The Implementation of Smart Home System Based on 3G and ZigBee in Wireless Network System

Liu Dan Chonnam National Univ. 2012 국내석사

Heterologous expression of B. Subtilis protoporphyrinogen oxidase in E. coli

Kim, Myojoung Chonnam National Univ. Graduate School 1999 국내석사

ppGpp-mediated regulation of salmonella virulence gene expression

송미령 Chonnam National Univ. 2007 국내박사

Chapter 1 We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1) during growth under the physiologically well-defined standard growth condition of LB medium with aeration. We found that the central regulator hilA and the genes under its control are expressed at the onset of stationary phase. Interestingly, two-component regulatory genes, hilC/hilD, sirA/barA, and ompR, known to modulate expression from the hilA promoter (hilAp) under so-called ‘inducing conditions (LB medium containing 0.3 M NaCl without aeration)’ acted under standard conditions at the stationary phase induction level. The induction of hilAp depended not on RpoS, the stationary phase sigma factor, but on the stringent signal molecule, ppGpp. In the ppGpp null mutant background, hilAp showed absolutely no activity. The stationary phase induction of hilAp required spoT but not relA. Consistent with this requirement, hilAp was also induced by carbon source deprivation, which is known to transiently elevate ppGpp mediated by spoT function. The observation that amino acid starvation elicited by addition of serine hydroxamate did not induce hilAp in a RelA+ SpoT+ strain suggested that in addition to ppGpp some other alteration accompanying entry into stationary phase might be necessary for induction. It is speculated that during the course of infection, Salmonella encounters various stressful environments that are sensed and translated to the intracellular signal, ppGpp, that allows expression of Salmoenella virulence genes including SPI1 genes. Chapter 2 Salmonella enterica is highly diverse in terms of genome structure, which is at least partly due to the horizontal transfer of genetic elements from various sources. In this study, we examined the expression profiles of such genes in Salmonella Pathogenicity Islands (SPIs) and the cob/pdu locus, horizontally acquired large DNA segments, during growth under physiologically well defined standard growth conditions. Transcripts from exponentially growing and early stationary phase Salmonellae were compared by cDNA microarray analysis. Nearly all genes encoded by SPIs and the cob/pdu locus were induced at the onset of stationary phase. Induction of this class of genes required the stringent signal molecule, ppGpp, but not the stationary phase sigma factor, RpoS. This finding was further verified by primer extension analysis of transcripts from major promoters in SPIs, and assaying for -galactosidase activity with cob::lac gene fusion constructs. Based on the results, we propose that these genes are stress-inducible, and that the stressful environment at entry into stationary phase during bacterial growth under standard conditions causes non-specific induction of these genes. This stress is sensed and translated to the intracellular signal, ppGpp, which in turn stimulates the expression of the stress-inducible genes encoded by horizontally acquired DNA. Chapter 3 In vitro studies have an advantage for the exclusion of indirect effects caused in vivo by cascades of events but also disadvantage with identifying the factors that does not bind directly. Use of crude cell extract, S-150, which contains transcription apparatus and also various cytosolic components, could be advantageous to examine regulation involving multiple unidentified components. We measured transcription regulation by ppGpp using DNA-directed transcription of S-150. Two model promoters employed were hisG promoter activated by ppGpp and leuV promoter (tRNAleu) repressed by ppGpp. In order to understand the interrelationship between ppGpp and DksA in ppGpp-dependent transcription activation or repression, we used these promoters as models. First S-150 dependent in vitro reaction was optimized including the concentrations of DNA templates and cations for each promoter: DNA 120 nM, K+ 200 mM, and Mg2+ 15 mM. Under this condition, we observed ppGpp-dependent activation of hisGp and repression of leuVp. The involvement of DksA in the regulation by ppGpp was demonstrated by using S-150 prepared from dksA mutant. Neither the activation nor repression was observed. It was suggested that the ppGpp-dependent regulation requires DksA. This result was further examined with purified RNA polymerase. We found that the ppGpp-dependent repression was observed in the presence of DksA but not the activation. It was concluded that an additional factor(s) might be needed in addition toDksA for the ppGpp-dependent activation. We propose that the molecular mechanism of ppGpp-dependent activation may be distinct from that of ppGpp-dependent repression.

