담즙을 이용한 담도암 관련 유전자 변이분석

왕홍은 전북대학교 의학전문대학원 2014 국내석사

Parthenolide의 항섬유화 효과

김인희 전북대학교 의학전문대학원 2010 국내박사

배경/목적: Parthenolide(PT)는 식물에서 유래한 sesquiterpene lactone으로 nuclear factor kappa B(NF-κB) 억제효과와 세포내 활성산소종(ROS)의 증가를 통해 여러가지 암세포들에서 세포사멸을 일으킨다고 알려져 있다. 본 연구에서는 PT가 백서 간성상세포에서 세포사멸을 유도함을 확인하고, thioacetamide 유도성 백서 간섬유화 모델에서 항섬유화 효과가 있음을 규명하고자 하였다. 방법: 백서 간성상세포주(RI-T 세포)에서 PT의 효과를 알아보기위해 세포성장억제, 세포사멸, NF-κB 결합 활성도, 세포내 ROS, 글루타치온(GSH) 등을 측정하였다. 또한, thioacetamide 유도성 백서 간섬유화 모델에서 PT의 항섬유화 효과를 평가하였다. 결과: PT는 MTT 측정법에서 RI-T 세포의 성장을 억제하였으며 유세포분석상 sub-G1 DNA 양의 증가, annexin V 염색세포의 증가 소견을 보여 세포사멸을 유도함을 알 수 있었다. PT에 의한 RI-T 세포의 세포사멸은 세포내 ROS와 superoxide anion (O2·-)치의 증가와 세포내 GSH치의 감소와 연관이 있었다. 항산화제인 N-acetyl-cysteine을 투여하였을 때 PT에 의한 세포사멸이 유의하게 감소하였으며, L-buthionine-sulfoxamine을 투여하였을 경우 더욱 증가하였다. PT는 Bax의 발현 증가, Bcl-2와 Bcl-XL의 발현 감소를 통해 미토콘드리아막전위의 소실과 caspase-3, 8의 활성화, poly (ADP-ribose) polymerase (PARP)의 분리를 유발하였다. 한편, PT는 RI-T 세포에서 TNF-α 유도성 NF-κB 결합 활성도를 억제하였으며 α-smooth muscle actin(α-SMA)과 collagen α-1의 발현을 감소시켰다. Thioacetamide 유도성 백서 간섬유화 모델에서 PT는 간섬유화를 유의하게 억제하였다. 이는 백서 간조직에서 α-SMA과 transforming growth factor-β1(TGF-β1)의 발현 감소, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) 염색 양성인 세포의 증가와 연관이 있었다. 결론: PT는 백서 간성상세포에서 세포사멸을 유도하고 thioacetamide 유도성 백서 간섬유화 모델에서 간섬유화를 유의하게 개선시켰다.

Interleukin-12에 의한 Nitric Oxide생성과 면역조절에 관한 연구

임주연 전북대학교 의학전문대학원 2011 국내석사

IL-12-activated lymphocytes secrete inflammatory cytokines such as IFN-γ and TNF-α that can induce nitric oxide (NO) synthesis via inducible NO synthase (iNOS) in macrophages. In this study, we investigated IL-12-activated lymphocyte-mediated macrophage apoptosis via nitric oxide (NO) synthesis. Culture supernatants of IL-12-activated lymphocytes induced NO synthesis in murine thioglycollate-elicited peritoneal macrophages. NO synthesis was markedly inhibited by blocking antibodies to IFN-γ and TNF-α suggesting the key role of these lymphocyte cytokines in mediating NO synthesis. Endogenous NO production inhibited macrophage proliferation, and induced apoptosis in concordance with the accumulation of p53, PTEN and DR5, and the decrease in the 32-kDa caspase-3 precursor, processes that were inhibited by NG-monomethyl-L-arginine (a competitive NO synthase inhibitor), and that were also suppressed in macrophages obtained from iNOS-knockout mice. The present study demonstrated a novel, non-contact-dependent mechanism of macrophage suppression by IL-12-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages due to endogenous NO synthesis induced by cytokines secreted from IL-12-activated lymphocytes.

