췌장 암 줄기세포의 동정과 특성 규명

Dept. of Medical Science/박사[한글] 췌장암은 서구에서 암에 의한 사망률 중 4위를 차지하며 초기진단이 어렵고 수술과 약물치료를 받은 뒤에도 재발이 잦기 때문에 5년 생존율이 4%에 그치고 있다. 췌장암은 한국에서 발생하는 전체 암의 2.2%를 차지하며 암으로 인한 사망의 5번째 원인이며 발병률이 상승추세를 보이고 있는 매우 치명적인 질병이다. 효과적인 진단법과 치료법을 개발하기 위해서는 췌장암의 보다 정확한 발암과정에 대한 분자생물학적 규명이 절실하게 필요하다.최근 암 발생 연구에서 이슈가 되고 있는 내용은 암 조직에도 일반 장기처럼 암 조직을 유지하고 재생하는 암 줄기세포가 존재하며, 이것이 암의 발생은 물론 암의 재발이나 전이에 핵심적인 역할을 한다고 알려져 있다. 2003년에 처음으로 Clarke 박사에 의해 고형 암인 유방암에서 CD24-/low/CD44+/Lin- 인 세포가 암 줄기세포의 표지인자라고 발표되면서 지금까지 많은 암 줄기세포 표지인자의 발굴을 위한 연구가 진행되고 있다. 소화기 암에서는 CD133+가 대장암과 간암의 표지인자라고 보고 되고 올해에 들어서는 CD24+/CD44+/ESA+ 와 CD133+/CXCR4+ 가 췌장 줄기세포의 표지인자라는 두 편의 논문이 발표 된 바가 있다.아직까지 췌장암의 기원세포는 무엇인지 잘 밝혀지지 않았는데 선방세포의 이행분화에 의해 만들어진 이행성 췌관세포 또는 정상 췌장속에 존재하는 centroacinar 세포가 췌장암 기원세포라는 가설이 주축을 이루고 있다. 췌장의 줄기세포 더욱이 암 줄기세포에 대한 연구는 암 발생기전을 밝히는데 도움이 될 것이다.이와 같은 배경을 바탕으로 본 실험의 최종목적은 췌장 암 줄기세포를 분리하여 이의 특성 분석을 통하여 새로운 암 줄기세포의 표지인자를 찾는 것이다. 새로운 암 줄기세포의 표지인자를 이용하여 새로운 조기 진단법 개발이 가능하며 보다 나은 수준에서 췌장 암 발생 기전을 밝힐 수 있다고 생각한다.최근에 인체의 고형 암뿐만 아니라 수립된 암 세포 주에서도 암 줄기세포가 존재한다 는 많은 연구가 보고 된 바가 있다. 본 실험에서는 암 줄기세포가 suspension 상태에서 embryo body 모양의 sphere 구조를 만드는 특성을 이용하여 수립된 췌장암 Hpac 세포 주에서 암 줄기세포를 성공적으로 분리 배양하였다. 이렇게 분리된 암 줄기세포는 줄기세포의 self-renewal 와 다른 특성의 세포로 분화하는 능력을 지니고 있으며 in vitro 와 in vivo 상에서 보다 강한 tumorigenecity를 보여주고 있다. 암 줄기세포의 특성 유지에 관련되는 signaling 분석을 통하여 암 줄기세포에서 특이적으로 발현하는 표지인자를 동정하여 후보 단백질로 선정하였다.앞으로 조직병리학적 방법으로 정상 췌장조직과 췌장암 조직에서의 후보 표지인자의 발현을 확인하고 생물학적 특성 비료를 통하여 표지인자를 가능여부를 판단한다. 이러한 표지인자들의 개발은 궁극적으로 새로운 진단법과 치료법의 개발에 도움이 될 것이다. [영문]Recent evidences suggest that cancer may arise from cancer stem cells, those have properties of self-renewal and abilities to generate differentiated progenies. Although cancer stem cells have been reported in many types of cancers, limitation in stem cell source and specific surface markers have been issued as an obstruction. In this study we sought to isolate and characterize cancer stem cells from pancreatic cancer cell lines using non-adherent cultivation method. We demonstrated that the Hpac cell line consists of a subpopulation of cells which has potential to form clonally derived tumorospheres. These cells display the cancer stem cell properties, such as capable of self-renewal to generate sub-tumorospheres, express self-renewal markers, show lower proliferative rate and were more chemoresistant than adherent cells. Moreover these cells are more tumorigenic and more aggressive than adherent cells when xenograft to NOD/SCID mice. Taken together, we assumed that tumorosphere cultivation is a useful tool for cancer stem cells expansion. More importantly, functional characterization of cells isolated from tumorospheres could help to identify novel candidate cancer stem cell markers. Better understanding in cancer stem cells is expected to be elucidated in the carcinogenesis.restrictio

