| FKBP51 is a 51-k Da immunophilin that belongs to the family of FK506-binding proteins,named after their capability to bind the immune suppressant FK506.FKBP51 belongs to the larger FKBP proteins and consists of a FKBP-type peptidyl-prolyl cistrans isomerase(PPIase)domain(called FK1),a FKBP-like domain(FK2)and a three-unit repeat of the tetratricopeptide repeat(TPR)domain,which can bind the MEEDV motif of other proteins.Due to its molecular structure,FKBP5 is a molecular chaperone of pleiotropic proteins.So far,the notable cochaperones of FKBP5 include stress related proteins(Hsp90),Steroid Hormone Receptors(GR,AR,PR),cell signaling proteins(Akt,IKK?,IKK?,MAPK),cytoskeleton proteins(TRAF2,Tau),etc.FKBP5 plays an important role in stress endocrinology and glucocorticoid signaling.The expression of FKBP5 can be robustly induced by stress and glucocorticoid hormones.Some FKBP5 SNP alleles associated with higher FKBP5 induction and prolonged cortisol responses are also accompanied higher risk for the stress related disorders.These SNPs were commonly tagged by rs1360780,rs9470080,rs9296158 or rs3800373.In recent years,an increasing number of clinical studies have demonstrated the interaction between FKBP5 genotype and stressors of various psychiatric disorders,such as depression,PTSD,and anxiety disorder.Clinical studies also showed an increased co-prevalence of osteoporosis in stress-related psychiatric disorders.Osteoporosis is the most common metabolic bone disease and a major public health threat.Severe osteoporosis represents a disease of high mortality and morbidity.Bone defect is a serious orthopedic disease and its treatment is challenging.Supercritical large area or large segment bone defect is still a difficult problem in clinical practice.Researchers are committed to the development of artificial bone materials,but in order to solve the problem of poor osteogenic effect of artificial bone materials,more and more attention has been paid to the application of mesenchymal stem cells in bone tissue engineering.Located in mesoderm and having osteogenesis potential,mesenchymal stem cells play a vital role in the bone development.It can differentiate into bone,cartilage,fat,nerve and myoblast cells and other interstitial cell subsets.Mesenchymal stem cells were first identified in bone marrow,but now researchers have found that MSC can also be isolated from placenta,umbilical cord,fat and other tissues.MSC can be used as seed cells to repair bone tissue and bring a new therapeutic strategy for bone defect and bone tissue injury repair.Clinical liposuction can produce 100?3000milliliters of adipose tissue each time,and the surgical trauma is small and the amount of adipose tissue obtained is large.This material is usually discarded as medical waste after surgery.Human adipose stem cells(hASCs),which are mesenchymal stem cells derived from adipose tissue,can be isolated and cultured from adipose tissue.They also have the ability of self-proliferation and multidirectional differentiation,making them ideal seed cells to repair tissue damage.However,there are still many limitations and challenges in the application of adipose stem cells to bone defects.For example,low efficiency of osteogenic differentiation in ischemic tissue.Therefore,how to improve the induction efficiency and make the bone differentiation more stable and controllable so as to increase the success rate of treatment is an urgent problem to be solved in clinical treatment.FKBP5 is expressed in many tissues,including brain,adipose tissue,bone marrow and so on,but the FKBP5 expression levels and regulatory patterns differ across tissues(http://biogps.org/).Located in mesoderm and having osteogenesis potential,mesenchymal stem cells play a vital role in the bone development.However,whether FKBP5 underlies the osteogenesis process of mesenchymal stem cells are unclear up to now.Therefore,we selected FKBP5 as our target molecule to study its effect and mechanism in the osteogenesis of stem cells.For this purpose,SAFit2,a selective inhibitor of the FKBP5 by an inducedfit mechanism was used.We found that the application of SAFit2 can make hASCs differentiate into osteoblasts efficiently and rapidly,the effect of SAFit2 on osteogenic differentiation of hASCs is partly mediated by GR pathway.Our results may provide novel strategy for balancing bone metabolism in clinical osteoporosis disorder and elevated efficiency of stem cell transplantation for bone infarct treatment.Part 1: Isolation,culture and identification of hASCs Objective: hASCs were extracted,isolated,cultured and identified from the waste adipose tissue of normal adult patients after clinical liposuction in vitro,which laid a foundation for subsequent experiments.Methods: Adipose mesenchymal stem cells were obtained by digestion,adherent culture,amplification and purification of hASCs from normal adult adipose tissue.Cell morphology and characteristic markers on cell surface were detected.The hASCs were collected,and the characteristic markers CD34(hematopoietic stem cell surface marker),CD11,DR,CD73,CD90(mesenchymal stem