악하선 심부 타석증에 대한 수술적 고찰

알탕후절자르갈 전북대학교 의학전문대학원 2011 국내석사

Submandibular salivary stones account for most symptomatic sialoliths and most are treated by adenectomy. Transoral removal of proximal or hilar stones is an alternative approach that preserves the functioning gland. Between 2005 and 2010, 120 consecutive patients with the symptomatic stones in the submandibular gland, who underwent stone removal, have been reviewed at Chonbuk National University Hospital. None of the patients had a prior history of stone ? removal surgery, and all patients had normal lingual nerve function prior to the surgery. All subjects were informed of their right to abstain from participation in the study. 55 (48%) of were male and 62 (52%), side of the stone(s), there were found amounts equal to 58 (48%) patients with LT, 54 (45%) patients with RT and 8 (7%) patients with both stones existent in both sides. In comparing the two alternatives, our results demonstrate that intraoral removal of proximal submandibular stones with preservation of the gland and ductal system is safe and efficacious. In addition, there was not objective evaluation for the function of salivary gland after surgery. Since mouth dryness and meal-time syndrome are symptoms that reflect the function of the salivary gland, patients have admitted not experiencing these symptoms so it is considerable that the salivary flow was intact after surgery.

프리온 분해 단백질 발견과 angiopoietin-like Proteinol 실험실 조건에서 조혈모세포 증식과 생존에 미치는 영향

샤히나아크터 전북대학교 의학전문대학원 2013 국내박사

Removal of misfolded prion (PrPSc) in brain and in vitro expansion of hematopoietic stem cells (HSCs) are the most and important challenges in biomedical sciences. During my PhD researches, I identified a serine protease that degraded specifically PrPSc, and studied in vitro HSCs expansion by angiopoietin-like proteins. Using the bipartite galactosidase-4-upstream activating sequence (GAL4-UAS) expression system, I generated transgenic Drosophila which pan-neuronally expressed wild-type (WT) mouse prion protein (mPrP) or the serine protease or co-expressed mPrP with the serine protease. Abnormal accumulation of PK-resistant PrPSc, reduced lifespan, locomotor dysfunction, and rough eyes were resulted in 40 to 60-day-old mPrP expressed Drosophila indicating a toxic dominant mechanism of PrP for the etiopathogenesis of prion diseases (PrD). By contrast, co-expression of the serine protease with PrP showed that the serine protease completely detoxified misfolded PrP-induced neurotoxicity by degrading PK-resistant misfolded PrP, thereby preserved the normal lifespan along with strong positive locomotion, and normal eye development. In vitro enzymatic assay also showed that the serine protease had proteolytic activity toward PK-resistant misfolded PrP and this proteolysis was inhibited by UCF-101, a serine protease specific inhibitor. However, transgenic flies expressing serine protease displayed no pathological or phenotypical abnormalities rather it showed increased lifespan along with strong negative geotactic response. Confocal microscopic studies showed that the serine protease clearly detoxified abnormally accumulated PrP in transgenic Drosophila and co-localizes with PrP in the endoplasmic reticulum (ER). In vivo analyses of mutated serine protease demonstrated that mutant serine protease failed to alleviate the abnormal accumulation of α-synuclein that causes not only PD but also plays a role in regulating abnormal accumulation of misfolded PrP. I studied the functional role of mouse Angptls 1, 2, 3, 4, 6, and 7 and growth factors SCF, TPO, IGF-2, and FGF-1 on purified mouse Sca-1+ HSCs. None of the Angptls stimulated HSCs proliferation, enhanced, or inhibited HSCs colony formation, but they did support the survival of HSCs. By contrast, any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3- to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone leading to an understanding of the basic function of Angptls and growth factors on signaling pathways for the survival as well as expansion of HSCs. Altogether, the results I demonstrate here addressing that the identified serine protease acts to detoxify misfolded PrP to protect PrD and the beneficial effects of Angptls on HSCs survival and expansion.

