Study Of The Relationship Between Autophagy And Age-related Changes Of BMMSCs In Senile Osteoporosis

As a metabolic disease, senile osteoporosis caused by imbalance between boneforming and bone resorption is characterized by decline of quantity and quality of bone,which presents decreased cortical thickness and mass of cancellous bone, a markedincrease in cortical porosity, decreased bone mineral density, bone fragility and anincreased incidence of fracture. Bone Marrow Mesenchymal Stem Cells (BMMSCs) aremesoderm-derived adult stem cells defined by their self-renew ability and multipotentdifferentiation ability, which play a key role in regulation of balance between boneforming and bone resorption. The degeneration of number and biological functions ofBMMSCs with advancing age plays a vital role in skeletal age-related changes. Autophagyis a process in which cellular components like misfolded proteins and damagedmitochodria are engulfed by autophagosome and delivered to lysosome to be degraded andrecycled to maintain cellular homeostasis. Autopahgy has been closely related with aging and age-related diseases, but how it functions in BMMSCs aging is still unclear. Therefore,we explored age-related changes of BMMSCs, change of autophagy in senescentBMMSCs and how autophagy worked in BMMSCs’s age-related changes, thus providinga new direction of understanding pathogenesis and finding possible treatment of senileosteoporosis.[Aim]1. To explore biological changes of BMMSCs during aging;2. To compare autophagy between young and senescent BMMSCs;3. To explore the role of autophagy plays in biological changes of BMMSCs duringaging.[Methods]1. Firstly, Micro-CT was applied to detect the morphology and quantity of distal femurtrabecular; Subsequently, we applied the method of whole bone marrow culture to isolateand purify BMMSCs of both young and senile mice and identified the cells by FCM;Clone forming assay was used to detect and compare the proliferation and self-renewability of BMMSCs from the two groups; Multipotent differentiation ability was tested byosteogenesis and adipogenesis induced culture; Finally, FCM was applied to detect andcompare proliferation ability, cell cycle and apoptosis between the two kinds of BMMSCs.2. Western Blot was performed to compare the expression of age-related proteins P53,P21, P16and TERT between young and senescent BMMSCs; ROS kit was used tocompare cellular ROS level of young and senescent BMMSCs.3. We compared the expression of both genes and proteins of Beclin1and LC3byRT-PCR and Western Blot; Immunofluorescent staining of LC3was also performed;Autophagosomes and microscopic structures of both cells were observed by TEM.4. Effective doses of rapamycin and3-MA were tested by Western Blot; RT-PCR wasperformed to detect changes of apoptosis genes and cell cycle genes in both cells whenstimulating by rapamycin and3-MA; Finally, we used ROS kit to test ROS changes ofboth cells stimulating by rapamycin and3-MA. [Results]1. Senile mice presented decreased bone quantity and characteristic of senileosteoporosis;FCM identification of BMMSCs showed that positive and negative markersare in line with characteristics of MSCs; CFU showed that senescent BMMSCs presentedsmaller and fewer colony-forming units than young BMMSCs, demonstrating a decreasedself-renew ability; Senescent BMMSCs also exhibited impaired osteogenesis ability andaccelerated adipogenesis ability;FCM test showed that senescent BMMSCs presented adecreased proliferation ability and higher apoptosis level compared with young BMMSCs.2. Wstern Blot showed that the expressions of P53, P21, P16in senescent BMMSCs werehigher than in young BMMSCs while TERT reversed. Laser Scanning ConfocalMicroscope showed that ROS of senescent cells was higher than young cells.3. RT-PCR and Western Blot showed that the expressions of Beclin1and LC3II/LC3I ofsenescent BMMSCs were lower than young BMMSCs.We also observed autophagosomesin both cells by TEM and the number in senescent cells is less than in young cells.4. We used Western Blot to confirm the effective doses of rapamycin as100nM and3-MAas5mM; RT-PCR showed that rapamycin and3-MA inhibited CCND1and CCND2expressions of both young and senescent BMMSCs, rapamycin did no effect of apoptosisgenes of young BMMSCs while promoted their expressions of senescentBMMSCs.Furthermore,3-MA inhibited apoptosis genes expressions of both young andsenescent cells. ROS test showed that rapamycin could reduce ROS triggered by H2O2ofyoung cells and physiological ROS of senescent cells while3-MA could increase ROSlevel of young cells.[Conclusions]1. Senescent BMMSCs showed biological changes during aging which includeddecreased proliferation and increased apoptosis,reduced osteogenesis ability and increasedadipogenesis ability,higher expression of P53,P21,P16and ROS and lower expression ofTERT.2. Autophagy decreased in BMMSCs during aging. 3. Increased autophagy could reduce ROS level in BMMSCs while decreased autophagyreversed; Regulation of autophagy affected cell cycle and apoptosis of BMMSCs.