Clinical and experimental study of residual shortening after legg-calve-perthes' disease

김명선 Chonnam National Univ. 2005 국내박사

In Legg-Calve-Perthes' disease (LCPD), residual shortening of the affected limb is the important complication causing the limping and the gait abnormality. Residual shortening in LCPD has been reported in many studies. However, the reported results are inconclusive and conflicting in regard to the factors responsible, the role of ipsilateral tibia, and the degree of the shortening. To clarify these issues, residual shortening of the affected limb was measured at skeletal maturity by the teleoro¨ntgenograms in 68 LCPD patients and special attention was paid to the length of the ipsilateral tibia. Of these 68, 38 patients were treated by abduction orthosis (AO) and 30 patients by femoral varus osteotomy (FVO). In addition, the experimental study of a piglet LCPD model was undertaken in order to clarify the developmental pattern of residual shortening in limbs with devascularization of the capital femoral epiphysis. Residual shortening in AO group was significantly greater than that in FVO group (p=0.007). In detail, the femoral lengths in both of these groups were similar; 12.5 mm in AO group and 10.1 mm in FVO group (p=0.261). However, in contrast, the tibial lengths were significantly different; 2.5 mm shortening in AO group and 0.9 mm lengthening in FVO group (p<0.001). In experimental study of a piglet LCPD model, the appearance rate of shortening at each postoperative week showed a tendency to gradually increase with time (r_(s)=0.975, p=0.005). Shortening of the operated femur in millimeters gradually increased with time until skeletal maturity was completed (r_(s)=0.627, p=0.001). In conclusion, the author determined the degree and the frequency of residual shortening in LCPD by measuring the discrepancy of leg length precisely by teleoro¨ntgenograms after the full maturation of the skeleton. Residual shortening in patients treated by FVO (mean 9.2 mm) was less than patients by AO (mean 15.0 mm). The difference is speculated to be caused by the overgrowth of the ipsilateral tibia. Legg-Calve-Perthes병에 있어서 이환된 하지의 단축은 파행과 보행장애를 일으킬 수 있는 중요한 합병증이다. 그러나 많은 연구보고에도 불구하고 하지 단축의 원인인자, 동측 경골의 길이 변화, 그리고 단축의 정도에 대하여서는 논란의 대상이 되어 왔으며 또한 정확한 보고도 매우 드물다. 저자는 이처럼 미해결된 분야를 명백히 하기 위해서 골성장이 완료된 환자 68명을 선택하여 이들을 대상으로 양하지를 한 장의 X-선 사진으로 촬영하는 원격 X-선 촬영법 (teleorontgenography)을 시행하였다. 1973년부터 1990년까지 전남대학교 병원에서 치료하였던 총 345명의 환자중 원격 X-선 촬영을 시행하고 골 성숙기까지 추시가 가능하였던 68명의 환자를 대상으로 하였다. 68명의 환자 중 30명은 수술 요법인 대퇴골 내반 절골술을 시행하고 다른 38명은 비수술적 보조기 요법인 외전 보조기를 착용하였다. 또한 하지단축의 점진적인 발생과정을 확인하기 위하여 생후 4 내지 5 주된 어린 돼지를 이용하여 대퇴 골두 골단의 혈관 경색을 유도하였다. 하지 단축은 외전 보조기군에서 평균 15.0 mm, 대퇴골 내반 절골술군에서 평균 9.2 mm로 외전 보조기군에서 의미있게 높았다 (p=0.007). 구체적으로 이들 양군에 있어서 대퇴골의 길이는 외전 보조기군에서 12.5 mm, 대퇴골 내반 절골술군에서 10.1mm로 두 군간에 의미있는 차이는 없었다 (p=0.261). 반면, 경골의 단축은 보조기군에서 2.5 mm 단축, 대퇴골 내반 절골술군에서 0.9 mm 연장으로 대퇴골 내반 절골술군이 외전 보조기군보다 유의하게 낮았다 (p<0.001). 돼지를 이용한 실험 결과에서는, 지단축은 수술 후 시간이 경과할수록 골성장 종료시까지 점진적으로 증가하였으며 그 증가 정도는 시기에 따라 차이를 보였다(rs=0.975, p=0.005). 시간의 경과에 따른 지단축의 정도는 대퇴골의 길이성장이 계속되는 동안 점진적으로 증가되었다 (rs=0.627, p=0.001). 결론적으로 저자는 골성장기에 도달한 환자를 대상으로 teleorontgenogram을 촬영하여 하지부동을 측정함으로써 지단축의 정확한 정도를 알 수 있었으며, 이러한 지단축은 골성장이 종료될 때까지 점진적으로 증가함을 실험을 통하여 확인하였다. 또한 지단축은 대퇴골 내반 절골술 환자군이 비수술요법인 외전보조기 치료군보다 더 양호함을 확인하였으며 이러한 치료군간의 지단축 차이는 수술 치료군에 있어서 동측 경골의 과성장이 원인임을 알 수 있었다.

