| [Aim]To investigate the expression of Ihh-PTHrP signaling axis in Precartilainous StemCells (PSCs) obtained from rats and to further study their regulation effects on cellsproliferation, apoptosis and differentiation, as well as the regulation mechanism andinteraction of each other.[Methods]1. Epiphyses tissues were obtained from postnatal rats (0 to 4 weeks) bymicrosurgery technique. HE stain was used to observe the cartilage structure, Ihh andPTHrP expression and localization were detected by immunohitochemistry.2. PSCs were obtained from the La Croix ring of perichondrium by microsurgerytechnique and immunomagnetic separation system. Then identificated by expression ofFGFR3 and Colâ…¡protein.3. PSCs were incubated with different concentration of cyclopamine, a specificinhibitor to block Ihh signaling; Plasmids pEGPF-N1-PTHrP containing the human fulllength PTHrP gene were transfected into PSCs to overexpress PTHrP signaling. Differentintervention groups were set to study the dependent or independent effects and mechanismof Ihh-PTHrP signaling axis on regulation PSCs proliferation and differentiation.4. RT-PCR was used to detected the expression levels of Hh family genes (Ihh, Shh,Dhh) and signaling relative genes (PTHrP, Ptch, Ihh, Smo, Colâ…¡and Col x), as well asBcl-2 and p21 genes which were involved in cells poliferation and apoptosis in differentintervention groups. Using Western-Blot to detect the protein expression of Ihh and Ptch.5. MTT assay and BrdU incorporation method were used to examine the cellsproliferation; and Annexin V-PI method was used to examine the cells apoptosis. 6. Immunocytochemistry shown Colâ…¡protein expression level and the sulfateglycosaminoglycan was measured by Alcian blue staining.[Results]1. Ihh and PTHrP were detected mainly in hypertrophic chondrocytes region close toprimary or secondery ossification center.2. PSCs were obtained successfully; identification showed the cells specificlyexpression FGFR3 and Colâ…¡protein.3. Ihh, Ptch and Smo were obviously expressed in PSCs, however, Shh and Dhhexpressed weakly; after cyclopamine treatment, PSCs living status and proliferation weredepressed acutely.4. Ihh signaling could be blocked by cyclopamine in dose- and time-dependentmanner; Plasmid pEGPF-N1-PTHrP was transfected successfully and high level of PTHrPmRNA expression could be detected after two weeks.5. Inhibition Ihh signaling could depress PTHrP expression. However,overexpression of PTHrP could down-regulate Ihh expression. Which indicated an negativefeedback ring between Ihh and PTHrP signaling.6. Blockade Ihh signaling way could inhibit PSCs proliferation and promote cellsapoptosis. However, overexpress PTHrP could not rescue this effection. This datasindicated that Ihh signaling play a positive role in regulation PSCs poliferation andapoptosis, which independent of PTHrP function.7. After Ihh signaling was blocked by cyclopamine, mRNA expression of Bcl-2were down-regulated, however p21 was up-regulated. So Ihh regulation of PSCsproliferation and apoptosis probably is mediated by Bcl-2 and p21.8. PCR and immunocytochemistry indicated PTHrP remained the mRNA and protein expression level of Colâ…¡, As well as Colâ…¹mRNA level was higher incyclopamine trentment group. Histological staining proteoglycan of Alcian blue showeddeposition of typical cartilage extracellular matrix in PTHrP and Ihh+PTHrP groups. Thedatas showed that PTHrP is a downstream element of Ihh signaling way and plays a notablerole in regulation PSCs hypertrophy differentiation process.[Conclusion]Our datas showed that Ihh and PTHrP signaling way are highly express in PSCs andmay play an important role in rats' epiphyses entochondrostosis process. They form anIhh-PTHrP signaling axis with an interaction of negative feedback ring. ThroughPTHrP-independent method, Ihh signaling promotes PSCs proliferation and inhibitsapoptosis, which maybe mediated by Bcl-2 and p21. On the other hand, Ihh signaling canstimulate PTHrP signaling and participate in regulating PSCs differentiation. Thesefindings will help us better understand the regulatory mechanism of PSCs proliferation anddifferentiation process, and will lay a theoretical foundation for the future use of PSCs toconstruct tissue-engineered cartilage.
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