초등학생의 환경감수성과 열등감 간의 관계 연구 : 서울지역을 중심으로

학위논문(석사) --서울대학교 대학원 :협동과정(환경교육전공),2009.2.Maste

Reversal of diabetes in rats using GLP-1-expressing adult pancreatic duct-like precursor cells transformed from acinar to ductal cells

Pancreatic injury induces replacement of exocrine acinar cells with ductal cells. These ductal cells have the potential to regenerate the pancreas, but their origin still remains unknown. It has been reported that adult pancreatic acinar cells have the potential to transdifferentiate to ductal progenitor cells. In this regards, we established novel adult pancreatic duct-like progenitor cell lines YGIC4 and YGIC5 and assessed the usefulness of these ductal progenitors in the cell therapy of diabetic rats. Acinar cells were cultured from pancreata of male Sprague Dawley rats and gradually attained ductal cell characteristics, such as expression of CK19 and CFTR with a concomitant down-regulation of amylase expression over time, suggesting transdifferentiation from acinar to ductal cells. During cell culture, the expression of Pdx-1, c-Kit, and vimentin peaked and then decreased, suggesting that transdifferentiation recapitulated embryogenesis. Overexpression of pancreas development regulatory genes and CK19, as well as the ability to differentiate into insulin-producing cells, suggests that the YGIC5 cells had characteristics of pancreatic progenitor cells. Finally, YGIC5 cells coexpressing Green fluorescent protein (GFP) and glucagon-like peptide (GLP)-1 under the activation of a zinc-inducible metallothionein promoter were intravenously infused to STZ-induced diabetic rats. Hyperglycemia was ameliorated with elevation of plasma insulin, and GFP-positive donor cells were colocalized in the acinar and islet areas of recipient pancreata following zinc treatment. In conclusion, after establishing pancreatic progenitor cell lines YGIC4 and YGIC5 under the concept of acinar to ductal transdifferentiation in vitro, we demonstrate how these adult pancreatic stem/progenitor cells can be used to regulate adult pancreatic differentiation toward developing therapy for pancreatic disease such as diabetes mellitus.ope

Study on the Optimized Assembly of High Functional Nanostructure and High Performance Bioreceptor: Construction of Ideal NanoBio Convergence Platform

고기능 나노구조체와 고성능 바이오리셉터의 최적 조립 연구: 이상적인 바이오나노 융합플랫폼 구현KGM108201

CD44-positive cells are responsible for gemcitabine resistance in pancreatic cancer cells.

Accumulating evidence suggests that tumors are composed of a heterogeneous cell population with a small subset of cancer stem cells (CSCs) that sustain tumor formation and growth. Recently, there have been efforts to explain drug resistance of cancer cells based on the concept of CSCs having an intrinsic detoxifying mechanism. In the present study, to investigate the role of CSCs in acquiring chemoresistance in pancreatic cancer, gemcitabine-resistant cells were established by exposure to serially escalated doses of gemcitabine in HPAC and CFPAC-1 cells. Gemcitabine-resistant cells were more tumorigenic in vitro and in vivo, and had greater sphere-forming activity than parental cells. After high-dose gemcitabine treatment to eliminate most of the cells, CD44(+) cells proliferated and reconstituted the population of resistant cells. CD44(+)CD24(+)ESA(+) cells remained as a small subset in the resistant cell population. Among ATP-binding cassette (ABC) transporters, which are known as the mechanism of drug resistance in CSCs, ABCB1 (MDR1) was significantly augmented during the acquisition of drug resistance. ABC transporter inhibitor verapamil resensitized the resistant cells to gemcitabine in a dose-dependent manner and RNA interference of CD44 inhibited the clonogenic activity of resistant cells. In human pancreatic cancer samples, CD44 expression was correlated with histologic grade and the patients with CD44-positive tumors showed poor prognosis. These data indicate that cancer stem-like cells were expanded during the acquisition of gemcitabine resistance and in therapeutic application, targeted therapy against the CD44 or ABC transporter inhibitors could be applied to overcome drug resistance in the treatment of pancreatic cancer.ope

