The Expression And Biological Function Of Corin In The Process Of Bone Metabolism

Part I Culture, identification, osteogenic and chondrogenicdifferentiation of human bone marrow mesenchymal stem cell in vitro.Objective:To establish and consummate a set of methods of isolating，culturing and identifyingmesenchymal stem cells (BMSCs) from bone marrow and to discuss the capacity of humanbone marrow mesenchymal stem cells osteogenic and chondrogenic differentiation in vitro.Methods:1. Culture of bone marrow mesenchymal stem cells (BMSCs): Bone marrows (5-10mL) with heparin anticoagulation were obtained by bone marrow puncture from posteriorsuperior iliac spine with informed consent of patients. And then take them to the preparedFicoll lymphocyte isolation liquid, centrifugation, the Mononuclear cells were primarycultured by basal medium (low-glucose DMEM medium with10%FBS,100U/mLpenicillin,100U/mL streptomycin), and per Petri dishes with1×106cells. The cellsmedium were changed for the first time after cultured for72h, and then the mediumchanged every3days.2. The identification of bone marrow mesenchymal stem cells (BMSCs): The thirdgeneration (P3) BMSCs were collected, and marked with the negative and positiveantibodies. The cell surface phenotypes (CD44, CD73, CD90) of BMSCs were identifiedby Flow cytometry.3. The osteogenic differentiation and identification of BMSCs: The third generation(P3) BMSCs were seeded in6-well plates (1×106cells per plate). The experiment wasdivided into4groups: the control group and the osteogenic groups with1,2,3-weeks. The control group cells were cultured with basal medium and the osteogenic groups cells withosteogenic medium (low-glucose DMEM medium with10%FBS,0.1μmol/Ldexamethasone,50mg/L ascorbic acid,10mmol/L beta-glycerophosphate sodium,100U/mL penicillin,100U/mL streptomycin) were all cultured for1,2and3weeks. Everygroup cells medium was replaced every3d. And then, the ability to differentiate intoosteoblasts was demonstrated by identifying the activity of ALP (alkaline phosphatase,ALP), Goromi, Von kossa, alizarin red staining, and the specific genes of osteoblastsdifferentiation were monitored by Reverse Transcription Polymerase Chain Reaction(RT-PCR).4. The chondrogenic differentiation and identification of BMSCs: The thirdgeneration (P3) BMSCs were seeded in6-well plates (1×106cells per plate). Theexperiment was divided into4groups: the control group and the chondrogenic groupswith1,2,3-weeks. The control group cells were cultured with basal medium and thechondrogenic groups cells with chondrogenic medium (1mmol/L sodium pyruvate,0.1mmol/L ascorbic acid,0.1umol/L dexamethasone,1%ITS,10ng/L of TGF-β1) were allcultured for1,2and3weeks. Every group cells medium was replaced every3d. And then,the ability to differentiate into chondrocytes was demonstrated by Alcian blue staining,Type II collagen immunohistochemistry and the specific genes of osteoblastsdifferentiation were monitored by Reverse Transcription Polymerase Chain Reaction(RT-PCR).Results:1． Phase contrast microscope observation: after the primary BMSCs culture mediumwas changed at72h, adherent cells can be observed, the cells are small and adherent, theappearance of cells are spindle; cultured7d, the spindle appearance significantly longer;2w, cells covered bottom, appear spiral or swirling; passage cells are stable, cell morphologydid not change.2． The surface phenotype of third generation BMSCs uniformly expressed positivefor CD44, CD73, CD90and negative for CD34, CD45, and so on. 3． The identification Of BMSCs osteogenic differentiation: after osteogenicinduction, the staining of Gomori, alizarin red, Von Kossa were positive, the ALPexpression of BMSCs is little, with the osteogenic induction of3d,6d,2w,3w,4w, ALPactivity was significantly enhanced, with the induction time growing, ALP activity alsoincreased. the gene expression of CollageⅠ, alkaline phosphatase (ALP), Osteocalcin(OCN), Bone sialoprotein (BSP), Osteopotin (OPN) and Osteonectin (ONN) have beendetected in osteogenic induct BMSCs by RT-PCR.4． The identification Of BMSCs chondrogenic differentiation: after chondrogenicinduction, the staining of Alcian blue was positive. The expression of type II collagen wasdetected by immunohistochemistry in microspheres cultured BMSCs. The chondrocyte-specific gene expression of Collage II, CollageX, the COMP and aggrecan have beendetected in osteogenic induct BMSCs by RT-PCR.Conclusions:1. We established a set of stable, mature method of