• Journal of Chest Surgery
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• v.37 no.4
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• pp.297-306
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• 2004

Current artificial heart valves have several disadvantages, such as thromboembolism, limited durability, infection, and inability to grow. The solution to these problems would be to develop a tissue-engineered heart valves containing autologous cells. The aim of this study was to optimize the protocol to obtain a porcine acellular matrix and seed goat autologous endothelial cells on it, and to evaluate the biological responses of xenograft and xeno-autograft heart valves in goats. Material and Method: Fresh porcine pulmonic valves were treated with one method among 3 representative decellularization protocols (Triton-X, freeze-thawing, and NaCl-SDS). Goat venous endothelial cells were isolated and seeded onto the acellularized xenograft leaflets. Microscopic examinations were done to select the most effective method of decellularizing xenogeneic cells and seeding autologous endothelial cells. Two pulmonic valve leaflets of. 6 goats were replaced by acellularized porcine leaflets with or without seeding autologous endothelial cells while on cardiopulmonary bypass. Goats were sacrificed electively at 6 hours, 1 day, 1 week, 1 month, 3 months, and 6. months after operation. Morphologic examinations were done to see the biological responses of replaced valve leaflets. Result: The microscopic examinations showed that porcine cells were almost completely removed in the leaflets treated with NaCl-SDS. The seeded endothelial cells were more evenly preserved in NaCl-SDS treatment. All 6 goats survived the operation without complications. The xeno- autografts and xenografts showed the appearance, the remodeling process, and the cellular functions of myofibroblasts, 1 day, 1 month, and 3 months after operation, respectively. They were compatible with the native pulmonary leaflet (control group) except for the increased cellularity at 6 months. The xenografts revealed the new endothelial cell lining at that time. Conclusion: Treatment with NaCl-SDS was most effective in obtaining decellularized xenografts and facilitate seeding autologous endothelial cells. The xenografts and xeno-autografts were repopulated with myofibroblasts and endothelial cells in situ serially. Both of grafts served as a matrix for a tissue engineered heart valve and developed into autologous tissue for 6 months.

• Journal of Acupuncture Research
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• v.24 no.2
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• pp.1-14
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• 2007

목적 : 이 연구는 Vipera lebetina turanica의 사독약침파(蛇毒藥鍼波)(Snake venom toxin, SVT)이 in vitro에서 $NF-{\kappa}B$의 활성억제와 apoptosis 관련 단백질의 발현 조절을 통하여 세포자멸사(Apoptosis)를 유도하는지 in vivo에서 또한 전립선 암세포주인 PC-3 세포의 성장을 억제하는지 살펴보고자 하였다. 방법 : SVT를 처리한 후 PC-3의 성장억제를 관찰하기 위해 WST-1 assay, CCK-8 assay를 시행하였고，Apoptosis evaluation에는 DAPI, TUNEL staining assay를 시행하였으며，Apoptosis regulatory proteins의 변화 관찰에는 western blot analysis를 시행하였고，apoptosis와 연관된 $NF-{\kappa}B$의 활성 변화를 관찰하기 위해 EMSA시행하였으며，SVT의 핵내이동을 관찰하기 위해 Immunofluorescence Staining, Confocal immunocytochemistry를 시행하였으며，전립암세포의 종양형성에는 흉선을 제거한 쥐에 Tumorigenecity study를 시행하였다. 결과 : PC-3 세포에 SVT를 처리한 후，전립선 암세포의 성장，Apoptosis의 유발，Apoptosis관련 단백질의 발현，$NF-{\kappa}B$의 활성，SVT의 PC-3세포 핵내 이동여부 및 흉선제거 후 PC-3 세포를 이식한 쥐의 종양형성과정에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. PC-3 세포에서 SVT를 처리한 후 세포성장이 억제되고，세포자멸사가 유도되며，조절인자인 p53, caspase-3, -9는 증가되었고，Bcl-2는 감소되었다. 2. PC-3 세포에서 SVT를 처리한 후 $NF-{\kappa}B$의 활성이 유의하게 감소되었다. 3. DAPI로 염색된 상태에서 SVT가 PC-3 세포의 핵내로 이통되는 것이 관찰되었다. 4. 흉선제거 후 전립선 암세포주를 이식한 쥐에서 SVT를 피내로 주입한 결과 전립선암의 크기와 무게가 유의하게 감소하였다. 결론 : 이상의 결과는 SVT가 $NF-{\kappa}B$의 활성 억제를 통하여 인간 전립선암세포주인 PC-3의 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것이며，이를 재확인한 생체 연구에서의 긍정적인 결과는 향후 SVT의 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 초석이 될 것으로 기대된다.

