기능성 유전자 탑재 임플란트의 생체적합성과 골 적합성 유도에 관한 연구

바타라이고빈다 전북대학교 치의학대학원 2013 국내박사

The objective of this study was to deliver the efficacy and safety of conjugated anti-inflammatory molecules PPARγ and transcription factor c-myb on implant surface Ti to reduce implant-induced inflammation and to increase the osseointegration. Ch-GNPs were conjugated with the PPARγ and c-myb cDNA through a coacervation process. Conjugation was cast over Ti surfaces by dipping, and cells were seeded on different sizes (6×6×0.1 cm and 1×1×0.1 cm; n=3) of Ti surfaces. The size of Ch-GNPs and surface characterization of Ti was performed by UV-vis spectroscopy, TEM and EDX. The DNA conjugation and transfection capacity of Ch-GNPs were simultaneously confirmed by agarose gel electrophoresis, β-galactosidase staining and immunoblotting. Results showed that Ch-GNPs were well dispersed and spherical in shape, with average size around 10~20 nm. Ti surfaces coated with Ch-GNPs/LacZ, as transfection efficacy molecule, showed strong β -galactosidase staining in MC-3T3 E1 cells. Cells cultured on Ch-GNPs/PPARγ coated Ti surfaces were able to inhibit implant-induced inflammation by simultaneously suppressing the expression of TNF-α, IL-1β, iNOS, COX-2 and MMP-2. The inhibition mechanism of Ch-GNPs/PPARγ was due to inhibition of both ROS and NO secretion (n=3; P<0.05). In addition, Ch-GNPs/PPARγ was able to increase expression of BMP-7 and RUNX-2. Further, ALP was also increased than in control (n=3; P<0.001). Whereas the expression of RANKL was decreased. To determine the effect of c-myb in implant integration, chitosan gold nanoparticles conjugated with plasmid DNA/c-myb coated Ti implants were installed in rat mandibles. One and four weeks post-implantation, mandibles were examined by microcomputed tomography, immunohistochemistry and hematoxylin & eosin staining. In vitro overexpressed Ch-GNPs/c-myb coated on Ti surfaces was associated with enhanced expression of osteogenic molecules OPN, RUNX-2 and BMP2/7 by MC-3T3E1 osteoblasts compared to uncoated surfaces. The microcomputed tomography analysis demonstrated that c-myb overexpression increased the density and volume of newly formed bone surrounding the implants. Further, c-myb increased the number of cells expressing BMPs and aided in the increase of enhanced osseointegration. These results support the view that c-myb overexpression accelerates osseointegration of Ti implants and can serve as a potent molecule in promoting tissue regeneration around dental implants. The recipient rat of this system provides an excellent in vivo model for studies of bone regeneration. This depicts that novel gene delivery materials, like Ch-GNPs, can carry the PPARγ cDNA and c-myb into the required areas of the implant surfaces, thus aiding to inhibit inflammation and promote osseointegration.

호장근으로부터 bioactivity-guide 방법을 이용한 Streptococcus mutans 산생성 억제 물질의 분리와 분리 물질의 효과 검증

손광진 전북대학교 치의학대학원 2010 국내박사

동의보감에 따르면 치과질환 예방 및 처치를 위해 호장근을 전통적으로 사용해왔다. 그러나 치의학 영역에서의 호장근에 대한 연구는 찾아보기 어렵다. 본 연구는 호장근으로부터의 bioactivity-guide 방법을 이용한 Streptococcus mutans 산생성 억제 물질의 분리와 분리 물질의 효과 검증을 목표로 시행되었다. 먼저, 호장근 메탄올 추출물을 70% aqueous methanol, n-hexane 및 ethyl acetate 분획으로 분리하였다. 위의 분획 중 항산생성효과가 가장 우수한 ethyl acetate 분획을 column chromatography로 다시 분리한 후 각각의 분획을 이용하여 다시 항산생성효과를 비교하였다. 분리된 각각의 분획은 high-performance liquid chromatography와 glycolytic pH-drop assay를 이용하여 성분확인과 항산생성효과를 비교하였다. 분리된 분획들 중 F3 분획이 가장 우수한 항산생성효과를 보였으며 그 효과는 농도 의존적으로 나타났다. F3 분획은 S. mutans의 산생성을 12.5 &micro;g/ml에서도 억제하였다 (p < 0.05). F3의 주요구성성분으로는 resveratrol과 emodin (C14H12O3 and C14H4O2(OH)3CH3)이었으며 전체 구성성분의 60 %를 차지하였다. 결론적으로 F3는 S. mutans의 산생성을 억제할 수 있기 때문에 향후 충치 예방약물로서의 가능성을 보여주고 있다. OBJECTIVES: The aim of this study was to separate the anti-acidogenic substances against S. mutans UA 159 from Polygonum cuspidatum. MATERIAL AND METHODS: The anti-acidogenic substances were separated by a series of liquid-liquid fractionations followed bynormal-phase silica gel liquid chromatography, based on high-performance liquid chromatography and glycolytic pH-drop assay. The effectiveness of the separated substances on the acidogenicity of Streptococcus mutans UA 159 was examined using sodium fluoride as a positive control. The chemical composition and quantities of the components of the substances was also assessed by qualitative&#8211;quantitative chromatographic analysis. RESULTS: Among the substances separated from P. cuspidatum, F3 showed the strongest inhibitory effect on the acidogenicity of S.mutans UA 159 in a dose-dependent manner without displaying any bactericidal activity. F3 decreased the acidogenicity of S. mutans even at 12.5 &micro;g/ml (p < 0.05). F3 consisted mainly of resveratrol and emodin (C14H12O3 and C14H4O2(OH)3CH3, respectively), which made up approximately 60 % of the weight of F3. CONCLUSION: F3 can be considered a promising agent for controlling the acidogenicity of S. mutans and subsequent dental caries formation.

