| Objective:1.To study the expression of Cx43 in the normal bone tissue and the effects of Cx43 mediated intercellular regulatory network on the metabolism,differentiation and function of bone tissue cells(b MSCs,OB,OC,endothelial cells),as well as the maintenance of normal bone metabolism and bone structure.2.To study the expression change of Cx43 during the occurrence and development of GCs-induced ONFH,the effect of GCs on the regulation of Cx43 on the intercellular network structure of bone tissue,the role of Cx43 in the occurrence and development of GCs-induced ONFH and its influence on bone tissue related cells and the pathogenesis of GCs-induced ONFH.3.Based on the above studies,this study aimed to promote the repair of osteonecrosis by studying the regulation to Cx43 and further regulation to normal metabolism,proliferation and function of bone tissue cells,and explore the early prevention and treatment protocols to ONFH from the molecular and cellular levels.Materials and Methods:Chapter I: Study on the expression of Cx43 in rat femoral head necrosis bone tissue and OB cells as well as the pathogenesis of ONFH.1.The rat model of steroid-induced ONFH was established.Micro-CT and HE staining were used to observe the degree of trabecular destruction and the incidence rate of empty lacunae,then the incidence of ONFH was further calculated.The Cx43 protein,PI3K/Akt signaling pathways related factors and osteogenic proteins in model group and control group were analyzed by RT-PCR and Western blot.2.Rat osteoblasts(OB)were isolated and cultured in vitro.After treated with Dex,western blot and immunofluorescence were used to detect the effect of Dex on Cx43 expression in OB cells;Western blot was used to detect the influence of GCs on the expression of p-PI3 K,p-Akt,and β-catenin;Akt activator(SC79)and inhibitor(LY294002)were employed to study the molecular regulation mechanism of Cx43 expression in OB cells after treated with Cx43;the regulatory relationship between β-catenin and Cx43 was studied by immunoprecipitation and si RNA technologies.Chapter II: Study about the effect and regulatory mechanism of Cx43 on proliferation and osteogenic differentiation of OB cells under the GCs treatment.1.The effect of Dex on osteogenic differentiation of OB cells was confirmed by Alizin red staining and ALP staining.Western blot was used to detect the effect of Dex on the expression of Cx43 and osteogenic related proteins(Runx2,ALP and COL)in OB cells.Furthermore,the localization of Cx43 in OB cells and the effect of Dex on the expression of Cx43 and osteogenic related proteins in OB cells were detected by immunofluorescence staining.CCK8,flow cytometry and Ed U staining were applied to study the effect of Dex on the proliferation of OB cells.The effect of Dex on the expression of PCNA and CDK4 in OB cells was detected by Western blot.2.To study the effect of Cx43 on the proliferation and osteogenic differentiation of OB cells,the lentivirus-mediated Cx43 gene overexpression plasmid(Lv-Cx43)was constructed and transfected into osteoblasts.The effect of Lv-Cx43 on Cx43 expression including m RNA and protein levels in OB cells were detected by RT-PCR and WB methods,respectively.CCK8 method,Ed U staining and flow cytometry were used to detect the effect of Lv-Cx43 on the proliferation of OB cells.The effect of LvCx43 on the m RNA and protein expression of osteoblast-related proteins(Runx2,ALP and COL)were examined by RT-PCR and WB techniques.Western blot was applied to examine the effect of Lv-Cx43 on the expressions of PCNA,CDK4 and p-ERK1/2.In order to further confirm whether the effect of Cx43 on the proliferation and osteogenic differentiation of OB cells through regulating ERK1/2 signaling pathway,we pretreated OB cells with PD98059,CCK8 method and Alizin red staining were used to observe whether PD98059 could reverse the effect of Lv-Cx43 on the proliferation and osteogenic differentiation of OB cells.Chapter III: Study about the effect and regulatory mechanism of OB cells with overexpressed Cx43 on b MSCs osteogenic differentiation and OCs osteoclast activity.1.Rat b MSCs were isolated and cultured in vitro,then its multi-differentiation ability was identified by osteoblasts,adipocytes and chondroblasts induction differentiation of P3 generation b MSCs,and its cell surface antigen was identified by flow cytometry;b MSCs was cocultured with supernatant of OB cells for 14 days,and the culture medium was changed every other day.Aizarin