• Journal of Chest Surgery
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• v.37 no.6
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• pp.482-491
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• 2004

Background: The xenogenic or allogenic valves after in Vitro repopulation with autologous cells or in vivo repo-pulation after acellularization treatment to remove the antigenicity could used as an alternative to synthetic polymer scaffold. In the present study, we evaluated the process of repopulation by recipient cell to the acellu-larized xenograft treated with NaCl-SDS solution and grafted in the right ventricular outflow tract. Material and Method: Porcine pulmonary valved conduit were treated with. NaCl-SDS solution to make the grafts acellularized and implanted in the right ventricular outflow tract of the goats under cardiopulmonary bypass. After evaluating the functions of pulmonary valves by echocardiography, goats were sacrificed at 1 week, 1 month, 3 months, 6 months, and 12 months after implantation, respectively. After retrieving the implanted valved conduits, histopathologic examination with Hematoxylin-Eosin, Masson' trichrome staining and immunohistochemical staining was performed. Result: Among the six goats, which had been implanted with acellularized pulmonary valved conduits, five survived the expected time period. Echocardiographic examinations for pulmonary valves revealed good function except mild regurgitation and stenosis. Microscopic analysis of the leaflets showed progressive cellular in-growth, composed of fibroblasts, myofibroblasts, and endothelial cells, into the acellularized leaflets over time. Severe inflammatory respon-se was detected in early phase, though it gradually decreased afterwards. The extracellular matrices were regenerated by repopulated cells on the recellularized portion of the acellularized leaflet. Conclusion: The acellularized xenogenic pulmonary valved conuits were repopulated with fibroblasts, myofibroblasts, and endothelial cells of the recipient and extracellullar matrices were regenerated by repopulted cells 12 months after the implantation. The functional integrity of pulmonary valves was well preserved. This study showed that the acellularized porcine xenogenic valved conduits could be used as an ideal valve prosthesis with long term durability.

• Reproductive and Developmental Biology
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• v.39 no.3
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• pp.59-67
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• 2015

Galactose-${\alpha}1,3$-galactose (${\alpha}1,3$-Gal) epitope is synthesized at a high concentration on the surface of pig cells by ${\alpha}1,3$-galactosyltransferase gene (GGTA1). The ${\alpha}1,3$-Gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesize $Gal{\alpha}$(1,3)Gal. To prevent hyperacute graft rejection in pig-to-human xenotransplantation, previously, we developed ${\alpha}1,3$-galactosyltransferase gene-knock-out somatic cell by homologous recombination. In this study, we established cell lines of ${\alpha}1,3$-GT knock-out expressing hDAF and hHT gene from minipig fibroblasts to apply somatic cell nuclear transfer. The hDAF and hHT mRNA were expressed in the knock-in somatic cells and ${\alpha}1,3$-GT mRNA was suppressed. However, the knock-in somatic cells were increased resistance to human serum-mediated cytolysis.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.28 no.1
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• pp.186-201
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• 2002

Fructan(Levan)은 식물체 및 미생물에서 발견되는 탄수화물로 이는 과당(fructose)이 $\beta$-2, 6 결합으로 연결되어 있는 polysaccaride 이다. 본 연구에서는 Fructan을 생성하는 미생물(Zymomonas mobilis)과 10% sucrose(기질), 1-2% 효모 추출물을 주성분으로 하는 배지를 사용하여 30-37$^{\circ}C$, pH 5.0-7.0에서 20-24시간 동안 배양한후 원심분리하여 균체를 제거하고 3배량의 알코올을 가하여 침전, 건조하여 얻은 Fructan의 화장품 원료로서의 가능성을 조사하였다. 보습효과에 있어서는 Hyaluronic acid와 유사하였으며, keratinocyte에 대한 세포증식 효과를 나타내었다. 또한 3-D culture에 의해 구축된 생인공 피부내에 0.05%의 sodium lauryl sulfate (SLS)를 사용하여 피부자극에 의한 초기 염증 반응을 유도한후 0.01mg/m1, 0.05mg/m1의 Fructan을 각각 처리하였을 때, SLS만을 처리한 생인공피부와 비교하여 세포증식효능을 보였고, SLS 자극물질로 유도된 전염증성 조절인자인 interleukin-l$\alpha$(IL-l$\alpha$)의 분비량을 조사 하였을때 0.01mg/ml, 0.05mg/ml의 Fructan을 처리한 생인공피부의 IL-l$\alpha$ 양이 Fructan을 처리하지 않은 것보다 상대적으로 감소하였다. 이러한 결과로 Fructan이 생인공 피부내 피부 세포의 증식효과를 나타낼 뿐만 아니라, 또한 피부자극물질에 의한 염증반응에 대해 자극완화효능이 있음을 알 수 있었다. 섬유아세포 및 동물을 이용한 안전성 시험에서도 독성이 없는 안전한 원료로 평가되었다.

