Elucidation of structural function and basic research for clinical application of dentin sialophosphoprotein in porcine pulp

我们通过离子交换（IE）色谱法从发育中的猪磨牙中分离出具有TGF-β样活性的DPP和DSP沿着，并在有或没有TGF-β受体抑制剂的情况下测定它们在人牙周（HPDL）细胞中的碱性磷酸酶（ALP）刺激活性。然后，我们通过使用ALP-HPDL系统的反相高效液相色谱（RP-HPLC）纯化TGF-β-未结合或结合的DPP和DSP。通过ELISA和LC-MS/MS分析，将与DPP和DSP结合的TGF-β同种型鉴定为TGF-β 1。我们将无载体的人重组TGF-β 1（CF-hTGF-β 1）与未结合TGF-β的DPP或DSP孵育，并使用ALP-HPDL系统在IE-HPLC上表征结合。DPP和DSP挽救了TGF-b1活性的丧失。约19%和10%的ALP刺激活性保留的结合TGF-β 1的DPP和DSP，分别。I型胶原很少与CF-hTGF β 1结合。我们得出结论，DPP和DSP有助于保留猪牙本质中的TGF-β 1活性。

We fractionated DPP and DSP along with TGF-b-like activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP) stimulating activity in human periodontal (HPDL) cells with or without TGF-b receptor inhibitor. We then purified TGF-b-unbound or -bound DPP and DSP by reverse phase high performance liquid chromatography (RP-HPLC) using ALP-HPDL system. The TGF-b isoform bound to DPP and DSP was identified as being TGF-b1 by ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-b1 (CF-hTGF-b1) with TGF-b-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. DPP and DSP rescued the loss of TGF-b1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-b1 to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGFb1. We conclude that both DPP and DSP help retain TGF-b1 activity in porcine dentin.