red staining was used to observe osteogenic differentiation of b MSCs in each group,and the expression of Runx2,ALP and COL of b MSCs in each group was detected by Western blot.2.In order to further study the mechanism of OB cells supernatant after overexpressed Cx43 to b MSCs’ osteogenic differentiation,scrape-loading and dye transfer(SLDT)were used to detect the opened gap junction hemichannel quantities of OB cells,and PGE2 concentration in OB cells supernatant was detected by ELISA analysis;the gap junction hemichannel was inhibited by Cx43-si RNA and a gap junction blocker 18α-glycyrhizic acid,and PGE2 concentration was examined by ELISA analysis.3.In order to clarify the effect of PGE2 on osteogenic differentiation of b MSCs,different concentrations of PGE2 were added to b MSCs.Western blot was used to detect Runx2 expression in b MSCs,and the impact of PGE2 on b MSCs’ osteogenic differentiation was detected by alizarin red method.After respectively pretreated b MSCs with EP1/2/3 inhibitor(AH6809),EP1/3 activator(Sulprostone)and EP2agonist(Butaprost),the effect of PGE2 on osteogenic differentiation of b MSCs was observed by Alizarin red staining.In the presence of PGE2,WB method was adopted to detect the expression changes of PI3K/Akt pathway related molecules and Runx2 in b MSCs.Then b MSCs were pretreated with PI3 K inhibitor LY294002 and Akt activator SC79,WB was used to detect the effect of PGE2 on the expression of Runx2.Immunofluorescence staining and Western blot were used to detect the effect of PGE2 on Cx43 protein expression in b MSCs,and SLDT technique was used to observe the effect of PGE2 on gap junction hemichannel opening level in b MSCs.Transwell cell migration assay was used to confirm the effect of OB supernatants on the migration ability of b MSCs,and WB was used to detect the expression of MMP2 in b MSCs.4.The total m RNA of transfected OB cells was extracted,and the m RNA expression of OPG and RANKL in each group were detected by RT-PCR.The supernatant of OB cells in each group was collected and cultured together with mononucleate macrophages collected from rat bone marrow for 14 days,then the modalism of OC cells in each group was scanned by microscope,and the OC differentiation ability of each group was examined by TRAP staining(cells count and total area measurement was used to reflect the differentiation level of OC cells),finally the expression of NFATC1 was detected by RT-PCR and Western blot.Chapter IV: Study about the effect and regulatory mechanism of OB cells with overexpressed Cx43 on h UVECs vascular-like structure formation.1.h UVECs cells were cultured in vitro,and the system of OB and h UVECs indirect co-culture was confirmed.This part was divided into blank,negative control and Cx43 overexpressed groups based on the difference of osteoblasts;The migration ability and vascular-like structure formation of h UVECs in each group were evaluated by cell scratch test and tube formation assay,and the MMP2 and CD31 protein levels in h UVECs were examined by Western blot.2.Different concentrations of PGE2 were applied to endothelial cells,and the effect of PGE2 on h UVECs tubulation ability was observed by matrix glue tube formation assay: Under the treatment with EP1/3 activator-sulprostone,EP2 agonist-butaprost,EP1/2/3 inhibitor-AH6809,EP4 agonist(ONO4819)and EP4 antagonist(AH23848),the effect of PGE2 on h UVECs tubulation differentiation was detected by tube formation assay.3.h UVECs were treated with different concentrations of PGE2 or ONO-4819,and the production of c AMP in h UVECs was detected by ELISA.Different concentrations of SQ22536(adenylate cyclase specific inhibitor)were prepared to observe its effect on endothelial cell tube formation.The effect of PGE2,ONO-4819 and SQ22536 on the phosphorylation level of vasodilation-stimulated protein phosphate(VASP)in h UVECs was detected by Western blot.The effect of H89(PKA specific inhibitor)on the VASP phosphorylation level and tube formation ability of h UVECs were studied by WB and tube formation assay.Chapter V: Study about Cx43 on regulating bone tissue intercellular homeostasis,maintaining bone metabolism,preventing and repairing steroid-induced ONFH of rats.1.Different ratios of hyaluronic acid-gelatin hydrogels(1:0,0:1,1:1)were prepared,which respectively represented: pure hyaluronic acid hydrogel(HA),Pure gelatin hydrogel(G),Hyaluronic acid-gelatin biomimetic hydrogel(HA-G),electron microscopy was used to observe the cross-sectional