단삼의 난소제거 흰쥐에 대한 항골다공증 효과

최언 전북대학교 의학전문대학원 2011 국내박사

Osteoporosis is characterized by compromised bone strength predisposing a person to an increased risk of fracture. Long-term administration of currently prevalent medicaments may lead to an increased risk of severe side effect like cancer. Accumulating evidence suggest Salvia Miltiorrhiza have anti-osteoporotic effect. To seek out natural products that can manage osteoporosis and concurrently have less side effects, we observed anti-osteoporotic property of Salvia Miltiorrhiza in ovariectomized rats by assessment of serum biochemical parameters, bone turnover markers, inflammatory cytokines and oxidative stress parameters, measurement of bone mineral density (by dual-energy X-ray absorptiometry), analysis of trabecular bone structural parameters (by microcomputerized tomography scanning) and histological analysis of bone and liver. Result of experiment: ovariectomy significantly reduced bone mineral density and trabecular bone architecture, increased bone turnover, inflammatory cytokines and oxidative stress parameters. ovariectomy also aggravated mononuclear cellular infiltration in liver portal area induced by aging. while SM treatment significantly ameliorated bone mineral density and trabecular bone mass decrease and increase in inflammatory cytokines and oxidative stress parameters by the 30 mg/kg body weight/day dosage. ovariectomy significantly increased bone turnover and hence induced trabecular bone loss. Salvia Miltiorrhiza treatment significantly attenuated decrease in bone mineral density and trabecular architecture deterioration induced by ovariectomy. The preventive effect of SM was presumably by its anti-oxidative stress and anti-inflammatory activity partly via modulating bone turn over rate. In current study, Salvia Miltiorrhiza is suggested as a promising osteoporosis therapeutic natural product.

Optical Imaging of the MMP Expression and Cancer Progression in an Inflammation-Induced Colon Cancer Model : 염증성 대장암 모델에서 matrix metalloproteinases (MMP)의 발현과 암 성장의 광학 영상화 연구

Jang, Doo-rye 전북대학교 의학전문대학원 2011 국내석사

Purpose: Human colorectal cancer is one of the most common fatal malignancies. The purpose of this study was to use a near-infrared (NIR) fluorescent cyclic His-Try-Gly-Phe peptide to characterize and image the expressions of matrix metalloproteinases (MMPs), which are correlated with cancer promotion, in a mouse model of inflammation-induced colorectal cancer (ICRC). Materials and Methods: My first explored the relationship between the development of colon cancer and the expression of MMPs at the same colonic sites in ICRC models. MMP-2 expression and β-catenin activation in colonic lesions were characterized by immunohistochemical (IHC) staining. After verifying the expressions of the two proteins in induced cancer cells in the colon, c(KAHWGFTLD)NH2 (C6) peptide was prepared by standard Fmoc peptide synthesis to target MMPs. To develop a mouse model of ICRC, mice were administered a single intraperitoneal dose (10 mg/kg body weight) of azoxymethane (AOM) and exposed orally to 2% dextran sodium sulfate (DSS) for one week. In vivo MMP-targeted imaging of Cy5.5-C6 was performed using an IVIS optical imaging system in the ICRC model. Results: The molecular weight of Cy5.5-C6 as determined by maldi-tof-ms analysis was 1954.78 (calculated MW = 1955.23). In the ICRC model, histological examination of colon cancer tissues was performed. After being treated with inducers for some time, cancerous lesions were found to express high β-catenin and MMP-2. The expressions of MMP profiles were correlated with MMP expression with β-catenin activation in the colonic lesions. The in vitro characterization of Cy5.5-C6 showed MMP binding specificity in a cell experiment. In vivo NIR fluorescence imaging showed high accumulation of Cy5.5-C6 in tumors with associated expression of MMP-2 in colonic lesions after intravenous injection. Conclusion: The MMP-2 specificity of Cy5.5-C6 was confirmed by successful inhibition of probe uptake by the tumor in the presence of excess C6 peptide. The use of Cy5.5-C6 to target MMP-2 has the potential to be developed into an effective molecular imaging agent to monitor ICRC progress.