Molecular mechanisms of stem cell proliferation under hypoxic condition

이상훈 Chonnam National Univ. 2010 국내박사

Embryonic stem (ES) cells are unique cell populations with the ability to undergo both self-renewal and differentiation into different lineages. Numerous studies have been performed to test hypoxic condition in maintenance of ES cell self-renewal. In addition, other studies demonstrated that ES cells have been coaxed to differentiate into varied cells, simply by changing the culture conditions in which the cells are grown. The results from these studies suggest that a number of mechanisms contribute to the changes in ES cell functions, which occurs in response to exposure to hypoxic condition. Therefore, understanding the effects of the hypoxic condition and its related mechanisms of these factors might help to comprehend the changes in the functions and of ES cell fate. Indeed, preimplantation embryos develop in vivo under conditions of low oxygen. Uterine oxygen concentration decreases to around 3-5% at the time of implantation in the hamster and rabbit. Consistent with this, lowering the oxygen concentration in the gaseous phase during embryo culture, from atmospheric levels to more physiological levels, has been associated with improved embryo development, in terms of blastocyst development rate and embryo cell number, in a number of species. These suggest that hypoxic condition plays important roles in the early-stage of mammalian embryonic development as well as in various physiological functions. Therefore, this study was focused on the effect hypoxic condition in stem cells proliferation. That is, this study investigated 1) Effect of hydrogen peroxide (H2O2) on stem cell proliferation/apoptosis, and its related signal cascades, 2) Effect of hypoxia with AA or hypoxic condition on stem cell proliferation, migration and migration its related signaling pathways. Results were as followings: 1. Effect of hydrogen peroxide (H2O2) on stem cell proliferation and apoptosis Reactive oxygen species generated by a variety of endogenous factors and their roles in ES cells has yet to be identified. Thus, we examined role of AA in H2O2-induced proliferation and apoptosis of mouse embryonic stem cells (mES) cells and its related signaling molecules. AA release was maximally increased in response to 10-4 M H2O2 for 1 hr. In addition, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) and the phosphorylation of protein kinase C (PKC), MAPKs and epidermal growth factor receptor (EGFR), which was blocked by the inhibition of MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H2O2-induced cytosolic phospholipase A2 (cPLA2) activation and AA release. H2O2 increased NF-κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)-2 proteins. Subsequently, H2O2 stimulated PGE2 synthesis, which was reduced by the inhibition of NF-κB activation. Moreover, each H2O2 or PGE2 increased DNA synthesis and the number of cells. However, 10-3 M H2O2 increased the release of lactate dehydrogenase (LDH) and DNA fragmentation but reduced the cell viability in a time-dependent manner (8 hr). Moreover, H2O2 decreased the level of DNA synthesis and the levels of the cell cycle regulatory proteins. These effects of H2O2 were inhibited by a pretreatment with dihydrotestosterone (DHT). However, a treatment with flutamide (androgen receptor inhibitor, 10-3 M) abolished the protective effects of DHT. This result was supported by the presence of the androgen receptor in mES cells. The activity of the antioxidant enzyme, catalase, were increased by the DHT treatment but not by a co-treatment with DHT and flutamide. DHT decreased the intracellular H2O2 levels but flutamide blocked this effect. H2O2 also increased the level of MAPK, and NF-κB phosphorylation, which were inhibited by the DHT and catalase pretreatment. DHT inhibited the H2O2-induced increase in caspase-3 expression and decreased the level of Bcl-2 and the cellular inhibitor of apoptosis protein (cIAP)-2. These effects were abolished by the flutamide treatment. 2. Effect of hypoxic condition on stem cell proliferation and apoptosis Hypoxic condition plays important roles in the early-stage of mammalian embryonic development as well as in various physiological functions. This study examined the effect of hypoxic condition on regulation of proliferation and interrelationship among the possible signaling molecules in mES cells. Hypoxic condition increased the level of [3H] arachidonic acid (AA) releases, and [3H] thymidine incorporation were further increased in hypoxic condition with AA treatment compare with normoxic condition. In addition, hypoxia increased the level of MAPKs and NF-κB phosphorylation and Wnt-1, Notch-1, and hypoxia inducible factor-1α (HIF-1α) expression. The expression of each signaling molecule induced an increase in Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) expression that was greater in hypoxia with AA than in hypoxia alone. The inhibition of IL-6 and VEGF expression using VEGF-targeted siRNA or IL-6 antibody and soluble IL-6 receptor decreased the hypoxia-induced increase in cell cycle regulatory protein expression, DNA synthesis, and cell number. In addition hypoxic condition increased level of PI3K, Akt, mTOR, eNOS, and MAPKs phospholylation and HIF-1α expression. The expression of each signaling molecule induced an increase in VEGF, fibronectin (FN) and integrin β1 (IN β1) expression. Moreover, in this hypoxic condition, focal adhesion kinase (FAK) and Src phosphorylation were increased in time-dependent manners. These increases were obviated by IN β1 antibody. In addition, hypoxia-induced increase of F-actin distribution and cell migration (activation of matrix metalloproteinase-2 and -9) was inhibited by FAK-targeted siRNA and IN β1 antibody. However long term hypoxic condition increased the release of LDH and DNA fragmentation, but reduced cell viability. These effects of hypoxic condition were inhibited by pretreatment with midkine (MK) (100ng/ml). However, low-density lipoprotein receptor-related protein-1 (LRP-1) antibody abolished the protective effect of MK. These results were supported by hypoxia itself and hypoxia with MK increase of LRP-1 expression. MK decreased hypoxia-induced intracellular H2O2 and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation as well as apoptosis indicating loss of mitochondrial membrane potential, release of cytochrome c from mitochondria to cytosol, increase of cleaved caspase-3 expression, and decrease of Bcl-2 and cellular inhibitor of apoptosis protein (cIAP-2) which were reversed by pretreatment of LRP-1 antibody. In addition, blockage of JNK/SAPK and p38 MAPKusing small interference RNA (siRNA) showed decrease of hypoxia-induced apoptosis. Hypoxia with MK increased Akt phosphorylation in early time and reversed hypoxia-induced decreased of heme oxygenase-1 (HO-1). Also, hypoxia with MK increased the expression of hypoxia inducible factor-1 alpha (HIF-1α) whereas decreased von Hippel-Landau (VHL). Inhibition of Akt and HIF-1α prevented MK-induced blockage of apoptosis, suggesting that the protective effect of MK was Akt and HIF-1α-dependent. In summary, 1) H2O2 increased AA release and PGE2 production by the upregulation of cPLA2 and COX-2 via Ca2+/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mES cells. However DHT prevent H2O2-induced apoptotic cell death via the activation of catalase and downregulation of p38 MAPK, JNK/SAPK, and NF-κB via the androgen receptor in mES cells. 2) Hypoxia with AA upregulates G1 phase regulatory proteins. In addition, AA potentiates hypoxic condition-induced VEGF and IL-6 expression in mES cells through the Notch-1, Wnt-1, MAPKs, NF-κB and HIF-1α pathways and Hypoxic condition increases proliferation and migration of mES via FN-IN β1 and VEGF production through the PI3K/Akt, mTOR, eNOS and HIF-1α, FAK pathways. Furthermore MK prevents the hypoxic condition-induced apoptotic cell death of mES cells through the activation of Akt via LRP-1.

