감태 추출물이 사람 치주인대 줄기세포의 골분화에 미치는 영향

학위논문 (박사) -- 서울대학교 대학원 : 치의학대학원 치의학과, 2020. 8. 정필훈.Introduction: In the field of tissue engineering, dental stem cells isolated from human teeth have been differentiated into various cells in order to promote regeneration of tooth structures and periodontal tissue. Furthermore, many researches have been conducted to discover materials, especially natural products with fewer adverse side-effects or toxicity, that promote stem cell differentiation and periodontal tissue regeneration. Recently, implementation of naturally occurring bioactive compounds from seaweeds has been carried out for stimulating proliferation and differentiation of stem cells. In this study, Ecklonia cava, an edible brown alga species found along the southern coast of Korea, was investigated as a possible osteogenic product. Various biological activities of Ecklonia cava have been reported including antioxidation, anti-inflammatory, anticancer, antihypertensive, and antidiabetic effects. However, no previous study has investigated the effects of Ecklonia cava on the differentiation of human periodontal ligament stem cells (hPDLSCs). Thus, the aim of this study was to investigate the effects of Ecklonia cava on the osteogenic differentiation of hPDLSCs. Materials and Methods: hPDLSCs were isolated and cultured from human third molars. To characterize the cultured hPDLSC, flow cytometric analysis was performed using mesenchymal stem cell (MSC) markers. Then, the multilineage differentiation capacity of hPDLSC was confirmed with Alizarin Red S stain, Oil Red O dye and Alcian Blue stain as indicators of osteogenic, adipogenic, and chondrogenic differentiation, respectively. To examine the effects of Ecklonia cava extract (ECE) on hPDLSC viability, hPDLSCs were incubated with various concentrations of ECE for 1, 3, and 7 days. Then, to investigate the effects of ECE on the osteogenic differentiation of hPDLSCs, gene expression levels of the osteoblastic marker genes, Col1, ALP, OPN, OCN, and Runx2, were examined by real-time PCR, and protein expression levels of OCN, OPN, Col1, and Runx2 were detected by Western blot. In addition, possible mechanism underlying the osteogenic differentiation process after ECE treatment was explored by measuring the expression levels of Smad4, p-Smad2/3, p-p38, p38, p-ERK1/2 and ERK1/2 using Western blot. To evaluate the capacity of hPDLSCs for bone formation in response to ECE, hPDLSCs were mixed with HA/TCP without or with ECE and then subcutaneously transplanted into the dorsal surface of immunocompromised mice. For histological analysis, samples were acquired 10 weeks after transplantation, and hematoxylin and eosin (H&E) staining and immunohistochemical analysis were used to evaluate the differentiated bone-like tissue. Results: Characterization of hPDLSCs as mesenchymal stem cells (MSCs) and multilineage differentiation potential of hPDLSCs were confirmed. For the effects of ECE on hPDLSC proliferation, ECE increased proliferation of hPDLSCs without any cytotoxicity after 1 and 3 days of culture. After 7 days of culture, cell proliferation declined below the control value starting