소포체 스트레스 조절자에서의 약동학과 약리학의 기전연구 : Pharmacokinetic and pharmacodynamic studies on endoplasmic reticulum stress regulators

마라하타아누 전북대학교 의학전문대학원 2014 국내박사

In my study, three parts are included. I have been focusing the pharmacokinetic profiles and its associated biological effect/dynamic effects. Therefore my thesis is composed of the following three parts. Part I: 4-Phenylbutyric acid regulates CCl4-induced Acute Hepatic Lipid Accumulation in a Mouse Model: A Mechanism-based PK/PD study We aimed to document ER stress as a possible clinical biomarker and to introduce 4-PBA as a therapeutic chemical for alleviation of ER stress-related diseases. Male C57B/L mice at eight weeks were used. Mice were divided into three groups: non-treatment, CCl4 alone, and CCl4+ 4-PBA. Treatment with 4-PBA inhibited ER stress by upregulating expression of GRP78 and CHOP, thereby preventing hepatic lipid accumulation. Further, liver triglyceride accumulation was lowered along with decreases in lipid peroxidation and cytochrome P450 2E1 activity. We also studied mechanism-based pharmacokinetic and pharmacodynamic profiles, introducing ER stress-related proteins GRP78 and CHOP, along with serum apolipoprotein B and triglyceride levels, as novel biomarkers for ER stress-induced hepatic lipid accumulation. ER stress and its clinical relevance for therapeutic approaches were well correlated with the activity of the ER stress regulator 4-PBA, which may be a promising drug for treatment of hepatic lipid accumulation. Part II: IC87114 alleviates asthma by inhibiting ER stress through IRE1 dependent pathways by modulating NF-B signaling pathways PI3K plays a pivotal role in the recruitment and activation of certain inflammatory cells. Using mice sensitized with ovalbumin (OVA) and LPS and challenged with OVA only, we investigated the effect of IC87114 on pathogenesis of asthma. Our data show that the increased numbers of inflammatory cells, increased airway hyperresponsiveness, levels and mRNA expression of IL-4, IL-5, IL-8, IL-13 and IL-17 in the bal fluid after LPS plus OVA inhalation were significantly reduced by administration of IC87114 in a time dependent manner. The LPS plus OVA mice showed that the expressions of ER stress markers in lungs were significantly increased compared with the control. IC87114 significantly reduced the increases in ER stress through IRE1 dependent specific pathway. Thus, inhibiting mRNA of spliced XBP1. Moreover, LPS plus OVA induced increases of NF-B were lowered by IC87114 treatment. The degradation of IB was also significantly decreased by IC87114 in LPS plus OVA treated mice. These results suggest that inhibition of PI3K relief asthma by decreasing UPR through IRE1/XBP1 specific pathway through modulation of NF-B/IB signaling pathway. Part III: Soybean greatly reduces valproic acid plasma concentrations: A food–drug interaction study The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively, while the tmax and MRT of VPA were not different between groups. In addition, the AUC of valproic acid decreased to 83% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration; however, this was less pronounced in the group that was pretreated with soy extract than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