Novel core-shell type nanoparticles based on polyelectrolyte complex formation between methoxy poly(ethylene glycol)-grafted chitosan and all-trans retinoic acid

김상효 Chonnam National Univ. 2005 국내박사

The goal of this study is to develope novel types of nanoparticles for delivery of drugs to the brain and to test it against brain tumor cell lines in vitro. Methoxy poly(ethylene glycol) (mPEG)-grafted chitosan (abbreviated as CP graft copolymer) were synthesized to make core-shell type nanoparticles with all-trans retinoic acid (ATRA) based on polyelectrolyte complexes formation. CP nanoparticles encapsulating atRA could prepared by mixing of ATRA into CP aqueous solution using ultra-sonication followed by dialysis of mixed solution. CP nanoparticles encapsulating atRA have spherical shapes with particle size below 200 nm and showed high loading efficiency. ¹H NMR showed core-shell structure formation between CP graft copolymer and ATRA since specific peak of ATRA was disappeared in D₂O. ATRA was continuously released from nanoparticles over 1 month. At cell cytotoxicity study in vitro, CP nanoparticles encapsulating ATRA showed almost similar cytotoxicity against 9L gliosarcoma, U343MG, U87MG, and U251MG cell lines. At migration test using U87MG cell lines, CP nanoparticles encapsulating ATRA efficiently inhibited migration of tumor cells rather than ATRA itself. 본 연구의 목적은 뇌종양을 치료하기 위한 새로운 나노입자를 개발하는 것이며 in vitro상에서 새로운 나노입자의 성능을 조사하였다. Methoxy poly(ethylene glycol) (mPEG)-grafted chitosan (abbreviated as CP graft copolymer) 을 합성하여 레티노익산을 봉입한 core-shell 형태의 나노입자를 제조하는데 이용하였다. 새로운 나노입자는 DMF에 녹인 레티노익산을 키토산 수용액에 혼합하여 간단하게 제조할 수 있으며 레티노익산과 키토산사이에 이온복합체를 형성하여 나노입자가 형성되는 것으로 이해된다. 레티노익산을 봉입한 CP nanoparticle은 구형의 형태를 보이고 200 nm 이하의 크기를 보였다. CP 그라프트 공중합체와 레티노익산이 core-shell 구조의 나노입자를 형성하는지를 알아보기 위해 ¹H NMR을 이용하였으며 DMSO를 용매로 사용하였을때 보인 레티노익산의 특정 피크가 D₂O상에서 사란진 것으로 보아 core-shell 구조의 나노입자가 형성됨을 알 수 있었다. 레티노익산은 CP 나노입자로 방출이 한달 이상 지속되었으며 약물의 봉입량이 적을 수록 빠르게 방출됨을 알 수 있었다. 9L gliosarcoma, U343MG, U87MG, U251MG 등의 뇌종양 세포주를 이용하여 레티노익산을 봉입한 CP 나노입자의 세포독성실험을 수행한 결과 나노입자가 기존의 레티노익산과 동등한 세포독성효과를 보였다. U87MG 세포주를 이용한 나노입자의 암세포 이동능 억제 효과에 대한 실험결과 CP nanoparticle 이 기존의 레티노익산에 비해 월등한 이동능 억제 효과가 있었음을 알 수 있었다. 결론적으로 신경교종의 침윤억제 치료에 효과적으로 응용할 수 있다고 생각된다.

