학술논문
ODREĐIVANJE PROGENITORNOG SVOJSTVA PERIVASKULARNIH STANICA KARAKTERIZIRANIH IN VIVO IZRAŽAJEM ALFA-GLATKOMIŠIĆNOG AKTINSKOG PROMOTORA – CRE REKOMBINAZEDefining in vivo progenitor potenital of perivascular cells characterized by expression of alpha-smooth muscle actin promoter – Cre recombinase“
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Croatian
Abstract
Ovim istraživanjem ispitana je sposobnost diferencijacije perivaskularnih stanica PDL-a koje izražavaju alfa-glatkomišićni akitnski promotor (αSMA) u zrele stanične loze parodonta in vivo. Za trajno obilježavanje stanica koje izražavaju αSMA promotor, kao i njihovog potomstva, korišten je αSMACreERT2/Ai9 transgenični konstrukt. Col2.3-GFP transgen je korišten za dokazivanje diferencijacije SMA9 obilježenih stanica u osteoblaste/cementoblaste, dok je za praćenje diferencijacije u PDL fibroblaste korišten Scx-GFP transgenični konstrukt. Tri tjedna starim αSMACreERT2/Ai9/Col2.3-GFP i αSMACreERT2/Ai9/Scx-GFP miševima apliciran je tamoksifen intraperitonealno kako bi se obilježile SMA9 stanice. 2. dan nakon obilježavanja, SMA9 pozitivne stanice zamijećene su unutar desni i u periapikalnoj regiji PDL-a. 17. dan SMA9 stanice su proliferirale te su detektirani dvostruko obilježeni αSMA/Col2.3-GFP pozitivni osteoblasti i cementoblasti. 7. tjedan nakon obilježavanja, SMA9 obilježene stanice su i dalje prisutne unutar parodonta, te su detektirane αSMA/Col2.3-GFP pozitivne stanice unutar staničnog cementa. Također, zamijećeni su brojni dvostruko obilježeni, SMA9/Scx-GFP pozitivni, PDL fibroblasti. Ti rezultati potvrđuju sposobnost SMA9 obilježenih stanica da se diferenciraju u zrele stanične loze parodonta: osteoblaste, cementoblaste i PDL fibroblaste tijekom rasta zuba. Identične linije transgeničnih miševa korištene su u eksperimentalnom modelu ozljede PDL-a. 7. dan nakon izvedene ozljede opažena je snažna ekspanzija SMA9 obilježenih stanica unutar desni i PDL-a ozlijeđenog korijena. Četiri tjedna nakon ozljede detektirani su SMA9/Col2.3-GFP pozitivni ostoblasti i cementoblasti. Također, zamijećene su brojne SMA9 obilježene stanice unutar PDL-a morfoloških karakteristika fibroblasta koje izražavaju Scx-GFP signal. Očito je kako se tijekom procesa regeneracije SMA9 stanice diferenciraju u zrele stanične loze parodonta. Protočnom citometrijom analiziran je izražaj hematopoetskog biljega CD45 i biljega endotelnih stanica CD31 na αSMA-GFP pozitivnim stanicama PDL-a. Pokazano je da αSMA-GFP pozitivne stanice ne izražavaju CD31 niti CD45 biljeg, odnosno zadovoljavaju neophodan uvjet tijekom fenotipske karakterizacije mezenhimskih matičnih stanica. Imunofluorescentnom metodom detekcije antigena dokazana je prisutnost PDGFRβ, biljega stanične loze pericita, na SMA9 obilježenim stanicama unutar parodonta, posebice unutar PDL-a. Zaključno, ovim istraživanjem in vivo je dokazana sposobnost diferencijacije SMA9 obilježenih perivaskularnih stanica u stanične loze parodonta: osteoblaste, cementoblaste i PDL fibroblaste tijekom rasta zuba, kao i tijekom procesa regeneracije parodonta.
