목 적: 조절마비제로 CyclogylⓇ 과 AtropineⓇ 이 사용되고 있으며, 조절마비제를 점안했을 때 결막세포 에 미치는 영향을 알아보고자 하였다. 방 법: 결막세포(clone 1-5c-4)를 배양하여 CyclogylⓇ, cyclopentolate HCl, AtropineⓇ, atropine sulfate를 각각 1%, 0.75%, 0.50%, 0.25% 농도로, 보존제인 benzalkonium chloride(BAC)는 0.01%, 0.0075%, 0.005%, 0.0025% 농도로 15분 처리하여 MTT assay 방법을 사용하여 세포 생존율을 측정하였다. 형태적 변화를 조사하기 위해 hematoxylin-eosin(H-E) 염색 후 광학현미경으로 관찰하고, 유세포분석기를 이용하여 세포자연사를 정도를 측정하였다. 결 과: Cyclopentolate HCl 0.25% 와 atropine sulfate 0.25% 에서만 세포 생존율이 70%를 넘는 것으로 나타났으며, H-E 염색한 결과 CyclogylⓇ 1% 와 AtropineⓇ 1% 에서 세포수가 감소하고 핵의 분절화를 관찰할 수 있었다. CyclogylⓇ 1% 와 AtropineⓇ 1% 에서는 대조군에 비해 A0 수치가 52.92% 와 31.36% 로 세포자연사가 나타났다. 결 론: 조절마비제인 CyclogylⓇ 와 AtropineⓇ 1% 에서는 세포 생존율이 30% 를 넘지 않았으며, 세포자 연사를 일으키는 것으로 나타났다.

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3∼4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

The nature of molecular mechanisms governing embryonic cell block is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to determine effects of programmed cell death on porcine oocytes development in vitro after parthenogenesis. Among the blastocysts matured in 3MA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of Cyst < 3MA < RP. However, Casp-3 and TNF-r RNA gene expression level decreased in the order of RP < 3MA < Cyst. Expression of mTOR within the RP-cultured blastocyst was the most highly to the inner cell mass, while 3MA-cultured blastocyst showed very lowest expression in inner cell mass. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. When the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each RP treatment group, with the level of another treatment group being relatively higher. Analyses of TIMP-2 and TIMP-3 revealed that their expression was higher in groups that did not receive RP treatment. More specifically, the level of TIMP-2 was not affected by Cyst treatment, while the level of TIMP-3 was higher in 3MA and RP treatment group. There was highly cell division activation efficiency of parthenogenesis on cultured system of RP supplement IVC medium. Therefore, these results suggest that embryo development was significantly increased in conditional culture medium with active autophagy as compared to common cultured condition. Further investigation of this distinction may enable the development of innovative improvements for the production of porcine somatic cell nuclear transfer.

The purpose of this study is to develop transgenic cell line expressing targeted human granulocyte colony stimulating factor (hGCSF) and green fluorescence protein (GFP) genes as well as production of Somatic Cell Nuclear Transfer (SCNT) embryos derived from co-expressed transgenic donor cells. Constructed pPiggy-mWAP-hGCSF-EF1-GFP vector was chemically transfected into bovine fetus cells and then, only GFP expressed cells were selected as donor cells for SCNT. Cleavage and blastocyst rates of parthenogenetic, SCNT embryos using non-TG cell and hGCSF-GFP dual expressed SCNT embryos were examined (cleavage rate: 78.0±2.8 vs. 73.1±3.2 vs. 70.4±4.3%, developmental rate: 27.2 ±3.2 vs. 21.9±3.1 vs. 17.0±2.9%). Result indicated that cleavage and blastocyst rates of TG embryos were significantly lower (P<0.05) than those of parthenogenetic and non-TG embryos, respectively. In this study, we successfully produced hGCSF-GFP dual expressed SCNT embryos and cryopreserved to produce transgenic cattle for bioreactor system purpose. Further process of our research will transfer of transgenic embryos to recipients and production of hGCSF secreting cattle.