• Tuberculosis and Respiratory Diseases
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• v.49 no.1
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• pp.18-29
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• 2000

호흡기 바이러스 감염은 모든 연령층의 천식에 상당한 영향을 미치는 데 영아에서 RSV는 천명을 야기하고 대부분 일시적이나 재발성 일수도 있다. 어릴 때 바이러스 감염은 면역체계 형성에 영향를 미쳐 알러지와 천식의 위험을 완하할 수있다고 한다. 또한 소아와 성인 천식에서 RV같은 감기 바이러스는 천식의 급성 증상을 유발한다. 호흡기 바이러스 감염에 대한 면역반응이, 기관지로 부터 바이러스 제거 기능외에 기도수축과 호흡기 증상에 관여한다고 한다. 이러한 변화가 일어나는 기전은 호흡기 바이러스가 proinflammatory 사이토카인과 매개체 생성을 유도하는 능력과 연관성이 있는 것 같고 이들이 상하기도 호흡기 증상 및 기도반응 변화에 관여하는 것으로 생각된다. 호흡기 바이러스 감염에 대한 면역반응을 요약하면 바이러스 감염으로 상피세포, 내피세포, 과립백혈구가 활성화되며, 상피세포는 사이토카인, 키모카인, 매개체들을 분비하여 항 면역 반응를 주도하다. 이와 같은 상피세포와 다른 기관지 세포들의 조기 활성화로 내피 세포에 유착분자 표현을 증가시켜 백혈구 동원 증가 및 혈관 투과성을 증가시켜 부종과 분비물을 증가시킨다. 바이러스 또는 바이러스 유발 사이토카인에 의해 활성화된 과립 백혈구, 대식세포, T세포들도 기도염증 증가, 기도폐쇄를 야기하고 기도반응을 증가시킨다. 세포독성 임파구에 의한 바이러스 감염세포의 분해, TGF-$\beta$ IL-10 같은 사이토카인에 의해 부분적으로 염증억제, 기도 remoldeling에 의한 기도구조의 재생등이 바이러스 감염후 기관지 기능의 지속적 변화를 결정한다. 끝으로 천식환자에서 RV 감염의 병인에 관한 기본적 문제는 RV감염이 정상인에서는 경한 증상을 나타내는 데 천식환자에서는 왜 심한 임상증상을 나타내는지 아직 완전히 밝혀지지 않았다. 항 바이러스에 대한 면역반응이 천식환자에서 손상되었는지 또는 천식환자에서 RV감염에 의한 중증의 임상증상은 어떤 다른 세포가 관여하는지? 이들에 대한 답은 기도염증이 천식에서 어떻게 조절되는지 또한 바이러스 감염에 의한 악화된 증상을 어떻게 치료할 것인가에 대한 방향을 제시해줄 것이다.

• Polymer(Korea)
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• v.32 no.1
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• pp.49-55
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• 2008

It is recognized that Schwann cells (SC) are essential for peripheral nerve development and regeneration. SIS (small intestinal submucosa) consists of some growth factors which can stimulate cell activity without immune rejection responges. SCs were harvested from the femurs and tibias of female Fischer rat and then suspended with $2{\times}10^6$ cell/sponge in SIS sponge. Fischer rat received an implant consisting of the SCs and the SIS sponge at the place of a 5 mm gap created by the sciatic nerve resection. Thin sections were stained with H &E staining and immunostaining of S-100, GFAP and NF after 1, 2, and 4 weeks. It was observed that the effects of the SIS sponge with SCs on neuroinduction(Group II, with scaffold & cell) are strong as much as uninjured model(Control I), and significantly stronger than SIS sponge model (Group 1, with scaffold only) and blank model (Control II). In conclusion, these results suggest that SIS sponge filled with SCs may have an important role for peripheral nerve regeneration of tissue engineering.