The goal of this study was to examine the ability of α-smooth muscle actin promotor (αSMA) expressing cells residing within periodontal tissues to generate mature lineages of the periodontium in vivo. For permanent labeling of αSMA expressing cells and their progeny αSMACreERT2/Ai9 transgene was used. To trace the differentiation of SMA9 labeled cells into osteoblasts/cementoblasts a Col2.3-GFP transgene was utilized, while expression of Scx-GFP was used to follow differentiation into PDL fibroblasts. To label SMA9 cells tamoxifen was administered intraperitonaly to the three-week-old αSMACreERT2/Ai9/Col2.3-GFP mice. Second day after labeling SMA9+ cells were observed within the gingiva and perivascular areas of PDL in the periapical region. On the 17th day SMA9+ cells proliferated, furthermore a double-label αSMA/Col2.3-GFP osteoblasts and cementoblasts were detected. Seven weeks after labeling there were still SMA9+ cells within periodontium, also SMA9/Col2.3-GFP cells were observed in cellular cementum in the apical region of the root. To evaluate differentiation of SMA9+ progenitor cells into PDL fibroblasts, similar lineage tracing in αSMACreERT2/Ai9/Scx-GFP mice was performed. Seven weeks after labeling, numerous double-label SMA9/Scx-GFP PDL fibroblasts were detected. These results confirm the ability of SMA9 labeled cells to differentiate into mature cell lineages of periodontium during tooth growh: osteoblasts, cementum and PDL fibrolasts. Identical transgenic lines of two-month-old mice were used for PDL injury experimental model. Seven days after injury there was a notable expansion of the SMA9+ population within gingiva and in perivascular areas of PDL of the injured root. Significant co-expression of Col2.3-GFP was not detected in SMA9+ cells at this timepoint. Four weeks after injury, co-expression of Col2.3-GFP was detected in SMA9+ cells, including osteoblasts lining the adjacent new bone and cementoblasts. The contributions of SMA9+ cells to PDL fibroblasts after injury were confirmed in αSMACreERT2/Ai9/Scx-GFP mice. Four weeks after injury SMA9+ cells expanded and differentiated into numerous cells with fibroblastic shape within the PDL co-expressing Scx-GFP, indicative of a PDL fibroblast phenotype. These observations in the injured periodontium provide evidence of contributions of SMA9+ cells to PDL fibroblasts, osteoblasts, and cementoblasts. Using flow cytometry an expression of the hematopoetic marker CD45 and endothelial marker CD31 was examined on αSMA-GFP+ cells isolated from PDL of the six-week-old mice. Flow cytometry indicated that they do not express hematopoietic or endothelial lineage markers, respectively they meet the necessary requirement for phenotypic characteristics of mesenchymal stem cells. Furthermore, immunostaining has shown that SMA9 labeled cells within periodontium, and especially within PDL, express high levels of pericyte marker PDGFRβ. This study provided in vivo evidence that the αSMA+ perivascular cells residing in the PDL have the ability to differentiate into mature cell types within the periodontium during growth and regeneration after injury.
The goal of this study was to examine the ability of α-smooth muscle actin promotor (αSMA) expressing cells residing within periodontal tissues to generate mature lineages of the periodontium in vivo. For permanent labeling of αSMA expressing cells and their progeny αSMACreERT2/Ai9 transgene was used. To trace the differentiation of SMA9 labeled cells into osteoblasts/cementoblasts a Col2.3-GFP transgene was utilized, while expression of Scx-GFP was used to follow differentiation into PDL fibroblasts. To label SMA9 cells tamoxifen was administered intraperitonaly to the three-week-old αSMACreERT2/Ai9/Col2.3-GFP mice. Second day after labeling SMA9+ cells were observed within the gingiva and perivascular areas of PDL in the periapical region. On the 17th day SMA9+ cells proliferated, furthermore a double-label αSMA/Col2.3-GFP osteoblasts and cementoblasts were detected. Seven weeks after labeling there were still SMA9+ cells within periodontium, also SMA9/Col2.3-GFP cells were observed in cellular cementum in the apical region of the root. To evaluate differentiation of SMA9+ progenitor cells into PDL fibroblasts, similar lineage tracing in αSMACreERT2/Ai9/Scx-GFP mice was performed. Seven weeks after labeling, numerous double-label SMA9/Scx-GFP PDL fibroblasts were detected. These results confirm the ability of SMA9 labeled cells to differentiate into mature cell lineages of periodontium during tooth growh: osteoblasts, cementum and PDL fibrolasts. Identical transgenic lines of two-month-old mice were used for PDL injury experimental model. Seven days after injury there was a notable expansion of the SMA9+ population within gingiva and in perivascular areas of PDL of the injured root. Significant co-expression of Col2.3-GFP was not detected in SMA9+ cells at this timepoint. Four weeks after injury, co-expression of Col2.3-GFP was detected in SMA9+ cells, including osteoblasts lining the adjacent new bone and cementoblasts. The contributions of SMA9+ cells to PDL fibroblasts after injury were confirmed in αSMACreERT2/Ai9/Scx-GFP mice. Four weeks after injury SMA9+ cells expanded and differentiated into numerous cells with fibroblastic shape within the PDL co-expressing Scx-GFP, indicative of a PDL fibroblast phenotype. These observations in the injured periodontium provide evidence of contributions of SMA9+ cells to PDL fibroblasts, osteoblasts, and cementoblasts. Using flow cytometry an expression of the hematopoetic marker CD45 and endothelial marker CD31 was examined on αSMA-GFP+ cells isolated from PDL of the six-week-old mice. Flow cytometry indicated that they do not express hematopoietic or endothelial lineage markers, respectively they meet the necessary requirement for phenotypic characteristics of mesenchymal stem cells. Furthermore, immunostaining has shown that SMA9 labeled cells within periodontium, and especially within PDL, express high levels of pericyte marker PDGFRβ. This study provided in vivo evidence that the αSMA+ perivascular cells residing in the PDL have the ability to differentiate into mature cell types within the periodontium during growth and regeneration after injury.