피부결손부위 복원을 위해 피부 테이프(Steri-strip)를 이용한 sutureless Burow's graft

조용선 전북대학교 의학전문대학원 2011 국내석사

피부암 등의 수술후 발생한 결손 부위의 복원 방법은 다양하다. 이중 Burow's graft는 인접피부를 공여부로 이용하는 편리한 방법으로, 이식편의 고정에 압박고정드레싱하는 방법이 흔히 이용되나, 저자들은 피부테이프를 이용하여 이식편을 고정한 후 결과를 평가하였다. 50세에서 84세(평균 72세) 사이의 25명(남자 9명, 여자 16명)의 환자들에서 발생한 26례의 피부종양을 제거한 후 발생한 결손부를 Burow's graft 로 재건하였으며, 이때 이식편은 피부테이프(Steri-strip)를 이용하여 고정하였다. 병변은 광선 각화증이 1예(3.8%), 사마귀모양암종이 1예(3.8%), 각화극세포종이 2예(7.7%), 기저세포암이 10예(38.5%), 편평상피세포암이 12예(46.2%) 였으며, 가슴에 위치한 1예를 제외한 25예는 얼굴에 위치하였다. 병변의 크기는 최대직경이 평균 1.0cm(0.4-2.4cm)이었으며, 결손부위의 크기는 최대직경이 평균 1.3cm(0.5-3.0cm)이었다. 결과에 대한 평가는 수술 후 1, 3, 6개월 시점에 추적관찰하여 임상 사진을 촬영하고 색감, 질감, 흉터 및 합병증의 관찰을 통하여 이루어졌으며, 만족스러운 결과를 보였다. 또한, 전통적인 방법에서 흔히 볼 수 있는 봉합에 의한 부작용이 없었으며, 시술시간이 단축되었다. 피부 테이프를 이용한 burow's graft 이식편의 고정은 고전적인 방법의 대안이 될 수 있을 것이라 생각한다. Background: There are several methods for reconstruction of defect after removal of skin tumor, and skin graft is commonly used. Classically, in full-thickness skin graft(FTSG), nylon basting sutures with a tie-over bolster dressing are used in securing skin graft to the recipient wound bed, but this method is complicated and time-consuming. Moreover, suturing in this method may pull the edges of the skin with too much tension, cause necrosis, crater-like deformity and suture marks. Recently to make up for these demerits of the classic method of FTSG, various other methods and materials are being attempted. Objective: The aim of this study was to evaluate the usefulness of an adhesive skin tape(Steri-strip) for securing a graft to recipient wound bed without suture when performing a Burow's skin graft, one of types of FTSG. Methods: Twenty five patients with twenty six surgical defects following Mohs'micrographic surgery of non-melanoma skin cancer and excision of benign tumor were included in this study. After graft in place, it was fixed by the Steri-strip and a light compressive dressing was applied over the graft. The cosmetic results were scored as excellent, good, fair or poor at the time of 1, 3, 6 months after the operation. Also the size of defect was measured and the run-time of operation for skin graft was checked. And we statistically evaluated these figures. Results: The mean age of the patients was 72.3 years old and 9 men and 16 women were enrolled. There were 12 cases of squamous cell carcinoma, and 10 cases of basal cell carcinoma, 2 cases of keratoacanthoma, 1 case of verrucous carcinoma and 1 case of actinic keratosis. 25 defects were located on the face(16 cases on the cheek, 4 cases on the lip, 3 cases of the nose, 2 cases on the forehead), and the other one was on the chest. The size of surgical defects ranged from 0.5 to 3.0 cm at the greatest diameter(mean: 1.3 cm). The mean run-time of operation for skin graft was 23.1 minutes. There were no graft failures or acute complications of skin graft such as infection or hematoma. The mean cosmetic results on the 6 months after the operation were satisfactory and there were no correlations between the size or location of the defect and the mean assessment scores statistically at all period(p>0.05). Conclusion: We suggest that sutureless Burow's graft with skin tape might be an easy and useful method to reconstruct the defect after skin surgery.

소금으로 유도된 고혈압과 대사성산증에서 Bax inhibitor-1의 역할의 연구 : The study of the role of Bax Inhibitor-1 in salt induced hypertension and metabolic acidosis

반다리비두르 전북대학교 의학전문대학원 2014 국내박사

Regulation of sodium balance is a critical factor in the maintenance of body fluid, and imbalance of renal sodium excretion could result in altered intravascular volume, such as hypertension. Since Bax Inhibitor-1, a recently studied Ca2+ channel-like protein, affects cationic balance including Ca2+ and Na+, it was applied to a sodium-induced hypertension model. Data shows that Na+/H+ exchange activity was higher in wild type mice compared to bax inhibitor-1 knock-out mice and remained elevated. Compared to the bax inhibitor-1 knock-out mice, brush border membrane vassicles from wild type mice exhibited a faster rate of acridine orange fluorescent recovery that was sodium dependent. Membrane levels of NHE3 protein and mRNA were also increased in a relatively similar proportion to the activity, suggesting that the relative over-abundance of protein in the apical membrane could account for the increase in Na+/H+ exchange activity. Apart from sodium balance regulation, the kidney also has an important role in the regulation of acid-base homeostasis. Renal acid production and excretion are essential for net acid excretion under basal conditions as well as during metabolic acidosis. Acid is excreted into the urine at the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH3+ diffusion coupled to H+ secretion. In this study, I was able show that this process is dependent on bax inhibitor-1. Bax inhibitor-1 knock out mice has abnormal urinary acidification due to impaired acid secretion in acid loading – a feature of distal renal tubular acidosis. I also found that bax inhibitor-1 regulates NaK-ATPase which concomitantly alters roles of V-ATPase to assist urine acidification during acid loading. These studies clearly assist to suggest critical role of bax inhibitor-1 in high salt induced hypertension and metabolic acidosis in mice.