structure of hydrogels in each group,and the rotating rheometer(AR 2000EX)was used to detect the rheological properties of hydrogels.The effect of hydrogels on the cell viability was observed by Live-dead staining.The spread of OB cells on the surface of hydrogel was observed by laser confocal microscope.2.The rat model of femoral head necrosis was established by lipopolysaccharide and methylprednisolone,after drilling the femoral head and neck for decompression,biomimetic hydrogel with different components were implanted into the femoral head in groups through decompression channels and study its repairation effect on osteonecrosis.This experiment was divided into five groups: Control group,biomimetic hydrogel(HA-G)group,non-transfected OB cells encapsulated with biomimetic hydrogel(HA-G +OB group),Lv-NC-OB encapsulated with biomimetic hydrogel group(HA-G +Lv-NC/OB group),Lv-Cx43-OB encapsulated with biomimetic hydrogel group(HA-G +Lv-Cx43/OB group).After the ONFH model was established,the above hydrogels were implanted respectively into the femoral head through the fenoral head and neck drilling channel.Six and twelve weeks after surgery,samples were collected and observed by Micro Fil perfusion about the presence of blood vessels in the femoral head of each group,Micro CT was applied to observe the growth of bone trabecula and the repairation of bone defect.HE staining and Masson staining were used to evaluate the growth of new bone tissue.Trap staining was used to observe the osteoclasts differentiation level in each group.WB was used to examine the protein expression of Cx43,Runx2,PI3K-Akt-GSK3β pathway related factors(pPI3K,p-Akt,p-GSK3β),p-ERK1/2,and angiogenesis related proteins(MMP2 and CD31)in each group.The positive expression of Cx43,Runx2,phospho-PI3 K,phospho-Akt,and phospho-ERK1/2 was detected by immunohistochemical staining,and the expression of CD31 was detected by histological immunofluorescence staining.Results:Chapter I: Study on the expression of Cx43 in rat femoral head necrosis bone tissue and OB cells as well as the pathogenesis of ONFH.1.The rat model of GC-ONFH was established successfully,and the expression of Cx43 in GC-ONFH model group was obviously lower than that in the control group,and Cx43 was positively correlated with the expression of PI3K/Akt signaling pathway molecules and osteogenic proteins Runx2,ALP and Collagen I Type(COL).2.OB cells of rats were successful isolated and cultured in vitro,then induced by Dexamethasone(Dex)to mimic GC-ONFH rat OB cells in vivo.Under the effect of Dex,the expression of Cx43,p-PI3 K,p-Akt and β-catenin gradually declined with the time prolonging.After pretreated with SC79,the inhibition effect of GCs to Cx43 was significantly reversed,but the inhibition effect was significantly enhanced after pretreated with LY294002.The immunoprecipitation results showed that β-catenin expression was related with the expression of Cx43,and deeper researches demonstrated that β-catenin si RNA down-regulated the expression of Cx43 significantly.Chapter II: Study about the effect and regulatory mechanism of Cx43 on proliferation and osteogenic differentiation of OB cells under the GCs treatment.1.The results of alizarin red staining and ALP staining showed that the osteogenic differentiation ability of OB cells decreased significantly under GCs treatment.WB and cell immunofluorescence staining results both showed that expression of Cx43 and osteogenic related proteins(Runx2,ALP,COL)significantly decreased under GCs treatment.CCK8,Ed U staining and flow cytometry results showed that OB cells proliferation ability decreased under GCs treatment,and proliferation related proteins(PCNA,CDK4)and p-ERK1/2 expression decreased significantly.It suggested that the inhibition of GCs to OB cells proliferation and osteogenic differentiation might be resulted from the decreased expression of Cx43.2.After Lentivirus-mediated Cx43(Lv-Cx43)gene was transfected into OB cells,RT-PCR and WB results showed that Lv-Cx43 significantly increased the expression of Cx43 in OB cells compared with the control and negative control group(Lv-NC);Flow cytometry and Ed U staining showed that overexpression of Cx43 significantly promoted the proliferation of OB cells.Alizarin red staining showed that Cx43 overexpression promoted the forming of calcium nodules in OB cells significantly.In addition,RT-PCR results showed that overexpression of Cx43 enhanced the expression of Runx2,ALP and COL m RNAs.WB assay showed