| Background: Orthodontic treatment mainly in children and adolescents. In recent years,with the continuous development of social economy and the improvement of people’s living standards, clinical adult orthodontic treatment ratio increased year by year. However,compared with adolescents, adult orthodontic treatment for a long time, and the treatment effect is not as good as teenagers. The reconstruction of periodontal tissue is the key to the orthodontic treatment and the speed of treatment. The periodontal ligament and alveolar bone play an important role in the remodeling of periodontal tissue. Periodontal ligament cells are the direct effects of orthodontic force, and play an important role in the remodeling of periodontal tissue. When the orthodontic force is transferred to the periodontal ligament, the precursor cells of periodontal ligament or periodontal ligament stem cells differentiate into osteoblasts in the tension side, resulting in bone deposition.Periodontal ligament stem cells (PDLSC) derived from periodontal tissue of adult stem cells that can be derived from the root or nest alveolar periodontal tissue, which has close relationship with the periodontal tissue, study showed that it has a good ability of proliferation, self-renewal ability and multi-directional differentiation ability, it can be differentiated into different cells in the induction of specific conditions. It not only plays an important role in the treatment of periodontal disease and the repair of soft tissue defects around implants, at the same time, it is involved in bone remodeling during orthodontic tooth movement, which has become the first selection of seed cells for periodontal tissue regeneration guided by tissue engineering and genetic engineering.Mechanical force plays an important role in the process of orthodontic alveolar bone remodeling. However, age is also an important factor affecting the remodeling of periodontal tissue during orthodontic treatment. Mesenchymal stem cells (MSCs) are one of the most widely distributed adult stem cells in vivo. Stem cells in the body is in a state of equilibrium, but with the growth of age, the function of stem cells are affected, the balance is destroyed. In the process of aging, mesenchymal stem cells self regulating mechanism changed, so that some of its biological characters no longer stable, the main performance is the decrease of the self resistance and the reduction of the response to the external environment, which evoked susceptibility to disease, resulting in death, thus increasing age may cause the body biological characteristics of stem cells change, thus affecting the final transplantation or therapeutic effect. It is found that the microenvironment of stem cells changes with the increase of age, which may lead to the change of some signal pathways.Wnt signaling pathway exists widely in the organism, play an important role in signal transduction in the embryonic development process and various organs in the regulation of many biological processes, such as cell proliferation, differentiation, apoptosis, immune stress and so on. It is found that Wnt signaling pathway plays an important role in the development of periodontal ligament, and it can regulate the proliferation and differentiation of periodontal ligament stem cells and promote the regeneration of periodontal tissue. In recent years, it has been suggested that Wnt signaling pathway is closely related to stem cell senescence. Recent studies suggest that Wnt signaling pathway is closely related to stem cell senescence. Some scholars have respectively cultured bone marrow mesenchymal stem cells by serum of young and aged rats, compared with the young group, the elderly group significantly increased the number of aging cells,up-regulated the expression of Wntβ-catenin blockers, but after Dkk-1 was added , the number of senescent cells decreased, suggesting that the serum microenvironment of aged rats can activate the Wnt/β-catenin signal pathway, and the Wnt/β-catenin signaling pathway plays an important role in the regulation of the aging of mesenchymal stem cells.However, the specific regulatory mechanism is not clear.Objective: To observe the effects of aging factors on the biological characteristics of PDLSC by means of isolation, culture and comparative analysis of PDLSC in teeth of different ages, and explore the relationship between Wnt signaling pathway and PDLSC during aging.Methods:Part 1: The periodontal ligament stem cells (PDLSC) were obtained from the teeth of different age groups (all the specimens were divided into three groups according to the donor’ s age: group A: 18-20 years; group B : 30-35 years; group C: above 45 years), and their biological characteristics were compared and analyzed. The difference of the proliferation ability of three groups of PDLSC was detected by MTT ;detected the clone formation ability; the