| ObjectivePurpose by detecting sources of different ages people the periodontal ligament stem cells(human periodontal ligament stem cells,hPDLSCs)proliferation,aging condition,the change of osteogenetic differentiation ability and lipogenic differentiation ability,explore the age factor of periodontal membrane stem cell biology behavior and the effect of periodontal treatment for senile patients with periodontal disease,and stem cell therapy of periodontal tissue reconstruction to provide theoretical foundation and reference basis.MethodsThe human periodontal ligament stem cells from three different age groups were selected and divided into Group A(18-20 years old),Group B(30-35 years old),Group C(50-55 years old).The human periodontal ligament stem cells of the three experimental groups were amplified,and P2 human periodontal ligament stem cells were further cultured and detected.The proliferation curve of human periodontal ligament stem cells in each age group was compared by MTT method,so as to compare the proliferation rate,namely the ability of proliferation and differentiation.After osteogenic induction,the osteogenic differentiation ability of human periodontal ligament stem cells in each experimental group was compared.Firstly,the human periodontal ligament stem cells in each experimental group were osteogenic induced by osteogenic induction solution for 7 days,and then the human periodontal ligament stem cells in this group were stained by ALP alkaline phosphatase staining method Observe and detect the difference of osteogenic differentiation ability of three experimental groups at 7 days after osteogenic induction by Image J software,and then induce the human periodontal ligament stem cells of each experimental group at 21 days,and then carry out alizarin red staining on them respectively,and observe with naked eyes and image J software was used to detect the number of mineralized nodules and the staining area of mineralized nodules in each experimental group and quantify them,so as to judge the change of bone differentiation ability of human periodontal ligament stem cells in each age group at 21 days after osteogenesis induction;After 14 days of lipogenesis induction,the stem cells in each group were stained with oil red O,and the staining area was detected by Image J software,so as to determine the changes in the lipid differentiation ability of human periodontal membrane stem cells in each age group at 14 days after lipogenesis induction;At the same time,SA-?-gal staining was used to detect the senescence of the three groups of human periodontal ligament stem cells,and to determine and confirm the difference of their senescence.Finally,RT-PCR was used to detect the expression results of the transcription factors Nanog and Sox2 related to the pluripotency of the three groups ofhuman periodontal ligament stem cells.ResultsThe human periodontal ligament stem cells in each group showed good proliferation ability.On the first four days of conventional culture,there was no significant statistical difference in the proliferation rate of the three groups.On the fifth day of culture,there was a statistical difference in the proliferation ability.The above results mean that the proliferation ability of human periodontal ligament stem cells is related to age factors With the increase of donor's age,the proliferation ability(proliferation rate)of human periodontal ligament stem cells showed a downward trend;However,the osteogenic induction of three groups of human periodontal ligament stem cells,whether ALP alkaline phosphatase staining after 7days of induction or alizarin red staining after 21 days of induction,showed that the osteogenic differentiation ability of human periodontal ligament stem cells depended on the donor's age;After 14 days of adipogenic induction,the human periodontal ligament stem cells in each group were stained with oil red O,indicating that their adipogenic differentiation ability increased with the increase of donor age;The SA-?-gal aging staining of human periodontal ligament stem cells in each group confirmed the difference of the aging degree of human periodontal ligament stem cells in the three experimental groups,and also suggested that the aging expression of human periodontal ligament stem cells increased significantly with the increase of age.The results of real-time quantitative PCR showed that,with the increase of age,the expression level of multi-functional transcription factors such as Nanog and Sox2 in human periodontal ligament stem cells also decreased.ConclusionHuman periodontal ligament stem cells can be obtained and cultured from donors of different ages,but their biological characteristics are closely related to age factors.Their proliferation ability(proliferation rate)and osteogenic differentiation ability decrease significantly with the increase of donor age.Their expression levels of pluripotent related transcription factors Nanog and Sox2 also decrease with the increase of donor age,but the adipogenic differentiation ability increased with the increase of donor age and their SA-?-gal decline the old expression level showed an age-related upward trend. |
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