마우스 삼차신경 척수로핵 미측소핵 아교질 신경세포에서 Bicuculline Methiodide (BMI)의 비 GABAA 수용체 매개성 반응

최순정 전북대학교 치의학대학원 2010 국내박사

Bicuculline is one of the most commonly used type A gamma aminobutyric acid (GABAA receptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide (BMB) are preferred. However, a number of studies have shown that BMI has non-GABAA receptor mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. However, little is known about non-GABAergic action of BMI on the SG neurons of Vc in mice. This study investigated whether BMI has a non-GABA receptor mediated activity on SG neurons of Vc in mice using a whole cell patch clamp technique. The majority of SG neurons tested were depolarized by application of BMI (20 μM) in condition of a high Cl- pipette solution (n=36/54, 66.7%). GABA (30-100 μM) also induced membrane depolarization of SG neuron. Although BMI is known to be a GABAA receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PIC), a GABAA and GABAC receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PIC or an antagonist mixture consisting of tetrodotoxin (TTX, Na+ channel blocker), AP-5 (NMDA receptor antagonist), CNQX (non-NMDA glutamate receptor antagonist), and strychnine (glycine receptor antagonist). In addition, BMI induced inward current in voltage clamp mode, however BMB (bicuculline methbromide) and BMC (bicuculline methchloride), another water-soluble derivative of bicuculline are induced outward currents on SG neurons of Vc in mice at dose dependent manner. These data demonstrate that BMI has non-GABAA receptor mediated action and suggest that the non-GABAA receptor mediated action can be mediated by iodide. Further studies are needed to investigate the precise action mechanism underlying the BMI-induced non-GABAA receptor mediated action on the SG neurons of Vc in mice.

세포치료를 위한 제대혈과 말초혈액유래 혈관내피전구세포의 단백체학적 분석

전영주 전북대학교 치의학대학원 2013 국내박사

Vasculopathy due to ischemia in damaged tissues is a major cause of morbidity and mortality. To treat these conditions, endothelial progenitor cells (EPCs) from various sources, such as umbilical cord or peripheral blood, have been the focus of the regenerative medicine field due to their proliferative and vasculogenic activities. However, the fundamental, molecular-level differences between EPCs obtained from different cellular sources have rarely been studied. In this study, I established endothelial progenitor cells derived from cord blood and peripheral blood (CB and PB-EPCs) and investigated their fundamental differences at the cellular and molecular levels through a combination of stem cell biology techniques and proteomic analysis. Our results suggest that specifically up-regulated factors such as STMIN 1, CFL 1, PARK 7, NME 1, GLO 1, HSP 27 and PRDX 2 in CB-EPCs as key elements that could be functionally active in ischemic regions. I also discussed functional behaviors important for inducing and maintaining long-lasting blood vessels under ischemic conditions. As a result, CB-EPCs retained a higher anti-oxidant and migration ability than PB-EPCs in vitro. Furthermore, CB-EPCs retained a higher therapeutic efficacy than PB-EPCs in a hindlimb ischemic disease model. The up-regulated expression pattern of STMIN 1, CFL 1, PARK 7, NME 1, GLO 1, HSP 27 and PRDX 2 was confirmed under several conditions in vitro and in vivo, indicating that the up-regulation of these molecules in CB-EPCs may be critical to the mechanism of healing in ischemic conditions and that CB-EPCs may be more appropriate for inducing neo-vessels. Thus, these results may aid in predetermining which cell sources will be of value for cell-based therapies of pathological conditions and identify several candidate molecules that may be involved in the therapeutic mechanism for ischemia.