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

Mushroom have long been valued as highly nutritious and tasty foods in many societies throughout the world. Itis known for biological activities including anti-inflammatory and anti-oxidative potential. However little is known about anti-cancer property. In this study, we investigated the anti-prostate cancer activity of mushrooms. For that, eight kinds ofmushrooms such as, T. matsutake, S. crispa, G. lucidum-US, G. lucidum-AS, C. cardinalis-BR, G. frondosa, P. linteus, U.esculenta were extracted with hot water. Among them, three kinds of mushrooms including T. matsutake, G. lucidum-US andC. militaris-BR extracts inhibited prostate specific antigen (PSA) expression in prostate cancer cell, LNCaP. These resultsdemonstrate that some of mushrooms inhibited PSA expression suggesting that the mushrooms might be a candidate for thetreatment of prostate cancer.

The objective of this study was to monitor health conditions of genetically identical somatic cells cloned Korean white cattle, endangered indigenous cattle (EIC) and indigenous cattle (IC) by analysis of hematologic characteristics. Naturally ovulated oocytes and donor cells were used for somatic cell nuclear transfer (SCNT). Donor cells and enucleated oocytes were followed by electric fusion, chemical activation and surgical embryo transfer into the oviducts of surrogate females. Two recipients became pregnant; two maintained pregnancy to term, and one live cattle were delivered by caesarean section. The cloned Korean white cattle were genetically identical to the nuclear donor cattle. As a result, the mean values of RBC and platelet of cloned cattle and white cattle were significantly decreased by age (P<0.05). The mean values of RBC, HCT, MCV and MCHC between cloned cattle and IC of the same age (1∼2 years) showed the statistical significance (P<0.05). Also, in the WBC of Korean white cattle, the estimated values were decreased according to the age from 12.0×103/μl under 1 year to 11.0×103/μl over 1 years respectively. Although clone-cattle had lower numbers of RBC than reference range, the most of RBC and WBC related heamatologic results of cloned cattle were not different when compared to reference range. This study suggests that cloned Korean white cattle derived from SCNT did not have remarkable health problems, at least in the growth pattern and hematological parameters. In addition, this study provides a valuable resource for further investigations of the preservation of rare genetic stocks underlying traits of interest in cattle.

Mushroom is known for anti-inflammatory and anti-oxidative potential. This study provides evidence that theinhibitory effect of mushroom on the expression of pro-inflammatory cytokines in human keratinocytes, HaCaT cells. To definethe underlying mechanisms of action, tumor necrosis factorα/IFNγ-activated human keratinocytes model was used. Mushroomsignificantly inhibited the expression of cytokines in HaCaT cells. Taken together, the results demonstrate that mushroom inhibitedinflammtion, suggesting that mushroom (DW extract: Grifola frondosa Cordyceps militaris), (Ethanol extract: Ganoderma lucidum,Lentinus edodes, Cordyceps militaris, Flammulina velutipes) might be a candidate for the treatment of skin inflammation.

광범위큰 B세포 림프종은 비호지킨림프종의 대표적인 아형으로 공격적 림프종이며 원발성으로 담낭에 발생한 림프 종은 일부 증례를 통한 보고가 있지만, 담낭에 단독 재발한 광범위큰 B세포 림프종은 국내에서 보고된 바가 없다. 본 증례는 상복부의 통증과 황달의 임상증상 및 복부컴퓨터단층 촬영 그리고 양전자 방출 단층촬영에서 만성 담낭염과 담낭 암으로 추정하여 수술 후 조직검사를 통해 재발한 광범위큰 B세포 림프종으로 진단할 수 있었다. 저자들은 과거 회장에 발생한 광범위큰 B세포 림프종으로 치료를 시행하여 완전완 화가 유도된 이후 다른 장기 혹은 림프절 전이 없이 단독으로 담낭에 재발한 광범위큰 B세포 림프종 1예를 경험하였기에 문헌 고찰과 함께 보고하는 바이다.