Mitochondrial DNA aberrations in patients with aplastic anemia

Gim, Jong-pil Chonnam National Univ. 2005 국내박사

This study was undertaken primarily to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia (AA). Non-isotopic RNase cleavage assay as a screening method was performed in 16 patient’s peripheral bloods (PBs). And complete mtDNA nucleotide sequence was analyzed in 6 and 8 bone marrow (BM) specimens from the patients with AA and control subjects respectively. 27 mutations (mean = 4.5) were found which comprised 25 substitutions and 2 deletions at the protein genes for NADH dehydrogenase (n = 10), cytochrome c oxidase (n = 6), cytochrome b (n = 5), non-coding region (n = 2), ATPase 6 (n = 2), 12S rRNA (n = 1) and tRNA (n = 1). The 13 mutations (48.1%) harbored amino acid changes. The number of affected mtDNA genes in patients with AA (mean = 17.8) was higher than those of controls (mean = 12.8). There was no predominant mutation (hot spot) of mtDNA genes among the patients with AA. For checking the heteroplasmy, buccal mucosa and PB were tested additionally. Eleven mutations from two patients (1 and 2) showed heteroplasmy, but the rest of mutations from three patients (3, 4 and 6) were homoplasmy. These findings might be explained by representing the major population of mtDNA or by clonally expanded mtDNA point mutations. Although there was not predominant mutation (hot spot) of mtDNA genes among the patients with AA, each patient had several mutations that would result in a significant change in the gene products. These mtDNA mutations in AA may be associated with pathophysiology of AA. 약물, 방사선 및 화학약품 등은 재생불량성빈혈(aplastic anemia; AA)을 일으키는 중요한 원인이다. 한편 이들은 미토콘드리아 DNA (mtDNA)의 유전변이를 일으키는 대표적인 인자로 도 알려져 있다. 따라서 본 연구에서는 mtDNA 유전변이와 재생불량성빈혈의 병태생리와의 연관성을 규명하고자 하였다. 먼저 16 명의 AA 환자 말초혈액 검체를 이용하여 non-isotopic RNase cleavage 분석법을 이용하여 AA와 mtDNA 유전변이와의 관련성으로 선별하였다. 다음으로 AA환자 골수 검체 6 예와 8 명의 건강인에서 채취한 골수 검체를 대상으로 전체 mtDNA 염기서열을 분석하였다. 환자군에서 2 개의 유전결손을 포함한 총 27 개의 mtDNA 유전변이가 검출되었다. 이들 mtDNA 유전변이의 분포는 NADH dehydrogenase (n = 10), cytochrome c oxidase (n = 6), cytochrome b (n = 5), non-coding region (n = 2), ATPase 6 (n = 2), tRNA (n = 1) 및 12S rRNA (n = 1)였다. 이중 13 개의 유전변이는 아미노산변이를 동반하였다. 환자군에서 ‘hot spot’ mtDNA 유전변이는 관찰되지 않았지만, 유전변이의 수 (평균 17.8)는 정상인 (평균 12.8)에 비해 높게 관찰되었다. 환자군에서만 관찰된 mtDNA 유전변이의 heteroplasmy 유무를 확인을 위하여 추가로 환자의 구강상피세포 및 말초혈액에서 mtDNA 유전변이를 검사하였다. 2 명의 환자(1 및 2)에서 검출된 11 개의 mtDNA 유전변이는 heteroplasmy 소견을 보였다. 3 명의 환자(3, 4 및 6)에서 관찰된 mtDNA 유전변이는 homoplasmy였다. 이상의 결과는 mtDNA 유전변이와 AA 의 병태생리와의 직접적인 연관성을 시사하였다.

Cisplatin-incorporated hyaluronic acid nanoparticles as a drug delivery vehicle for brain tumor

김성택 Chonnam National Univ. 2006 국내박사