Terrein 처리에 의한 티타늄 표면에서 골아세포의 생체활성 향상에 관한 연구

오영택 전북대학교 치의학대학원 2010 국내박사

Titanium is biocompatible with bodily tissues. However, the formation of reactive oxygen species(ROS) on the titanium surfaces might have negative response of the activity of the surroundings cells. Terrein was isolated from Penicillium sp. 20135 and found to reduce the effects of LPS-induced inflammation. This study examined the role of Terrein on the biocompatibility of titanium to determine if it can help improve osseointegration. MC-3T3 E1 cells were grown on titanium surfaces. The biocompatibility of Terrein was examined by adding it directly to the culture media at the indicated concentration. The cells on the titanium surface produced excessive ROS and decreased the activity of Cu/ZnSOD and MnSOD. Moreover, the cells had higher activity towards oxidative stress molecules, such as MAPK, FAK and iNO expression. In addition, MC-3T3 E1 osteoblast-like cells promoted osteoclast differentiation but reduced osteoblast differentiation and mineralization on the titanium surface. Interestingly, the cells given the Terrein treatment showed higher resisting toward oxidative stress through the up-regulation of ERK1/2 and FAK activity but the down-regulation of SAPK/JNK and iNO activity. Moreover, Terrein promoted osteoblast differentiation and bone mineralization to elevate the activity of ALP, SPARC and down-regulate RANKL expression after blocking NF-kB translocation from the cytosol to the nucleus. In conclusion, the presence of Terrein on titanium surfaces increases osteoblast cell growth with out inflammation. Moreover, Terrein, as a putative antioxidant agent, enhances osseointegration by decreasing the level of ROS and having a potentially synergistic effect on osteoblast differentiation.

X선 조사가 생쥐 두개골 골모세포의 분화 및 무기질 축적에 미치는 연구

박순선 전북대학교 치의학전문대학원 2013 국내박사

Radiotherapy is considered to cause detrimental effects on bone tis-sues eventually increasing bone loss and fracture risk. However, there is a great controversy on the real effects of irradiation itself on osteoblasts. The mechanisms by which irradiation affects osteoblast differentiation and min-eralization are also not completely elucidated. This study explored how does X-ray radiation influences differentiation and bone-specific gene expression using mouse primary calvarial osteoblast cultures. Irradiation at 2 Gy not only increased differentiation and mineralization of the cells, but also increased the synthesis of collagenase-digestible proteins at early stages of differentia-tion. Irradiation at the same dose also up-regulated the mRNA expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin mostly at early stages of differentiation. However, irradiation at higher doses (> 2 Gy) did not stimulate osteoblast differentiation, rather it suppressed DNA synthe-sis by the cells without cytotoxicity at a significant level. Additional experi-ments revealed that transforming growth factor-beta 1 and runt-related tran-scription factor 2 play important roles in the irradiation-stimulated bone dif-ferentiation by acting as the up-stream regulators of the bone-specific mark-ers. Collectively, these results suggest that irradiation at relatively low doses (≤ 2 Gy) may stimulate osteoblast differentiation and mineralization in pri-mary cultured osteoblasts, whereas it higher than the doses leads to harmful effects on osteoblastogenesis.

한국성인의 제1대구치, 중정치 생존여부에 영향을 미치는 요인에대한 로지스틱회귀분석

김종배 전북대학교 치의학대학원 2010 국내박사

과산화수소 유도성 치주인대섬유모세포의 증식과 조골세포 분화 촉진에 관한 기전 연구

최영지 전북대학교 치의학전문대학원 2013 국내박사

Numerous studies have shown that hydrogen peroxide (H2O2) inhibits proliferation and osteoblastic differentiation in bone-like cells. Human peri-odontal ligament fibroblasts (PLF) are capable of differentiating into osteo-blasts and are exposed to oxidative stress during periodontal inflammation. However, the cellular responses of PLF to H2O2 have not been identified. The aims of this study were to examine how H2O2 affects the viability and proliferation of PLF by exposing the cells to glucose oxidase (GO) or direct addition of H2O2 and to explore the effects of GO on the osteoblastic differ-entiation of PLF and the mechanisms involved. The viability and prolifera-tion in PLF were increased with the addition of 10 mU/ml GO but not by volumes greater than 15 mU/ml or by H2O2 itself. GO-stimulated DNA syn-thesis was correlated with the increase in cyclin E protein levels in the cells. Osteoblastic differentiation of PLF was also augmented by combined treat-ment with GO, as evidenced by the increases in alkaline phosphatase activity, mineralization, collagen synthesis, and osteocalcin content in the cells. The inductions of runt-related transcription factor 2 and osterix mRNA and pro-teins were further increased in PLF incubated in combination with GO com-pared to those in untreated cells. These results demonstrate that the continu-ous presence of H2O2 stimulates the proliferation of PLF and augments their potential to differentiate into osteoblasts through the up-regulation of bone-specific transcription factors. Collectively, the present findings suggest that H2O2 may elicit the functions of PLF in maintaining the dimensions of the periodontal ligament and in mediating a balanced metabolism in alveolar bone.