cell expression marker),CD105 on the surface of hASCs were identified by flow cytometry.Results: The hASCs were successfully extracted.Microscopic observation showed that the cells showed a swirling,adherent growth and long spindle shape.Flow cytometry results showed that the expression of CD34,CD11 and HLA-DR was low,and the expression of CD73,CD90 and CD105 was high,which was in line with the expression markers of mesenchymal stem cells.Conclusion: In this part of the experiment,hASCs were successfully extracted,providing a reliable and stable cell source for subsequent experiments.Part 2: Effect of FKBP5 on osteogenic differentiation of hASCs Objective: To observe the dynamic changes of FKBP5 in the process of osteogenic differentiation induced by hASCs,and to clarify its influence on osteogenic differentiation of hASCs.Methods:(1)The P3 generation of hASCs were selected and conventional osteogenic induction solution(dexamethasone,glycerin ?-phosphate and ascorbic acid)was added.Cells were collected at different time points from 0 to 72 h,respectively.Real-time quantitative polymerase chain reaction(RT-q PCR)and Western Blot were used to detect the expression levels of FKBP5 gene and protein during osteogenesis.The cells were collected at different time points within 0-72 h of the osteogenic differentiation induced by hASCs,and the changes of FKBP5 gene and protein levels during the osteogenic differentiation induced by hASCs were detected by RT-q PCR,and Western Blot.(2)The P3 hASCs were differentiated into osteogenic hASCs,and the small molecular compound SAFit2(500 n M)was selected to specifically inhibit the function of FKBP5.The experiment was divided into 4 groups:(1)hASCs medium group(PM group);(2)hASCs medium + SAFit2 group(PM+S group);(3)Osteogenic induction fluid group(OM group);(4)Osteogenesis inducement + SAFit2 group(OM+S group).Alizarin red staining was used to detect the difference of extracellular calcium in osteogenic differentiation of hASCs at cytochemical level.RT-q PCR and Western Blot were used to detect the expression differences of osteogenic related genes and proteins in the process of osteogenic differentiation of hASCs,and to observe whether FKBP5 had an effect on osteogenic differentiation of hASCs.Results:(1)In the early stage of osteogenic differentiation of hASCs(2-72 h after induction),FKBP5 gene and protein expression were significantly up-regulated.(2)SAFit2 can promote the osteogenic differentiation of hASCs.(3)The addition of SAFit2 increases the expression of the early osteogenic related gene alkaline phosphatase(ALP);(3)The addition of SAFit2 increases the expression of the late osteogenic related gene Osteopointin;(4)The addition of SAFit2 increased the expression of early osteogenic related protein RUNX2.Conclusion: In the early stage of osteogenic differentiation of hASCs,FKBP5 expression at mRNA and protein levels were dramatically increased in the osteogenesis process of human adipose stem cells,those results hinted that FKBP5 may play important role in this process.Using conventional osteogenic induction fluid,hASCs can be successfully induced to differentiate into osteocytes,adding FKBP5 selective inhibitor SAFit2 promotes the osteogenic differentiation of hASCs.In the cytochemical level,mRNA level and protein level confirmed that FKBP5 has a certain inhibitory effect on the osteogenic differentiation of hASCs.Part 3: The mechanism of FKBP5 inhibiting osteogenic differentiation of hASCs Aims: To explore the possible mechanism of FKBP5 inhibiting osteogenic differentiation of hASCs.Methods: We firstly examined the FKBP5 and GR expression status in hASCs under the osteogenesis induction.hASCs were incubated in the osteogenesis medium,and collected at different time points after induction by RT-q PCR and Western Blot.Co-immunoprecipitation(Co-IP)method was used to detect whether GR was a co-chaperone of FKBP5 in the osteogenic induction system.Different doses of dexamethasone(Dex 0.1um,1?m)were used in OM group and OM+S group,respectively,to observe whether there was a Dex dose-dependent effect of SAFit2 on promoting the osteogenic differentiation of hASCs.Results:.(1)The decrease of GR expression in early osteogenic differentiation of hASCs was inversely correlated with the increase of FKBP5;(2)GR is the molecular chaperone of FKBP5 in the ossification process of hASCs;(3)SAFit2promotes the osteogenic differentiation of hASCs in a dose-dependent manner.Conclusion: The effect of FKBP5 on the osteogenic differentiation of hASCs is mediated partly through the GR pathway.Conclusion of the full study:1.This experiment successfully extracted hASCs from human adipose tissue;2.hASCs can be successfully differentiated into bone cells by induction;In the early stage of osteogenic differentiation of hASCs,the expression of FKBP5 gene was up-regulated.The addition of FKBP5 inhibitor SAFit2 can effectively and rapidly differentiate hASCs into osteoblasts.3.The effect of FKBP5 on the osteogenic differentiation of hASCs is partly mediated by the GR pathway. |
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