Hyaluronic acid is a linear polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, and provides a matrix that facilitate invasion at brain. The aim of this study is to prepare cisplatin-incorporated nanoparticles based on an ion complex formation between hyaluronic acid (HA) and cisplatin for antitumor drug delivery. To prepare nanoparticles using HA, bulk HA was degraded by hyaluronidase. Cisplatin-incorporated HA nanoparticles were prepared by mixing cisplatin with an aqueous solution of HA and then the nanoparticle solution was dialyzed to remove trace elements. Since glioma tumor cell lines are able to secrete hyaluronidase, extracts from U343MG and U87MG cell lines were used to test the release of cisplatin from the nanoparticles. HA degraded by hyaluronidase has a weight average molecular weight (Mw) of 102,500 and a number average M.W. (Mn) of 92,400. The morphological examination of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size around 100-200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67 ~ 81 % (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the release rate of cisplatin from the nanoparticles increased when hyaluronidase was added to the release medium. In the results of the The study of HA zymography showed that the U343MG cells secreted hyaluronidase, while the U87MG cells did not. When the extracts from U343MG cells were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG cells did not significantly affect the release rate of cisplatin. In conclusion, cisplatin-incorporated nanoparticles have sufficiently small particle sizes to use as a drug targeting system. The release of cisplatin from the nanoparticles was responsive to the secretion of hyaluronidase by brain tumor cells. These nanoparticles may be suitable vehicles for an antitumor drug targeting system. 이 연구의 목적은 히알루론산을 이용해서 뇌종양에 선택적으로 적용할 수 있는 시스플라틴 나노입자를 만들고 이를 뇌종양 세포 주에서 분비된 히알루론산 분해효소를 이용하여 평가하는 것이다. 나노입자를 만들기 위해 분자량이 큰 히알루론산을 히알루론산 분해효소를 이용하여 분해하였다. 시스플라틴이 담지 된 나노입자를 만들기 위해서 시스플라틴과 물에 녹인 히알루론산을 혼합하여 3일간 교반시킨 후 투석하여 미량원소와 담지 되지 않은 약물을 제거하였다. 뇌종양 세포주로부터 분비된 히알루론산 분해효소가 약물방출에 미치는 영향을 조사하기 위해 U343MG-A와 U87MG세포의 배지를 냉동건조 후 약물 방출 배지에 첨가하여 방출실험을 행하였다. 분해된 히알루론산은 중량평균 분자량이 102,500 이하이며 기존의 히알루론산보다 수용액에 잘 용해되었다. 분해된 히알루론산과 시스플라틴을 혼합하여 100 ~ 200 nm의 크기를 가진 나노입자를 제조하였으며 투과전자 현미경으로 관찰한 결과 구형의 형태를 보였다. 담지 효율은 약 67 ~ 81 % (w/w) 로 시스플라틴이 4일 동안 서서히 방출되었다. 특히 배지에 히알루론산 분해효소를 첨가한 결과 약물의 방출속도가 더 증가하였다. 히알루론산 zymography 결과 U343MG-A세포 주는 히알루론산 분해효소를 분비하지만 U87MG세포 주는 분비하지 못하며 이를 각각 약물 방출실험에 적용한 결과 U343MG세포의 배지에서 추출한 추출물을 첨가한 경우에만 약물의 방출속도가 증가하였다. 이 연구 성적은 시스플라틴이 히알루론산 나노입자가 히알루론산 분해효소반응성이므로 히알루론산 분해효소를 분비하는 뇌신경교종에 표적성 약물 전달시스템으로 이용될 수 있음을 시사한다.