Hippo signaling is one of the signal transduction pathways revealed in Drosophila and mammalian. This signaling is known to control proliferation and growth of normal cells or cancer cells, in which many signaling proteins form a network. In this network, tumor suppressor kinases include MST and LATS while YAP and TAZ exist as oncoproteins. Combined with transcription factor, YAP the oncoprotien starts to secrete growth factors such as CTGF, FGF, and Cry61 regulating the growth of cells or the organ sizes. The YAP is also associated with the development of early embryo and the regeneration of the skin wound as well as abnormal growth of cancers in case of over-expression. Although many previous studies have found various tumors with YAP over-expressed, the expression of YAP is not yet clearly identified in oral cancer. The aim of this study was to check the expression level of YAP in oral squamous cell carcinoma. So we performed PCR and Western blot to check YAP expression in mRNA and protein level respectively. In result, all of the 13 cell lines examined has presented the expression of YAP, and especially in HSC2 and KOSCC11 cell lines has been observed the remarkable level of expression. In conclusion, we confirmed the overexpression of YAP cell line in oral squamous cell carcinoma, it will be a great help to the study carried out in the future. Once you understand the mechanism of oncoprotien YAP in oral cancer cells, it seems possible to research and development of targeted tumor therapy agents in oral cancer.

양파는 Allium에 속하는 조미채소로 한국을 포함한 동아시아 지역에서 경제적으로 중요한 작물이다. 본 연구에서는 슈퍼생생과 뉴마르스 양파 2개 품종의 인경 부위를 이용하여 kinetin 농도 별 백색 체세포배 캘러스 형성율을 조사하였다. 양파의 백색 체세포배 캘러스를 얻기 위하여 실시된 배양 조건은 1mg/L 2.4-D를 첨가한 B5배지를 기본으로 25℃ Dark에서 배양하였다. 관찰하고자 한 kinetin의 농도는 0mg/L, 0.1mg/L, 0.3mg/L, 1mg/L, 3mg/L로 정하여 실험하였으며, MSB배지에서 배양된 2개 품종의 양파인경을 얇게 잘라 B5배지에 치상하였다. 양파의 인경 조직 중 가장 안쪽 부분부터 반응이 시작되어 백색 체세포 배 캘러스가 형성되는 것을 kinetin 농도 별로 배양한 2주 후부터 관찰할 수 있었다. 양파 2개 품종의 인경 조직을 B5배지에 배양한 8주 후에 백색 체세포 배 캘러스 형성율을 조사하였다. 그 결과 슈퍼생생과 뉴마르스의 인경 조직에서 1mg/L 2,4-D와 0.3mg/L 그리고 1mg/L kinetin이 혼용처리 된 B5배지에서 73%와 66.7%로가장 높게 나타나는 것을 알 수 있었다. 본 연구에서 확립된 슈퍼생생과 뉴마르스의 인경 조직을 이용한 백색 체세포배 캘러스 형성 조사 결과는 추후 양파의 식물체 재분화 유도 및 대량증식 연구분야에 활용이 가능할 것으로 기대된다.

동물의 장기를 인간에게 이식하게 되면 초급성거부반응(Hyperacute rejection, HAR)이 일어난다. 초급성거부반응은 면역계의 구성요소 중 보체(complement)에 의해 일어나는 거부반응으로 돼지의 혈관세포 표면에 있는 Galα(1,3)Gal 당분자에 인간의 항체가 즉각 반응하기 때문에 일어나며, α1,3-galactosyltransferase(α1,3-GT) 유전자는 돼지 혈관세포 표면의 Galα(1,3)Gal 당분자 생성에 관여한다. 따라서 인간에게 돼지의 장기를 이식하기 위해서는 α1,3-galactosyltransferase 유전자를 제거하는 것이 필요한 것으로 알려져 있다. 본 연구실의 이전 연구에서, 시카고 미니돼지 귀체세포에서 상동 재조합(Homologous recombination)을 통해 α1,3-galactosyltransferase 유전자가 제거된 체세포를 개발한 바 있으며, 이 체세포를 통하여 α1,3-GT 유전자가 제거된 돼지도 생산된 바 있다. 본 연구에서는, human serum 처리 시 돼지 세포를 보호해 준다고 보고되고 있는 human complement regulator인 human Decay-accelerating factor(hDAF)와 human α1,2-fucosyltransferase(hHT)유전자를 α1,3-GT 유전자 위치에 gene targeting하여 동시에 hDAF와 hHT가 발현하는 체세포를 개발하였다. Knock-in vector는 hDAF와 hHT 두 유전자가 발현할 수 있도록 IRES로 연결하였으며, α1,3-GT 유전자의 start codon을 이용하여 발현할 수 있도록 구축하였다. 구축한 vector는 electroporation을 통해 미니돼지 체세포에 도입하였으며, PCR 결과, α1,3-GT 유전자 위치에서 상동 재조합이 일어났음을 확인하였다. Positivenegative 선별 방법을 통해 얻은 gene targeting 된 체세포는 RT-PCR에 의해 hDAF와 hHT 유전자의 발현이 확인되었으며, 대조군(NIH minipig)에 비해 α1,3-GT 유전자의 발현이 감소하였다. 또한 이들 세포에 100% human complement serum을 처리하였을 때 knock-in 세포가 대조군에 비해 30% 정도 더 높은 생존율을 보였다. 따라서 개발된 체세포는 이종간 장기이식을 위한 돼지 생산과 함께 이를 이용한 이종간의 장기 이식 시 초급성 거부반응을 억제하는 데 사용될 수 있을 것으로 생각된다.