that the expression of osteoblast-related proteins(Runx2,ALP and COL),proliferation-related proteins(PCNA and CDK4)as well as p-ERK1/2 significantly increased after Cx43 overexpressed.It suggested that Cx43 regulated and promoted OB cells proliferation and differentiation through activating ERK1/2 signaling pathways.In order to further confirm the above results,OB cells which Cx43 was overexpressed were pretreated with PD98059(ERK1/2 inhibitors),CCK8 and alizarin red staining results demonstrated that the positive impact of Lv-Cx43 to osteoblasts’ proliferation and osteogenic differentiation could be decreased obviously by PD98059(ERK1/2inhibitor).Chapter III: Study about the effect and regulatory mechanism of OB cells with overexpressed Cx43 on b MSCs osteogenic differentiation and OCs osteoclast activity.1.Alizarin red,oil red O,toluidine blue and Safranin O staining identified that b MSCs which extracted from rats could be induced into osteoblasts,adipocytes and chondroblasts differentiation,respectively.Cell surface antigen identification results showed that CD29 and CD90 expression rates were 98.18% and 99.2%,and the CD45 and CD34 were 0.12% and 0.04%,respectively.After indirect co-cultured with b MSCs,the supernatant of OB cells which transfected by Lv-Cx43 could obviously promote calcium nodules formation of b MSCs and the expression of b MSCs osteogenic proteins(Runx2,ALP and COL).2.SLDT assay results showed that the quantity of opened hemichannel of OB cells with overexpressed Cx43 was obviously higher compared with blank and negative control group.ELISA experiment demonstrated that PGE2 concentration in supernatant of OB cells which transfected by Lv-Cx43 was significantly higher than that of blank or negative control group.Both Cx43-si RNA and the gap junction blocker18α-glycyrrhetinic acid could significantly decrease the PGE2 concentration in the supernatant of OB cells.All these results indicated that the release of PGE2 is mainly controlled by the gap junction hemichannel.3.WB assay and alizarin red staining results showed that PGE2 could significantly promote the expression of Runx2 and the formation of calcium nodules in b MSCs.Under the treatment of PGE2,AH6809 could significantly inhibit the formation of calcium nodules,instead butaprost played the opposite role.WB results showed that PI3K/Akt/GSK3β pathway related molecules expression in b MSCs increased under PGE2 treatment,and LY294002 could inhibit the effect of PGE2 on phosphorylation of PI3K/Akt/GSK3β pathway,while SC79 played the opposite role.In addition,LY294002 could significantly inhibit the positive regulation of Runx2 by PGE2,while SC79 could significantly promote the positive regulation of Runx2 by PGE2.Immunofluorescence staining and WB assay results showed that PGE2 significantly promoted the expression of Cx43 in b MSCs,and SLDT results showed that PGE2 could significantly promote the opening of cell junction hemichannel in b MSCs.The results of Transwell and WB experiments showed that the supernatant of OB cells in which Cx43 overexpressed could promoted the migration of b MSCs and the expression of MMP2 protein significantly.4.RT-PCR demonstrated that OPG expression(m RNA level)in OB cells which tranfected with Cx43 gene was higher compared with blank or negative control group,while the expression of RANKL m RNA was lower obviously compared with blank or negative control group;The rat bone marrow mononucleate macrophages was successfully induced and indirect co-cultured with OB supernatant in vitro,we found that under the induction of OB supernatant with overexpressed Cx43,the number of osteoclasts and the total area of osteoclasts were lower compared with control and negative control group.Moreover,the m RNA and protein levels of NFATC1 in the supernatant induced group of osteoblasts transfected with Cx43 gene were lower compared with blank and negative control group.Chapter IV: Study about the effect and regulatory mechanism of OB cells with overexpressed Cx43 on h UVECs vascular-like structure formation.1.Cell scratches assay and WB results showed that the supernatant of OB cells with overexpressed Cx43 significantly promoted h UVECs cell migration,and MMP2 protein expression was more than blank and negative control group.Tubular assay and WB results demonstrated that OB supernatant with overexpressed Cx43 promoted the tube formation and CD31 expression of h UVECs compared with blank or negative control groups.2.As the concentration of