Transcription factor AP-4 acts as a transcriptional repressor to the developmental expression of BAI1-AP4 gene in the brain

김미영 Chonnam National Univ. 2005 국내박사

Previously, it was reported that brain-specific angiogenesis inhibitor 1-associated protein 4 (BAI1-AP4), a novel brain-specific protein is developmentally upregulated in the mouse adult brain. A novel DNA element (52 bp putative repressor element, PRE) in the BAI1-AP4 upstream sequence was identified to mediate the repression of reporter expression. To provide insight into the transcriptional regulation of BAI1-AP4 gene, the author tried to find the DNA binding proteins that bind to the PRE using yeast one-hybrid assay. The author have found that transcription factor AP-4 (TFAP4) binds to PRE and geminin, an ubiquitous cell cycle replication licensor, also interacts with TFAP4 but dose not bind to PRE. The similar patterns of developmental down expressions were observed in the brain between TFAP4 and geminin, and this down regulation pattern inversely correlated with that of BAI1-AP4. In human embryonic kidney (HEK293T) cells, overexpression of TFAP4 repressed the luciferase activity of BAI1-AP4 promoter-reporter construct. In contrast, transfection of antisense geminin construct into HEK293T cells relieved from the depression of the BAI1-AP4 protein expression. In the primary cultured neuronal cells isolated from BAI1-AP4 promoter-lacZ transgenic mice, transfection of geminin decreased β-galactosidase reporter mRNA. The administration of geminin to the intracerebro ventricular space of the transgenic mouse repressed the β-galactosidase reporter expression. These results indicate that TFAP4 and geminin act as a transcriptional repressor and a corepressor to the developmental down expression of BAI1-AP4 gene in the brain, respectively. 뇌 특이 신생혈관 억제자1-결합 단백4 (BAI1-AP4)는 새로운 뇌 특이적 단백으로서 발생이 진행될수록 성숙쥐의 뇌에서 발현이 증가된다. BAI1-AP4 유전자 염기서열의 상위부위에서 새로운 DNA부위 (52염기 억제부위; PRE)가 발견되었는데, 이 부위는 일시적 형질전환 실험에서 표지유전자의 발현을 억제한다. BAI1-AP4 유전자의 전사조절 기전을 알아보기 위해 본 연구자는 yeast one-hybrid 실험을 수행하여 PRE에 결합하는 전사인자를 찾아보고자 하였다. 본 실험에서 전사인자 TFAP4가 PRE에 결합함을 확인하였고, 세포주기에서 복제 조절자인 geminin은 PRE에 직접 결합하지는 않았지만, TFAP4와 단백간에 결합함을 확인하였다. 생쥐의 배아 뇌에서 성숙 뇌까지 발생 단계에 따른 TFAP4와 geminin의 발현이 점점 감소하는 반면, BAI1-AP4의 발현은 점점 증가하였다. 인간 배아 신장세포인 HEK293T 세포에서 TFAP4를 과발현시켰을 경우 BAI1-AP4 프로모터 활성이 억제되었다. 이와 반대로, antisense geminin 유전자를 HEK293T 세포에 형질전환 하였을 경우, BAI1-AP4의 전사억제가 회복되어, 이 단백의 발현이 증가되었다. 또한, BAI1-AP4 프로모터에 lacZ유전자를 연결시켜 만든 형질전환 생쥐에서 얻어낸 신경세포에 geminin을 과발현 시키면 β-galalctosidase 표지 유전자의 mRNA양이 감소하였다. 또한, 이 형질전환 생쥐의 뇌에 geminin유전자를 직접 도입시키면 역시 표지 유전자의 mRNA및 단백양이 줄어듦을 확인하였다. 본 연구 결과는 TFAP4와 geminin이 뇌내 BAI1-AP4의 발생단계에서 전사 억제자로 작용하고 있음을 제시한다.

