs growth and viability; andthen utilized flow cytometry to identify the specific surface CD29、CD34、CD45、CD90markers. Mouse bone marrow mesenchymal stem cells were induced todifferentiate to adipogenic and osteoplasts, which were determined by oil red O andalizarin red stain. We observed the survival rate and growth state of BMSCs withfreeze thawing, and observed the efficiency of1,1’-dioctadecyl-3,3,3’3’-tetramethylindocarbocyanine perchlorate(DiI) labeling BMSCs and the impact of DiIon BMSCs.2. The third generation of BMSCs were divided into three groups randomly, the blankcontrol group(control group),2D-clinostat modeled microgravity intervention group(MMG group)，and the normal gravity intervention group(NG group). The cells wereexposed to microgravity and gravity environment for72h and then cultured inendothelium cells medium for six days. Subsequently, BMSCs were observedFlk-1and vWF expression with microscope, immunofluorescence, flow cytometry,real-time PCR and Western blot. ELISA assays the concentration of vascularendothelial growth factor, basic fibroblast growth factor concentration. UsingDil-AC-LDL uptake and Matrigel angiogenesis assays the fouctional differences ofinduction cells in vitro.3. Setting up a model of mouse right femoral artery ligation, BMSCs were transplantedafter surgery in one day by normal gravity group and microgravity intervention groupfor72hours after application of Dil labeled. After surgery1d,10d,21d, we use laserDoppler perfusion imaging detected limb blood flow perfusion，respectively. It waslasted for21days. The mice were sacrificed. Hereafter, we used that immune-histochemistry HE staining detected tissue injury and repair; the flow cytometry detected ischemia musclecontained Dil positive cells; immunofluorescence doublestaining and western blot analysised the expression of vascular endothelial cellconversion efficiency and the expression amount of vWF.4. The third generations of BMSCs were divided into four groups randomly: normalgravity group (0h group), microgravity intervention for4hours group (4h group),microgravity intervention for72hours group (72h group), and microgravityintervention for10days group (10d group). We used that inverted microscopeobserved cell morphology; the flow cytometry analysised apoptosis;immunofluorescence assay cytoskeletal changes of α-actin and RhoA positioning inBMSCs; Western blot detected the activity change at different times aftermicrogravity intervention. Lately, we bulid RhoA plasmid transfection vector andidentified its transfection efficiency; BMSCs after transfection under microgravityintervention72h, we observed the formation of F-actin, then induced the cells inendothelial culture medium, investigated the influence of active RhoA and knoweddown RhoA in BMSCs proliferation and tube formations.Result:1. The BMSCs which culture by bone marrow adherence adherented growth, passagedon9～12day, which the proliferated rate become fast after passaged, and werepurified at3rd generation. The third generations of BMSCs showed the morphologyof firoblasts and proliferated in the culture medium. Flow cytometry of isolated cellsshowed that CD29, CD90positive, CD34, CD45negative, which is supposed to bespecific characteristics of BMSCs. Cultured cells could also be induced intoadipocytes and osteoplasts. The survival rates of BMSCs thawed after frozen wererespectively90%and85%and grow well. The efficiency of DiI labeling BMSCs was100%. DiI had no influence on the morphology and growth of BMSCs.2. The cells were exposed to microgravity and gravity environment for72h and thencultured in endothelium cells medium for six days, we observed the results:endothelial like cells expressed Flk-1and vWF significantly higher than that innormal gravity group by immunofluorescence detection, flow cytometry, Real-time PCR and Western blot levels. The DiI-Ac-LDL phagocytic and Matrigel angiogenesisin vitro experiment also proved that the induced cells with endothelial cell function.3. We successfully established the mouse right femoral artery ligation model. The laserDoppler perfusion index was significantly higher in the MMG group on day10aftertreatment and showed further improvement afterwards on day21. Twenty-one daysafter BMSCs transplantation, MMG group showed an effect of significant protectivein ischemic limb atrophy and muscle necrosis injury by immunohistochemistry HEstaining. We utilized flow cytometry to assay the amount of Dio positive BMSCs tosites of ischemic hindlimb, which was in line with the observation byimmunofluorescence microscope. The results demonstrated that the numbers ofDil-labeling BMSCs in the ischemic hindlimb can survival and differentiated intovascular endothelial cells in vivo, which were increased compared with that of NGgroup. We performed western blot of vWF to show that there was increasedexpression at ischemia hindlimb in MMG group.4. Microgravity intervention0h,4h,72h and10d, BMSCs present from spindle to roundshape. However, there were no significant differences among all groups in flow cytom-etry apoptosis. The cytoskeletal F-actin markers were detected by immunofluorescen-ce. The most obvious change happened on72h, which F-actin tension and expressionwas significantly decreased. For RhoA, it is mainly distributed in the cytoplasm andmembrane in BMSCs. With RhoA plasmid transfection, the effects of transfectionwere verified by western blot. Under microgravity active RhoA promoteddifferentiation of BMSCs, but shRhoA could inhibit it. In the endothelial inducemedium, active RhoA in the BMSCs enhanced the expression Flk-1、vWF, tubeformations, but knock down of RhoA could inhibit it.Conclusion:1. The purification of BMSCs can be harvested by method of whole bone marrowadherent.These BMSCs have the potential of isolation and proliferation. DiI can labelthe membrane of BMSCs effectively and has no obvious influence on the morphologyand growth of BMSCs. 2. The differentiation capacity of the BMSCs under MMG intervention is stronger thanthat of the normal gravity.3. MMG intervention combined with BMSCs transplantation can enhance thedifferentiation into endothelial cells and promote angiogenesis.4. The probable mechanisms of MMG intervention72h promoting BMSCsdifferentiation is that the RhoA regulate cell morphology, affected the cytoskeletondepolymerization and participated in endothelial cells differentiation andneovascularization.5. BMSCs exhibit morphology of circle under the guidance of Theory of Body andSpirit of Integration of spirit and god, so that it is in the initial state for stemcell.We think modeled microgravity stimulation provide a kind of harmony, balanceand suitable environment of their differentiation, improving the differentiationcapability better of BMSCs.