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

Immunoprecipitation-based high performance liquid chromatography (IP-HPLC) is a type of modified enzyme-linked immunosorbent assay (ELISA) that uses protein A/G (or antibody)-conjugated beads instead of the antibody-conjugated wells used in ELISA. In order to determine the fidelity of IP-HPLC, the author used 83 antisera to identify protein expression changes caused by cisplatin treatment in KB human oral cancer cells. KB cells were cultured for 12 or 24 hours with 10 ug/mL cisplatin. The results obtained by IP-HPLC were comparable with published cisplatin data, although ELISA was not conducted in the present study. Cisplatin dominantly reduced the levels of proteins associated with cell proliferation, transcription factors, growth factors, cytoskeletal proteins, and cellular differentiating factors, but on the other hand, apoptosis-related factors, oncogenes, and protective proteins were usually up-regulated, presumably to address cisplatin-induced DNA damage. In particular, cisplatin directly inactivated genomic DNA by down-regulating histone H1 and demethylase and by up-regulating deacetylase. Cisplatin also rapidly induced p53 overexpression and mitochondria-mediated endogenous apoptosis occurred after 12 hours of cisplatin treatment, although this was almost completely replaced by FASL/FAS-mediated exogenous apoptosis after 24 hours. This preliminary study was conducted to investigate the anticancer effect of cisplatin on the KB human oral cancer cells and to determine the fidelity of IP-HPLC data. It was concluded that IP-HPLC is useful for identifying profile changes of genome wide essential proteins and signaling changes of major molecular pathways.

Previous reports revealed that DMfree (green tea extract) inhibited expression of the IL-6 gene in Mycobacterium lepraeinfected MSCs (mesenchymal stem cells). This study aimed to measure IL-6, IL-1β, TNF-α and PGE2 production in M. leprae-infected MSCs using ELISA. To confirm the effect of DMfree on IL-6 and signal transduction, a western blotting test was performed. DMfree inhibited the expression of IL-6 in the MSCs and the heterodimer of STAT3, which also affects the expression of multiple genes. Though DMfree pre-treatment of control MSCs produced a baseline level of IL-6, it significantly inhibited the production of IL-6 in M. leprae-infected MSCs. There was no significant difference in IL-6 production between 1 and 7 day treatment groups. M. leprae-infected MSCs produced more IL-1β, TNF-α and PGE2, but DMfree could not inhibit their production at a physiological concentration. This is different from other reports that used higher concentration of EGCG treatment, resulting in significant inhibition of the cytokines. The inhibition appears to be related to the concentration of EGCG. These results indicate that DMfree can alleviate inflammation involving IL-6.