isolating, culturing, amplificatingof bone marrow mesenchymal stem cell, which did not loss the genetic stability fromgeneration to generation, and provide a reliable reference for the clinical application ofBMSCs.2. BMSCs have good osteogenic and chondrogenic differentiation characteristics,and can transplant themselves, and no immune rejection, may become the ideal seed cellsfor tissue engineering. Part II The expression and biological function of corin in theprocess of bone metabolismCorin is a type II transmembrane serine protease, which was first found in the heart. Itcan thansfer the pro-atrial natriuretic peptide (pro-ANP) into the activity of ANP, and thenregulates blood pressure and cardiac function. Recent studies have reported that corinexpressed in the developing bones. The process of bone metabolism includes boneformation (osteoblasts) and bone resorption (osteoclasts), which always maintains thehomeostasis. Osteoporosis is the most common form of the metabolic bone diseases, themain reason of the formation of osteoporosis that the bone resorption exceeds the boneformation, thereby becoming a reduction in bone mass. Therefore, the study of serumsoluble corin of the patients with osteoporosis and the expression of corin in the process ofbone metabolism, could help help us to understand the corin structure and function and thephysiological and pathological significance of bone metabolism, which corin would takepart in.Objective:In vitro, the process of bone formation in vivo was simulated, in order to explore theexpression of corin in the process of bone formation. And the serum soluble corin levels inpatients with osteoporosis were detected, to explore the relation and clinical significancebetween the expression of corin and osteoporosis, and then explore the expression of corinand biological function in the process of bone metabolic (bone formation and boneresorption).Methods:1. Culture, identification, osteogenic and chondrogenic differentiation of humanbone marrow mesenchymal stem cell in vitro, and the extraction of mRNA in the processof differentiation. It has been elaborated in the first part.2. The expression of corin mRNA of BMSCs, osteoblasts and chondrocytes wasdetected by RT-PCR. 3. The expression of corin mRNA of BMSCs, osteogenic and chondrogenicdifferentiation (1w、2w、3w) was detected by qRT-PCR.4. The blood of normal persons, simple fracture, osteopenia and osteoporosispatients was punctured in vein, and centrifuged at3,000g for10min in2hours, and thentaken the upper serum Aliquoted.5. The serum soluble corin of normal persons (125), simple fracture (102),osteopenia (53) and osteoporosis (101) patients was detected by enzyme-linkedimmunosorbent assay (ELISA).Results:1. The expression of corin mRNA was all detected in BMSCs, osteoblasts and adultchondrocytes.2. With the gradual increased time of osteogenic and chondrogenic differentiation ofBMSCs, the expression of corin mRNA also showed a growing trend.3. Serum corin levels with patients with fractures and osteopenia or osteoporosiswere significantly lower than healthy controls and patients with simple fractures (510±228and478±183pg/mL vs.695±248pg/mL and706±345pg/mL, P value <0.001),howerever,(68.1±9.6and68.1±16.4vs.32.4±10.7and52.2±20.5years old, respectively, Pvalues <0.01).4. In patients with osteoporosis group, regardless of male and female patients, theserum corin levels were significantly lower than that of controls (894±257vs.643±198pg/mL, in. males, P <0.05;595±172vs.438±155pg/mL in females, P <0.001);5. In patients with osteopenia and osteoporosis groups, there is a linear relationshipbetween serum of corin levels and bone mineral density (P=0.03).6. Multiple linear regression analysis showed that the patient medical history(hypertension, P <0.05) is a relevant factor. In addition, gender is also an independentfactor of the level of serum corin levels (P <0.001).Conclusion:1. The expression of corin was all detected in BMSCs, osteoblasts and chondrocytes. Moreover, the expression of corin is upregulated in the process of bone and cartilageformation.2. The soluble corin levels with patients of osteoporosis were significantly lowerthan the normal controls.3. In patients with osteopenia and osteoporosis groups, there is a linear relationshipbetween serum of corin levels and bone mineral density. And serum corin level appearsprogressive decreased with the severity of disease. It suggesting that serum soluble corinmay be used as a biomarker to help diagnose and monitor osteoporosis.