Radiosensitization effect of STI-571 on pancreatic cancer cells in vitro

PURPOSE: To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. METHODS AND MATERIALS: Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor beta (PDGFRbeta) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. RESULTS: Dramatic phosphorylated (p)-c-Kit and p-PDGFRbeta attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment in view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRbeta by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRbeta, p-PLCgamma was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. CONCLUSION: STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRbeta, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRbeta or K-ras mutation status.ope

Loss of the tight junction protein claudin 4 correlates with histological growth-pattern and differentiation in advanced gastric adenocarcinoma

The expression of E-cadherin located in the adherens junction varies with disruption of glandular morphology and loss of differentiation. It has been suggested that the loss of tight junction function is related to tumor differentiation, but little is known about the roles of major proteins, such as claudin 4 and ZO-1, in gastric cancer. The aims of this study were to examine the differences in expression of E-cadherin, claudin 4, and ZO-1 proteins according to the pathological and clinical features of advanced gastric cancer. The expression of E-cadherin, claudin 4, and ZO-1 was analyzed immuno-histochemically using formalin-fixed, paraffin-embedded tissues obtained from 49 patients who underwent radical resection for advanced gastric cancer. Western blot analysis and RT-PCR were performed in representative tumors of the diffuse or intestinal type. Immunostaining for E-cadherin, claudin 4, and ZO-1 was reduced in 69, 69, and 37% of cancers, respectively. Similar patterns of expression were noted for E-cadherin and claudin 4, but ZO-1 expression differed. According to the Lauren classification, the reduced expression of E-cadherin and claudin 4 was more frequent in diffuse than intestinal type tumors (p<0.001). The reduced expression of E-cadherin and claudin 4 correlated with poor differentiation (p<0.05). Western blot analysis and RT-PCR also showed decreased claudin 4 expression in diffuse type tumors and poorly-differentiated adenocarcinoma. The reduced expression of claudin 4 and E-cadherin correlates with disruption of glandular structure and loss of differentiation, which suggests that the dysfunction of claudin 4 may play a role in the disruption of cell-to-cell adhesion in diffuse type gastric cancer and in a loss of differentiation.restrictio

System and Method for Capturing and Assaying Hazardous Biomaterials Using Hybrid Nano-structures

본 개시 내용의 구체예에 따르면, 제1 고분자 재질의 나노 필라(pillar) 어레이의 표면에 제2 고분자 재질의 나노 웹을 형성한 복합 나노구조체 상에 각종 병원체, 감염성 미생물 등의 바이오물질을 효율적으로 포집하여 이를 세포 용해(lysis)시켜 뉴클레아제를 방출시키고, 방출된 뉴클레아제에 대하여 응답성을 갖는 프로브의 절단에 의한 형광 신호를 검출함으로써 간편하면서 정확하게 시료 내 바이오물질의 포집 및 진단을 수행하는 시스템 및 방법이 기재된다.국

Frequent mutations of human Mad2, but not Bub1, in gastric cancers cause defective mitotic spindle checkpoint

Since the underlying mechanism for the high incidence of aneuploidy in gastric cancer has not clarified, we screened 49 gastric cancers and five gastric cancer cell lines for mutations in the mitotic spindle checkpoint genes, Bub1 and Mad2, and we analyzed the functional consequences of these mutations. The presence of mutations in Bub1 and Mad2 coding sequences was primarily detected by RT-PCR–SSCP and subsequently confirmed by automatic sequencing of either the RT-PCR products and/or the PCR products from genomic DNA. Mad2 was mutated in 44.9% of gastric cancer tissues and one gastric cancer cell line, N87, but not Bub1. Of these, three mutational hotspots at codons 156, 165 and 182 were identified. Mutations at codons 165 and 182 led to amino acid substitutions, whereas the mutation at codon 156 was a silent one. Overexpression of mutant Mad2 in HeLa cells led to the appearance of aneuploid cells in the presence of nocodazole, and this indicated that these mutations caused a defect in MAD2 protein. Wild type and mutant MAD2 protein displayed distinct mobility on two-dimensional gel electrophoresis. Novel mutational hotspots in human Mad2 genes were discovered for the gastric cancers and these mutations caused the functional defects in the spindle checkpoint suggesting that these mutations might be involved in the development and progression of gastric cancer.restrictio