본 연구에서는 만성질환의 주요 위험인자인 비만을 예방 하기 위한 식품소재로서 3T3-L1세포를 이용하여 탈피한 도 토리의 항비만 효과를 알아보고자 하였다. 3T3-L1 세포에서 생존율(MTT assay)을 측정한 결과, AE와 AW 시료 모두 500 μg/mL 농도에서는 다소 생존율의 감소를 보여, 300 μg/mL를 최종 농도로 정하였다. 3T3-L1 세포의 지질축적 억제 효과를 측정한 결과, 농도 100 μg/mL로 처리하였을 때 두 시료 모두 지질축적량의 증가를 보였으나, 200 μg/mL 처리농도에서 AE 시료는 82%로, AW 시료는 74%로 감소되다가 300 μg/mL 농 도에서는 두 시료 모두 약 53% 수준까지 지질축적이 억제되 었다. 3T3-L1 세포에서 중성지방 억제 효과를 확인한 결과, AE 시료의 경우 200 μg/mL 농도에서 11%의 감소율, 300 μg/mL 농도에서 42% 수준의 감소율을 보였다. AW 시료도 200 μg/ mL 농도에서 5%의 감소율과 300 μg/mL에서 41%의 감소율 을 보였다. 3T3-L1 세포의 ROS 생성량을 측정한 결과, 시료 농도 200 μg/mL에서 AE는 42%, AW는 33%로 300 μg/mL에 서는 AE는 58%, AW는 52%로 ROS 생성량의 억제를 보였다. 3T3-L1 세포에서 mRNA 발현에 미치는 영향을 대조군과 비 교하였을 때 두 시료(AE와 AW) 모두 300 μg/mL 농도에서 PPAR-γ은 54%와 38%, aP2는 40%와 18% 수준의 발현을 억 제시키는 것으로 나타났다. 이상의 결과로 볼 때 탈피한 도토 리는 3T3-L1 세포의 분화를 억제함으로써 새로운 항비만 소 재로의 가능성이 있는 것으로 판단된다.

본 연구는 흰 민들레의 기능성 식의약품 소재로서의 활용 가능성을 탐색하기 위하여, 흰 민들레 꽃, 잎, 뿌리의 부위별 총 폴리페놀, 총 플라보노이드, 항산화 활성 및 암세포에 대 한 세포독성 효과를 분석하였다. 흰 민들레 부위별 에탄올 추 출조건에 따른 추출수율은 꽃, 잎, 뿌리가 각각 32.15±3.21%, 31.63±0.63%, 27.48±2.47%로서, 꽃 > 잎 > 뿌리의 순으로 나 타났다. 흰 민들레 부위별 에탄올 추출물에서의 총 폴리페놀 함량은 꽃 추출물에서 61.29±2.11 mg/g으로 가장 높게 나타 났으며, 그 다음이 잎(43.52±2.34 mg/g), 뿌리(11.36±1.87 mg/g) 순으로 나타났다. 흰 민들레 부위별 에탄올 추출물에서의 총 플라보노이드 함량은 총 폴리페놀 함량과 같은 경향으로 꽃 (46.11±1.88 mg/g), 잎(24.89±1.20 mg/g), 뿌리(6.31±1.22 mg/g) 의 순으로 각각 나타났다. DPPH 라디컬 소거활성으로 분석 한 흰 민들레 부위별 에탄올 추출물의 전자공여능은 추출물 농도 의존적으로 나타났으며, 꽃, 잎 및 뿌리 에탄올 추출물 1.0 mg/mL의 농도에서 각각 89.99±2.83%, 85.29±2.22%, 37.88± 2.34%로 꽃 에탄올 추출물이 가장 높게 나타났다. 이러한 결과는 폴리페놀 및 플라보노이드 함량이 DPPH 라디컬 소거능 과 관련이 있음을 보여주었다. MTT법에 의한 흰 민들레 부 위별 에탄올 추출물 400 mg/kg 첨가시 위암 세포주(AGS), 폐 암 세포주(A-549) 및 대장암 세포주(HCT-116)에 대한 세포독 성 효과를 확인한 결과, 위암 세포주(AGS)에서 꽃(62.85± 4.63%), 잎(47.83±4.22%), 뿌리(4.73±0.89%)의 순으로, 대장암 세포주(HCT-116)에서 꽃(69.89±3.44%), 잎(54.14±2.82%), 뿌 리(9.42±1.11%)의 순으로, 폐암 세포주(A-549)에서 꽃(85.72± 4.17%), 잎(71.79±2.98%), 뿌리(19.10±2.04%)의 순으로 모든 암 세포주들에 대하여 꽃 부위 에탄올 추출물이 가장 높았으 며, 뿌리에서 가장 낮은 것으로 나타났고, 모두 농도 의존적 으로 항암활성이 증가하는 것으로 나타났다. 이와 같은 결과, 국내에서 재배되고 있는 흰 민들레는 향후 기능성 식의약품 소재로서의 이용에 있어 유용한 기초자료로 활용될 수 있을 것으로 기대된다.