at 100 µg/ml of ECE. Real-time PCR showed that expressions of ALP, Col1, OCN, OPN, and Runx2 increased in ECE-treated group compared to untreated group, and protein expression levels of OCN, OPN, Col1, and Runx2 increased as well. In addition, Western blot analysis demonstrated that ECE may stimulate osteogenesis by activation of p38 and ERK1/2 signaling pathways. For in vivo study, histological analysis with H&E staining showed increased area of bone-like tissue layer in the ECE-treated group, and immunohistochemical analysis confirmed the generation of bone-like tissue with increased OSX, OPN, and OCN expressions. Conclusion: In this study, the effect of ECE on osteogenic differentiation of hPDLSCs was investigated in vitro and in vivo. The results of the experiments confirmed that ECE-treated group had greater ability to promote osteogenic differentiation of hPDLSCs compared to the control group. Therefore, ECE is suggested to be a potential natural compound with therapeutic properties against bone-related complications and diseases that urge further studies to elucidate its detailed mechanism of action and bioavailability.치아의 여러 부위에서 줄기세포를 추출하여 다양한 세포로 분화시키고 이를 이용한 치아 및 치주조직의 재생에 대한 연구가 많이 진행되어 왔다. 더 나아가 줄기세포의 분화 및 치주조직의 재생을 촉진하는 인자들, 특히 부작용이나 독성이 적은 천연물에 대한 연구도 활발하게 이루어지고 있다. 본 연구에서 사용한 감태는 다시마목 미역과의 갈조류로 우리나라 동해안과 제주도 근해에 널리 자생하고 있다. 현재까지 보고된 감태에 대한 연구로는 항산화, 항염증, 항암, 항고혈압 및 항당뇨 효능 등이 보고되어왔지만 사람치아줄기세포에 미치는 영향에 대한 연구는 이루어진 바가 없어 본 연구에서는 감태 추출물이 치주인대 줄기세포에 어떠한 영향을 주는지 알아보고자 한다. 성인 미성숙 제3대구치로부터 추출한 치주인대 줄기세포를 배양한 뒤 중간엽 줄기세포의 표지자 확인을 위해 유세포분석법을 시행하였다. 추출된 세포를 골아세포, 지방세포, 연골세포로 분화시킨 후 각각의 염색을 시행하여 치주인대 줄기세포의 다분화능력을 확인하였다. 세포 증식 및 독성에 대한 영향을 조사하기 위하여 감태 추출물을 농도별로 처리하여 1일, 3일 및 7일 동안 배양한 후 세포 생존능 분석을 시행하였다. 적정 감태 추출물의 농도를 확인한 후 골분화가 유도된 세포를 대상으로 quantitative RT-PCR을 통해 조골세포 분화 유전자인 Col1, ALP, OPN, OCN 및 Runx2의 발현 정도 및 Western blot으로 OCN, OPN, Col1 및 Runx2 단백질 발현 정도를 확인하였다. 또한 Western blot으로 Smad4, p-Smad2/3, p-38, p38, p-ERK1/2 및 ERK1/2의 발현 정도를 측정함으로써 감태 추출물 처리 후 골형성 분화 과정의 기본이 되는 메커니즘을 조사하였다. 체내에서의 분화를 확인하기 위해 치주인대 줄기세포를 HA/TCP와 혼합하여 면역 억제된 쥐의 등에 10주 동안 이식하였다. 감태 추출물에 의한 치주인대 줄기세포의 골분화를 조사하기 위해 조직학적 분석과 면역조직화학법을 이용한 분석을 시행하였다. 유세포분석법을 통해 성인 미성숙 제3대구치로부터 추출한 치주인대 줄기세포가 중간엽 유래 줄기세포의 특징을 갖고 있고 또한 Alizarin Red S 염색, Alcian Blue 염색, Oil Red O 염색을 통해 골조직, 연골조직, 지방조직으로 분화가 가능한 것을 확인하였다. 감태 추출물을 농도별로 처리하였을 때 배양 1일 및 3일 후에는 세포 독성이 나타나지 않았으며 배양 7일에서는 100 μg/ml 농도부터 세포 독성이 나타나는 것으로 관찰되었다. Quantitative RT-PCR을 통해 감태 추출물 처리군에서 ALP, Col1, OCN, OPN 및 Runx2의 발현이 처리되지 않은 군에 비해 증가하고, OCN, OPN, Col1 및 Runx2의 단백질 발현도 증가함을 보여 주었다. 또한, Western blot 분석은 감태 추출물이 p38 및 ERK1/2 신호 경로의 활성화에 의해 골형성을 유도 할 수 있음을 입증 하였다.면역 억제된 쥐를 이용한 생체내 실험에서 H&E 염색을 사용한 조직학적 분석은 감태 추출물 처리 군에서 뼈-유사 조직층의 면적이 증가한 것으로 나타났고, 면역조직화학 분석은 OSX, OPN 및 OCN 발현의 증가로 뼈-유사 조직의 생성을 확인하였다. 본 연구에서는 생체외 및 생체내 실험을 통하여 감태 추출물이 사람 치주인대 줄기세포의 골분화를 촉진하는 것을 확인하였다. 결론적으로 이 실험의 임상적 의미는 감태 추출물은 치조골 재생에 효과적인 영향을 미칠 것으로 사료되며 향후 감태 추출물의 정확한 작용기전 및 생체 이용률을 밝히기 위한 추가 연구가 필요할 것으로 생각된다.I. Introduction 1 II. Materials and Methods 5 III. Results 14 IV. Discussion 18 V. Conclusion 25 References 26 Table 34 Figures 35 Abstract in Korean 45Docto

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