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EN
Multiple studies showed that the cessation of TWIST1 expression is the prerequisite for osteoblasts' maturation. However, recent reports revealed that the function of TWIST1 is different in the dental pulp stem cells (DPSCs), where a high level of TWIST1 expression promoted DPSCs' differentiation. The aim of the study was to investigate the impact of TWIST1 and ID1 on the differentiation process in the human DPSCs. Methods: TWIST1 and ID1 expression in the DSPCs was modulated by lentivirus transduction. Genes expression was assessed with qRT-PCR. The proteins level was evaluated by Western blot. The DPSCs differentiation was assessed with the proliferation, alkaline phosphatase (ALP) activity, and calcium concentration assays. Results: TWIST1 silencing suppressed the expression of ID1 and both the early and late markers of odontoblasts' differentiation detected at the transcript and protein level. The forced overexpression of ID1 increased the expression of the late markers of odontoblasts differentiation but diminished the expression of the early markers. DPCSs with the silenced TWIST1 and subsequent ID1 overexpression displayed an increase in the expression of the late markers of odontoblasts differentiation. Cells with silenced TWIST1 and overexpressing ID1 had increased activity of ALP, higher calcium concentration and decreased proliferation rate. The high level of ID1 expression might be a critical factor stimulating DPSCs differentiation and it might compensate the repressed differentiation of DPSCs caused by TWIST1 silencing. Conclusion: The mutual correlation between the expression level of TWIST1 and ID1 might be a critical factor driving the process of the human odontoblasts' differentiation.
EN
Introduction. Recent data point to unerupted third molars as a promising source of stem cells (SC). Tooth derived SC are clonogenic and present a capacity for self-renewal and colony formation. Additionally, an environmental stimulation induces an in vitro differentiation of SC into multiple lineages, including odontoblasts. Aim of the study. The aim of the study was to evaluate the in vitro potential for proliferation and colony formation by stem cells derived from both dental pulp and apical papilla cultured in both standard medium (control and primary group) and medium modified with ingredients that stimulate mineralization (experimental group). Material and methods. Right after odontectomy the dental pulp and apical papilla were digested with dispase and collagenase type I. DPSCs and SCAPs were sorted using anti STRO-1, CD146, CD34, CD45 antibodies by means of the MACS method. Thereafter, the cells from the initial and control groups were cultured in a standard medium. The medium of the experimental group was additionally modified with ingredients that stimulated mineralization. To assess the cells commitment, the rate of proliferation and colony formation were examined. Results. The analysis showed that SCAPs from all the examined groups proliferated faster and formed more numerous and larger colonies compared to DPSCs. Environmental stimulation reduced proliferation and the ability to form colonies in both the DPSCs and SCAPs lineages. Conclusion. Faster proliferation and a higher ability to form colonies indicates the lower commitment of SCAPs compared to DPSCs. Additionally, the slower proliferation of stem cells from the experimental group suggests their more advanced commitment and differentiation. Although the SCAPs and DPSCs present different degrees of maturation, both cell lineages seem to be promising sources of stem cells.
PL
Wstęp. Niewyrznięte zęby mądrości są źródłem komórek macierzystych, które przy odpowiedniej stymulacji środowiska hodowlanego in vitro różnicują się w liczne linie rozwojowe m.in. odontoblasty, neurocyty, chondrocyty, adipocyty. Cel pracy. Porównanie in vitro potencjału proliferacji i tworzenia kolonii przez komórki izolowane z miazgi ludzkiego zęba (DPSC) oraz brodawki wierzchołkowej (SCAP), hodowanych w środowisku stymulującym mineralizację (grupa doświadczalna) oraz w warunkach standardowych (grupa wyjściowa i kontrolna). Materiał i metody. Po zabiegu odontektomii miazgę komorową oraz brodawkę wierzchołkową trawiono roztworem dyspazy i kolagenazy typu I, a pozyskane komórki hodowano w pożywce αMEM uzupełnionej surowicą bydlęcą, L-glutaminą oraz antybiotykami. Pożywkę w grupie doświadczalnej dodatkowo wzbogacano składnikami stymulującymi mineralizację. Mezenchymalny fenotyp komórek określono metodą MACS z użyciem przeciwciał przeciwko STRO-1 i CD146, a komórki hematopoetyczne i leukocyty eliminowano selekcją negatywną z użyciem przeciwciał przeciwko CD34 i CD45. Potencjał proliferacyjny szacowano pomiarem szybkości rozkładu soli tetrazolowej do formazanu, natomiast zdolność tworzenia kolonii oceniono barwieniem Giemsy. Wyniki. Analiza hodowli komórek DPSC i SCAP wykazała, że komórki SCAP we wszystkich badanych grupach dzieliły się szybciej oraz tworzyły większe i bardziej liczne kolonie w porównaniu z komórkami DPSC. Ponadto, promineralizacyjna stymulacja środowiska hodowlanego powodowała obniżenie zdolności formowania kolonii i spadek potencjału proliferacji w obu liniach komórkowych. Wnioski. Wysoki potencjał proliferacji oraz zdolność tworzenia licznych kolonii przez komórki SCAP dowodzi ich niższej dojrzałości komórkowej w porównaniu do komórek DPSC. Jednak zarówno SCAP jak i DPSC hodowane w środowisku promineralizacyjnym ulegają szybszemu różnicowaniu komórkowemu o czym świadczy ich niższy potencjał do samoodnawiania i spowolnienie procesów proliferacyjnych. Pomimo różnic w dojrzałości komórkowej komórek pochodzących z obu linii komórkowych, zarówno miazga zęba jak i brodawka wierzchołkowa niewyrzniętych zębów ósmych są cennym źródłem mezenchymalnych komórek macierzystych.
EN
Adjuvant chemotherapy with 5-fluorouracil remains the basic treatment for patients with advanced colorectal carcinoma. The major obstacle in successful treatment is the ability of CRC cells to acquire chemoresistance. Here we examined the impact of ID1 silencing on the sensitivity of CRC cells to 5-FU. To suppress ID1 expression in HT-29 and HCT-116 cells the cells were transduced with a lentiviral vector carrying the ID1 silencing sequence. Cells with silenced ID1 showed altered expression of epithelial and mesenchymal markers and exhibited increased proliferation rate compared to the parental cells. HCT-116 cells with suppressed ID1 became sensitized to 5-FU and this was not observed in HT-29 cells. Silencing ID1 resulted in altered expression of genes encoding enzymes metabolizing 5-FU. HT-29 cells with suppressed ID1 had significantly reduced mRNA level for thymidine phosphorylase, uridine-cytydine kinase 2 and dihydropyrimidine dehydrogenase. ID1 suppression in HCT-116 cells resulted in an increase of mRNA level for thymidine phosphorylase, thymidine kinase and uridine-cytydine kinase 2 with concurrent drop of dihydropyrimidine dehydrogenase and thymidylate synthetase mRNA levels. In conclusion, ID1 expression impacts the sensitivity of colon cancer cells to 5-FU and may be considered as a potential predictive marker in CRC treatment.
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