본 연구는 인체 유래 유방암세포인 MDA-MB-231 세포 증식을 억제하고 세포사멸을 유도하는 천연소재 발굴을 목적으로, 북한산 두릅 추출물을 MDA-MB-231 세포에 처리하여 세포사멸 및 작용기전을 규명하였다. 실험 결과, 두릅 추출물 처리 농도가 증가할수록 세포증식이 감소하였고, 세포질의 응축과 핵이 분절되는 등 apoptotic bodies를 형성하였다. 또한 유세포 분석기를 사용하여 MDA-MB-231의 세포주기가 억제되었고, 세포사멸의 특징인 sub-G1 수치가 증가되는 것이 관찰되었고, 두릅 추출물 농도가 증가함에 따라 apoptotic 세포가 유의적으로 증가하였으며, 특히 early apoptosis 보다 late apoptosis가 더 많이 진행됨을 확인할 수 있었다. 이를 바탕으로 MDA-MB-231 세포사멸과 관련된 유전자를 확인하고자 RT-PCR과 western blotting을 수행한 결과, 세포사멸의 주요한 조절인자인 anti-apoptotic인 bcl-2 발현이 두릅 추출물 처리 시 농도 의존적으로 감소되었고, 반대로 Bax의 발현은 증가됨을 확인하였다. 이를 통해 불활성화 형태로 존재한 pro-caspase-9과 pro-caspase-3의 발현이 시료 농도가 증가할수록 감소되었고, 활성화된 형태인 cleaved caspase-9과 cleaved caspase-3의 발현은 두릅 추출물 처리 농도에 의존적으로 증가하였다. 뿐만 아니라, 세포 사멸 과정의 주요 인자인 PARP 또한 시료 농도가 증가할수록 절단 현상이 비례적으로 증가하였다. 이상의 실험 결과를 근거로, 북한산 두릅 지상부의 70% ethyl alcohol 추출물이 MDA-MB-231 인체 유방암 세포의 증식을 억제하는 효과가 있음을 확인하였고, 세포사멸과 관련된 활성기전을 규명하였다. 추후 암 예방/치료제로서 두릅을 활용하기 위해서는 활성 본체와 안정성 규명 및 임상실험 등 추가 연구가 필요할 것으로 사료된다.

본 연구는 능소화를 대상으로 화분형태 및 RAW264.7 대식세포에서의 세포생존에 미치는 영향을 조사하여 유해성 여부를 파악하고자 한다. 주사전자현미경을 이용한 화분 형태를 확인해 본 결과, 화분립 형태는 장구형이고 발아구의 형태는 3공구형이며 화분 표벽 (extine)은 망상의 형태이고 외부로 돌출된 돌기는 관찰되지 않았다. 능소화 70% MeOH 부위별 (꽃, 잎, 줄기) 추출물 및 화밀을 24, 48시간 동안 농도별 (10, 20, 50, 100 μg mL-1)로 RAW264.7 대식세포에 처리한 후 MTT assay로 측정하였다. 그 결과, 능소화 추출물 및 화밀을 농도별로 24시간 처리하였을 때 모든 농도에서 99.0% 이상의 세포생존율을 보여 세포독성은 나타나지 않았다. 48시간 처리하였을 때 꽃, 잎, 줄기 추출물은 50 μg mL-1농도 이하에서 세포독성은 나타나지 않았으나, 화밀의 경우 저농도인 10 μg mL-1부터 농도의존적으로 세포독성이 높아지는 것을 확인하였다.