PGE2 increased,the influence on the tube formation ability of h UVECs becomes more obvious,and 100 n M PGE2 has the most significant effect;ONO-4819,the agonist of EP4 receptor,enhanced the tubularity effect of PGE2,while AH23848,the inhibitor of EP4 receptor,could inhibite the tubularity effect of PGE2,while the effect of EP2 agonist butaprost and EP1/2/3 receptor inhibitor AH6809 was not significant.The EP4 receptor agonist ONO-4819 could significantly promote the tubularity effect of OB supernatant overexpressed Cx43 on h UVECs,while the EP4 receptor inhibitor AH23848 played the opposite role.3.ELISA results showed that PGE2 or ONO-4819 promoted the c AMP production in h UVECs,while SQ22536 could inhibit the c AMP production.Tube formation assay showed that SQ22536 inhibited the formation of PGE2 or the ONO-4819 induced tubulation in a dose-dependent manner.WB results showed that PGE2 or ONO-4819 promoted the phosphorylation of VASP in h UVECs in a dose-dependent manner,while SQ22536 inhibited the positive regulation.H89 also inhibited the positive regulation of PGE2 or ONO-4819 on VASP phosphorylation in a dosedependent manner;Tube formation assay demonstrated that H89 inhibited the promoting tube effect of PGE2,and the higher concentration of H89,the more obvious the inhibition effect.Chapter V: Study about Cx43 on regulating bone tissue intercellular homeostasis,maintaining bone metabolism,preventing and repairing steroid-induced ONFH of rats.1.The hydrogel scanning electron microscopy images showed that the three groups all owned three-dimensional interlocking porous structures.The rheological test results showed that the mechanical properties of the HA-G group were better than those of the HA and G groups.Live-dead staining results showed that the toxicity of the three groups to OB cells was low.The results of phalloidine staining showed that the cell spread in HA-G group was significantly better than that in HA and G groups.2.Micro Fil perfusion results showed that the distribution density of blood vessels in the femoral head of Lv-Cx43/OB group which encapsulated with HA-G biomimetic hydrogel was significantly higher than that in other groups;In the femoral head and neck drilling channel of rats with HA-G biomimetic hydrogel encapsulated LvCx43/OB implanted group,new bone formation was observed obviously in the bone detect of osteonecrosis area.Trap staining results revealed that the osteoclast active properties were lower than that of other groups,and WB results demonstrated that the Cx43,Runx2,PI3K-Akt-GSK3β pathway related factors(p-PI3 K,p-Akt,p-GSK3β),p-ERK1/2,and angiogenesis related proteins(MMP2 and CD31)expressions in this group were obviously higher compared with non-transfected group and negative control group.Histological IHC and IF staining showed that Cx43,Runx2,p-PI3 K,pAkt,p-ERK1/2 and CD31 expression were significantly higher than the other two groups.Conclusion:1.The expression of Cx43 decreased significantly in ONFH tissue and OB cells,which may be attributed to the inhibition of the expression of Cx43 by GCs through the inhibition of PI3K/Akt/β-catenin signaling pathway.2.Cx43 promoted the proliferation and osteogenic differentiation of OB cells through ERK1/2 signaling pathway.3.Cx43 hemichannel could mediate the intercellular transduction of PGE2 signaling molecule,PGE2 played an important role in the regulation of homeostasis between OB and b MSCs or OB and OC cells.4.Cx43 hemichannel could mediate the intercellular transduction of PGE2 signaling molecules,PGE2 played an important role in the regulation of homeostasis between OB and h UVECs cells;PGE2 regulated the h UVECs angiogenesis through activating c AMP-PKA signaling pathway which mediated by EP4 receptor.5.After the OB cells which overexpressed Cx43 and encapsulated with biomimetic hydrogel were implanted into the femoral head,it promoted osteogenesis and vascularization in bone defect areas,inhibited osteoclast activity,promoted bone tissue repairing in osteonecrosis area,maintained the homeostasis in bone tissue intercellular network,and inhibited the destruction to the structure of bone tissue cell network,which could repair the bone defect area of steroid-induced ONFH partly.All the results above will provide a new idea for study on the pathogenesis of GCs-induced ONFH,and also provide a new target and theoretical basis for the prevention and treatment of early GCs-induced ONFH. |
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