• Journal of Korean Ophthalmic Optics Society
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• v.13 no.3
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• pp.95-101
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• 2008

Purpose: Hydrogen peroxide which is one of the reactive oxygen species has been seen to cause various diseases, various cellular disinfections, gene transformation and cell death. The goals of this study were to determine the protective effect of EGCG against $H_2O_2$-induced apoptotic death in conjunctival cell lines. Methods: We measured cell viability by MTT assay and analyzed DNA fragmentation to check up a distinctive feature in cell death and measured the removal ability of free radicals by DPPH free radical scavenging assay and evaluated the oxygen free radical's quantity in the cell by DCFH-DA assay. The mRNA expression in the cell were examined by RT-PCR. Results: Cell viability and free radical scavening activites was significantly increased in dose dependently after cell was exposed to EGCG. And DNA fragmentation and intracellular ROS was decreased. It was showed the mRNA expression which increase of bcl-2, bcl-xL expression and decrease of bax expression. Conclusions: From these results, it suggests that EGCG has an antioxidant effect and protects conjunctival cell lines from the $H_2O_2$-mediated apoptosis through the modulation of the mRNA expression.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.21-27
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Recipient bovine and porcine oocytes were obtained from slaughterhouse and were matured in vitro according to established protocols. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS and primary fibroblast cultures were established in TCM-199 with 10% FBS. The matured oocytes were dipped in D-PBS plus 10% FBS ＋ 7.5 $\mu$ g/ml cytochalasin B and 0.05M sucrose. Enucleation were accomplished by aspirating the first polar body and partial cytoplasm which containing metaphase II chromosomes using a micropipette with an out diameter of 20∼30 $\mu$m. A Single donor cell was individually transferred into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were electric fusion with 0.3M mannitol fusion medium. After the electrofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. And porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6 ∼8 day at $39^{\circ}C, 5% CO_2 $in air. Interspecies nuclear transfer by recipient bovine oocytes were fused with electric length 1.95 kv/cm and 2.10 kv/cm. There was no significant difference between two electric length in fusion rate(47.7 and 44.6%) and in cleavage rate(41.9 and 54.5%). Using electric length 1.95 kv/cm and 2.10 kv/cm in caprine-porcine NT oocytes, there was also no significant difference between two treatments in fusion rate(51.3 and 46.1%) and in cleavage rate(75.0 and 84.9%). The caprine-bovine NT oocytes fusion rate was lower(P＜0.05) in 1 pulse for 60 $\mu$sec(19.3%), than those from 1 pulse for 30 $\mu$sec(50.8%) and 2 pulse for 30 $\mu$sec(31.0%). The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(53.3%) and 2 pulse for 30 $\mu$sec(50.0%), than in 1 pulse for 60 $\mu$sec(18.2%). The caprine-porcine NT oocytes fusion rate was 48.1% in 1 pulse for 30 $\mu$sec, 45.2% in 2 pulse for 30 $\mu$sec and 48.6% in 1 pulse for 60 $\mu$sec. The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(78.4%) and 1 pulse for 60 $\mu$sec(79.4%), than in 2 pulse for 30 $\mu$sec(53.6%). In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer and 30.6% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than parthenotes(37.4%).

• Maxillofacial Plastic and Reconstructive Surgery
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• v.19 no.3
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• pp.300-310
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• 1997

In order to make in vivo model of human oral squamous cell cancer, we brought up BALB/C nude mice in specially designed housystem, and maintained some kind of human oral squamous cancer cell lines ; KB, SCC-4, SCC-9, SCC-15, SCC-25. Various concentration of cancer cells were inoculated subcutaneouly into flank area of nude mice. We observed each nude mouse more than 5 weeks after tumor inoculation. We appraised the results, measured the tumor size, and calculated the growing tumor volumes after tumor inoculation according to cancer cell line and concentration of cancer cells in media. Some cancer cell lines were rapidly growing in nude mice, but some cancer cell line couldn't grow in nude mice and resorbed completely. And in some cancer cell line, some nude mice showed continuously growing tumor, but other didn't show any tumor growing. And as a new try, we implanted specially disigned caps on the back of nude mice, and cancer cell lines were brought into the caps with media. We removed the cap after 1 week, and observed over 4 weeks. The shape and size of growing tumor were observed.