Ezetimibe and simvastatin combination treatment reduces neointimal hyperplasia and restenosis after drug-eluting stent implantation in a porcine coronary restenosis model

조정선 Chonnam National Univ. 2010 국내박사

The aim of this study is to examine the anti-proliferative and anti-inflammatory effects of ezetimibe/simvastatin (E/S) after drug-eluting stent (DES), either sirolimus or paclitaxel-eluting stents implantation in a porcine coronary restenosis model. Stents were deployed with oversizing (stent/artery ratio 1.3:1) in porcine coronary arteries. Pigs were randomized into two groups in which the coronary arteries (23 pigs) had DES. Fifteen pigs were taken 10/20 mg of E/S and eight pigs were not taken E/S. Histopathologic analysis was performed at 28th days after stenting. In neointima, most inflammatory cells were lymphohistiocytes. Lymphohistiocyte counts were not different between two groups (337±227 vs. 443±366 cells, P=0.292), but neointima area was significantly smaller (1.00±0.49 mm2 vs. 1.69±0.98 mm2, P=0.021) and percent area stenosis was significantly lower (23.3±10% vs. 39±19%, P=0.007) in E/S group compared with control group. There were no significant differences in fibrin score (1.99±0.79 vs. 1.81±0.88, P=0.49), endothelial score (1.75±0.66 vs. 1.80±0.59, P=0.79), and the percent of endothelium covered lumen (43±21 % vs. 45±21 %, P=0.84) between E/S group and control group. Combined treatment with ezetimibe and simvastatin inhibits neointimal hyperplasia, but does not inhibit inflammatory infiltration and arterial healing after DES implantation in a porcine coronary restenosis model. 본 연구는 돼지 관상동맥에 약물 용출성 스텐트 (DES)를 삽입 후 재협착 모델에서 ezetimibe/simvastatin (E/S)의 항염증성, 항증식성 효과에 대해 알아보고자 하였다. 23마리의 돼지를 대상으로 두 군으로 무작위 배정하여 DES를 삽입하였다. 스텐트는 돼지의 관상동맥 크기에 비해 확대시켜 재협착 모델을 만들었다 (stent/artery ratio 1.3:1). 15 마리의 돼지는 10/20 mg E/S을 투여하고, 8마리의 돼지는 E/S를 투여하지 않고 대조군으로 만들었으며, DES 시술 후 28 일째에 해부조직 검사를 시행하였다. 신생내막에는 출현하는 염증세포는 대부분 Lymphohistiocyte이었고 그 수는 양군간 차이를 보이지 않았으나 (337±227 vs. 443±366 cells, P=0.292), 신생내막 면적은 E/S 투여군에서 적었다 (1.00±0.49 mm2 vs. 1.69±0.98 mm2, P=0.021). 면적 협착율 (percent area stenosis) 역시 대조군에 비해 의의있게 적었다 (23.3±10 % vs. 39±19 %, P=0.007). Fibrin score (1.99±0.79 vs. 1.81±0.88, P=0.49), endothelial score (1.75±0.66 vs. 1.80±0.59, P=0.79) 등은 양군간의 차이를 보이지 않았으며, 내피로 덮인 내강 면적은 (43±21 % vs. 45±21 %, P=0.84) 양군간 의의 있는 차이를 보이지 않았다. 결론적으로 ezetimibe/simvastatin의 복합 치료는 돼지 관상동맥에 DES 시술 후에 신생내막 증식을 억제하였으나, 염증세포의 억제나 약물 용출 스텐트 시술 이후 혈관치유 등을 개선시키지는 못하였다.