국내 헌혈지원자에서의 혈색소 분포

Background:The current donor selection criteria need to be revised to ensure a reliable blood supply and for donor protection. This study was conducted to analyze the distribution of hemoglobin (Hb) levels of blood donors and to estimate the change of eligible donors when using the revised Hb criterion. Methods:The Hb levels of all the blood donors who visited the Korean Red Cross Blood Center (KRCBC) between November 9th, 2010 and November 15th, 2010 were measured with a portable hemoglobinometer (HemoCue). The Hb levels of all the eligible donors and some of the deferred donors who visited the Hanmaeum Blood Center (HBC) from April 26th, 2010 to April 30th, 2010 and from November 9th, 2010 to November 15th, 2010 were measured with a portable hemoglobinometer (Hemo_Control). Results: A total of 7,521 donors (6,500 eligible donors and 1,021 deferred donors) were enrolled. The donation eligibility rate at the KRCBC, which is where all the donors were examined, was 84.2% (3,409/4,049) and the deferral rate was 15.8% (640/4,049). The percent of blood donors whose Hb level was less than 12.5 g/dL was 2.1% of the men (44/2,145) and 34.9% of the women (664/1,904), respectively. The percent of female deferred donors with a Hb level of 12.0∼12.4 g/dL was 19.3% (109/564) and the percent of male eligible donors with a Hb level of 12.5∼12.9 g/dL was 2.6% (54/2,069). At the HBC, and with some deferred donors being excluded, the deferral rate of males and females was 2.0% (36/1,799) and 20.6% (345/1,673), respectively. Conclusion: About 20% of the female deferred donors could be expected to participate when a less strict Hb criterion (≥12.0 g/dL) is applied. This study is thought to be helpful in order to determine the number of donors according to the Hb criteria and to create improved criteria.배경 :안정적인 혈액 수급과 헌혈자 보호를 위해 현행의 헌혈자 선별기준의 개선에 대한 필요성이 제기되어왔다. 이 연구는 공급혈액원에 방문한 헌혈지원자의 혈색소(Hb) 분포를 분석하여 Hb 수치에 대한 기준이 변경되었을 때 헌혈자의 증가 또는 감소되는 수를 추정하는데 도움을 주고자 하였다. 방법 : 대한적십자사 혈액원(KRCBC)의 서울서부, 동부 및 남부 혈액원을 방문한 모든 헌혈지원자의 Hb을 휴대용 혈색소 측정기 HemoCue로 측정하였으며, 그 기간은 2010년 11월 9일부터 15일 사이의 일주일 동안이었다. 또한 한마음 혈액원(HBC)의 서울과 경기도 지역의 헌혈카페를 방문한 헌혈지원자 중 헌혈적격자와 일부 헌혈부적격자들의 Hb 수치를 휴대용 혈색소 측정기 Hemo_Control로 측정하였으며, 그 기간은2010년 4월 26일부터 30일까지의 5일간과 2010년 11월 9일부터 15일까지의 일주일간이었다. 결과 :총 7,521명의 헌혈지원자 중 헌혈적격자는 6,500명이었고, 헌혈부적격자는 1,021명이었다. 모든 헌혈지원자를 대상으로 조사한 대한적십자사 혈액원의 경우 헌혈 적격율은 84.2%(3,409/4,049), 부적격율은 15.8% (640/4,049)로, Hb 12.5 g/dL 미만에 해당하는 헌혈지원자는 남성의 2.1% (44/2,145), 여성의 34.9% (664/1,904)이며, 여성 헌혈부적격자 중 19.3% (109/564)가 Hb 12.0∼12.4 g/dL에 해당하였으며, 남성 헌혈적격자 중 2.6% (54/2,069)가 Hb 12.5∼12.9 g/dL에 해당하였다. 일부 헌혈지원자를 대상으로 조사한 한마음혈액원의 경우 헌혈지원자 중 헌혈부적격자의 비율은 남성 2.0% (36/1,799), 여성 20.6%(345/1,673)였다. 결론 :여성의 전혈 헌혈시의 혈색소 기준을12.5 g/dL 이상에서 12.0 g/dL 이상으로 낮출 경우 여성 헌혈부적격자의 약 20%가 헌혈에 참여할수 있을 것으로 예상된다. 이 자료를 바탕으로 헌혈자 선별기준 변경시 헌혈자 수의 증감을 예상할 수 있으며, 본 연구는 현행 헌혈자 선별 기준을 개선시키는 데 도움이 될 것으로 생각된다