4종류 인산염 함유 전해액으로 양극산화 및 열수처리한 티타늄의 표면 특성

전우용 전북대학교 치의학대학원 2007 국내박사

This study was performed to evaluate the surface characteristic of titanium modified by anodic spark oxidation and hydrothermal treatment. Electrolytic compositions of the experimental groups are as follow; GA: 0.015 M DL-α-glycerophosphate disosium salt hydrate (DL-α-GP-Na2) and 0.2 M calcium acetate (CA), GB: anodized in 0.015 M β-GP-Na2 and 0.2 M CA, GC: anodized in 0.015 M GP-Na2 and 0.2 M CA, and GD: anodized in 0.015 M GP-Ca and 0.2 M CA. Anodic spark oxidation was performed at 30㎃/㎠ to 290V. In addition, the anodized samples were treated hydrothermally at 300℃ for 2h in a autoclave system. The results obtained in this study were summarized as below; 1. Regardless the electrolytic compositions, the anodic oxide films showed the pores of ~5㎛ in size on the titanium surface and the size of diameter was larger at the bulgy parts than that at the hollow parts. 2. The main crystal phase of anodic oxide layer was anatase phase, and additionally mixed weak rutile phase. 3. HA crystals were precipitated on the porous titanium oxide layer by the hydrothermal treatment, Moreover. the shape of HA crystals was shown to be the dense fine needle shape but different by electrolytic composition. 4. Mean surface roughness (Ra) of group GB was 0.437㎛, and it was highest value. Ra values of hydrothermally treated group was increased to about 0.14㎛에서 0.2㎛ rather than anodized groups. 5. Anodic spark oxidation and the hydrothermal treatment resulted in increasing corrosion potential and decreasing corrosion current density, which means the improvement of corrosion resistance. 6. In Hanks' solution, surface activities decreased in descending order as following: GD, GA, GB, and GC.

Actinobacillus actinomycetemcomitans cytolethal distending toxin subunit CdtA의 유전자 클로닝과 단백질 발현

고선영 전북대학교 치의학대학원 2007 국내석사

Cytolethal distending toxin(CDT)은 세포 주기 중 G2에서 M 기로의 전환을 막아 세포의 증식을 억제할 수 있는 세균 단백 독소의 일종이다. 구강 미생물 중 유일하게 Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans)만이 이 CDT를 생성 할 수 있는 것으로 알려져 있다. A. actinomycetemcomitans는 Localized Aggressive Periodontitis (LAP)의 원인균으로 여겨지며 비 운동성의 그람 음성 구형 간균이고 37℃, 5% CO_(2) 하에 성장이 왕성하다. A. actinomycetemcomitans 의 CDT는 3개의 인접한 유전자인 cdtA, cdtB, cdtC 에 의해 형성 되며 각각의 유전자에 대한 단백질의 기능은 아직 완전히 밝혀지지 않았다. 현재까지 연구에 의하면 cdtA는 CDT의 세포부착과 관련이 있는 것으로 여겨지며 이 유전자의 기능 이상 시 CDT 의 독성 효과가 현저히 감소한다고 알려져 있다. 따라서 본 연구는 A. actinomycetemcomitans 의 cdtA 유전자를 클로닝, 단백질 발현하여 향후 치주질환의 발병 과정에서 CdtA의 역할을 규명하고 질환의 예방 및 치료법에 도움을 주고자 하였다. A. actinomycetemcomitans Y4 균주를 cdtA 유전자 클로닝을 위해 사용하였다. A. actinomycetemcomitans 의 genomic DNA는 genomic DNA 추출 kit를 사용하여 분리하고 cdtA에 특이적인 primer를 이용하여 PCR을 통해 cdtA 유전자를 증폭하였다. 증폭된 cdtA 유전자를 T-vector에 클로닝 하였으며, 클로닝 된 cdtA 유전자는 단백질 발현을 위해 pRSET A vector에 서브클로닝 한 후 발현 균주인 BL21(DE3)에서 발현되었다. 발현 후 Ni-NTA AP conjugate를 이용한 Western blot을 통해 pRSET-CDTA를 확인하였다.