osteogenic and adipogenic differentiation experiments were carried out, and the osteogenic differentiation ability was evaluated by alizarin red staining, the adipogenic differentiation ability was evaluated by oil red O staining. Cell cycle and apoptosis were detected by flow cytometry; and the difference of cell senescence was analyzed by SA-β-gal staining.Part 2: Periodontal ligament stem cells have the ability of multi-directional differentiation.In order to explore the influence of aging factors on the osteogenic differentiation ability of PDLSC, the PDLSC of three experimental groups were induced by osteogenic differentiation. After osteogenic induction of 7d, the corresponding RNA and protein were extracted, and ALP staining of PDLSC was performed. Real time quantitative PCR was used to detect the expression of key genes ALP, Runx-2 and COL-1, and the expression of ALP and OCN was detected by Western blot.Part 3: In order to explore the relationship between periodontal ligament stem cells and Wnt signaling pathway under aging condition, PDLSC was induced in three experimental groups, and RNA and protein were extracted after osteogenic induction of 7d. Real time quantitative PCR was used to detect the expression of Wnt related genes Wnt/p-catenin,P38 and NLK, and the expression of Wnt related protein Wnt/β-catenin, P38 and NLK was detected by Western blot.Results:Part 1: Changes of biological characteristics of PDLSC under aging factors1. PDLSC can be isolated from donors of different ages.2. The clone formation rate of PDLSC decreased from the young group to the age group,which was 36.67%, 26.67%, 9.33%, respectively, the difference was statistically significant;3. MTT assay showed that the three experimental group had strong proliferation ability, the proliferation capacity of PDLSC differed from the fifth day, the difference was statistically significant; the cell cycle showed that the proportion of PDLSC in the S+G2/M phase decreased from the young group to the age group, respectively 38.73%, 29.88%, 18.25%;4. Periodontal ligament stem cells were induced 21 days after osteogenic induction,alizarin red staining showed that three experimental groups have mineralized nodule formation, but the size and number of mineralized nodules from the young group (group A)to aging group (group C) gradually decreased; after 14 days of adipogenic induction, oil red O staining showed that lipid droplets were formed in the experimental group, but the number of lipid droplets increased from he young group (group A) to aging group (group C);5. From the young group (group A) to aging group (group C), the proportion of PDLSC in the early and late stage of apoptosis was increased by 1.57%, 4.56%, 5.84%, respectively;6. SA-β-gal staining showed that the number of positive staining cells increased from the young group (group A) to aging group (group C), the staining area was 4.14%, 16.39%,50.38%, respectively , the difference was statistically significant;Part 2: Effects of aging factors on osteogenic differentiation of PDLSC1.Three groups of PDLSC were stained with ALP staining after osteogenic induction, and the staining area decreased from the young group (group A) to aging group (group C);2.Real time quantitative PCR detection showed that the expression of ALP, COL-1 and Runx-2 after osteogenic induction decreased from the young group (group A) to aging group (group C) after osteogenic induction;3. Western blot further confirmed that after osteogenic induction, the expression of ALP and OCN were significantly higher than those in the control group, but the expression of the gray value decreased from the young group (group A) to aging group (group C) after osteogenic induction.Part 3: Regulation of Wnt pathway on osteogenic differentiation of PDLSC by aging factors.1. Real time quantitative PCR detection showed that the expression of Wnt/β-catenin increased with age, and the expression was enhanced after osteogenic induction.2. Real time quantitative PCR analysis showed that the expression of NLK in the non-canonical Wnt pathway increased with age, and the expression decreased after osteogenic induction.3. Real time quantitative PCR detection showed that the expression of P38 Mark decreased with the increase of age, and the expression was decreased after osteogenic induction.4. Western blot further confirmed that the expression of Wnt/β-catenin increased with age,and the expression was enhanced after osteogenic induction.Conclusion:1. With the increase of age, the proliferation ability of PDLSC was decreased, the ability of adipogenic differentiation was enhanced,and the level of apoptosis was increased.2. With the increase of age, the osteogenic differentiation ability of PDLSC decreased.3. With the increase of age, the expression of β-catenin in classical Wnt signal pathway was enhanced... |
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