• Clinical and Experimental Reproductive Medicine
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• 제36권1호
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• pp.23-34
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• 2009

목 적: 중간엽 줄기세포를 임상에 적용하기 위해서는 체외 배양을 통한 세포증식 과정이 필요하나, 오랜 기간 동안 체외 배양을 하게 되면 노화되어 특성이 변하고 분화 능력 또한 감소하게 된다. 따라서 현재까지는 초기 계대배양의 세포만이 임상에 적용되고 있는 실정이며 체외에서의 세포 배양이 세포의 특성에 미치는 영향에 대한 연구와 함께 세포의 특성 변화 없이 체외증식이 가능하도록 하는 연구들이 골수 및 지방유래 중간엽 줄기세포에서 보고되고 있다. 그러나 현재 탯줄유래 줄기세포의 체외 배양에 따른 특성 변화 분석 연구는 아직 잘 이루어지지 않고 있다. 본 연구의 목적은 탯줄유래 줄기세포의 체외 배양 시 계대배양 증가에 따른 줄기세포의 특성 변화를 분석하고자 하였다. 연구방법: 사람의 탯줄유래 줄기세포 (human umbilical cord-derived stem cells, HUC)를 분리하여 in vitro에서 계대배양하였다. 계대배양에 따른 세포의 형태와 population doubling time (PDT)을 조사하고 RT-PCR 방법을 이용하여 mRNA 분석을 하였으며 면역세포화학 염색법을 이용하여 단백질 발현을 분석하였다. 결 과: 탯줄유래 줄기세포는 평균 10번의 계대배양 후 senescence를 나타냈다. 세포의 형태는 7번째 계대배양 이후 세포질이 넓어지고 세포의 크기가 커지는 변화를 나타냈으며 PDT가 증가하기 시작하였다. 계대배양 4, 8, 10번째 시기의 세포의 mRNA 변화를 분석한 결과 Oct-4, HNF-4${\alpha}$, mRNA는 10번째 계대배양까지 지속적으로 발현하였으나 nestin, vimentin mRNA는 지속적으로 발현이 감소하였고 SCF mRNA는 지속적으로 발현이 감소하였다. 이에 반해 HLA-DR${\alpha}$, Pax-6, BMP-2 mRNA는 모든 계대배양 시기의 세포에서 발현되지 않았다. 면역세포화학 분석법을 통한 3, 6, 9번째 계대배양 세포의 단백질 발현 분석 결과 SSEA-3와 SSEA-4는 3, 6, 9번째 계대배양 세포 모두에서 발현하였으나 ICAM-1과 HLA-ABC는 계대배양이 증가함에 따라 발현이 증가되었다. Thy-1 단백질은 p9에서 발현이 증가되었으며 이와 반대로 TRA-1-60와 VCAM-1 단백질은 p6과 p9 시기에 발현이 감소되었다. HLA-DR 단백질은 모든 계대배양 시기에 발현되지 않았다. 결 론: 본 연구결과 탯줄유래 줄기세포는 체외 배양 시 줄기세포 특성이 일부 변하는 것을 관찰하였다. 앞으로 줄기세포의 특성을 유지할 수 있는 체외 배양법의 발달을 위한 연구들이 수행 되야 할 것으로 사료된다.

• 한국가축번식학회지
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• 제17권4호
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• pp.331-337
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• 1994

처녀발생은 난자의 세포질성숙을 투명대경화나 체외수정에 있어 난자 외적요소의 문제점을 배제하고 측정할 수 있는 지표이다. 본 실험에서는 체외성숙시간에 따른 소 난자의 처녀발생활성을 조사하였다. 도살장 난소로부터 회수한 미성숙난포란을 15% 소 태아혈청이 첨가된 TCM 199에서 6시간 간격으로 24~48시간까지 성숙시킨 후 7% ethanol로 7분간 활성화시켰다. 핵성숙과 세포질성숙은 rapid staining에 의해 핵형태와 전핵의 형성 유무로 판정하였다 핵성숙율은 24~48시간 사이 각각 81, 89, 72, 60 및 60%로 체외성숙 36시간에 성숙율이 최고였으나, 반면 감수분열 중기 II 염색체이상은 36시간부터 증가(0~30%)하였다. 에탄올처리에 의한 전핵형성율은 체외성숙 24~48시간에 각각 67, 68, 73, 84 및 87%였고, 그 중 이배체율은 각각 4, 5, 10, 16 및 20%로 성숙시간이 증가함에 따라 증가하였다. 위 실험의 결과 난자의 체외성숙 연장에 따라 전핵형성과 이배체수가 증가되는 것으로 나타났으며, 정상적인 핵성숙에 비해 세포질성숙은 더 많은 성숙시간이 필요한 것으로 나타났다. 이러한 결과들은 소 초기배 체외생산시와 핵치환용 핵수용란 생산시 적정 성숙시간 결정에 유용하게 이용될 수 있을 것이다.

• The Journal of Advanced Prosthodontics
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• 제7권4호
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• pp.312-316
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• 2015

PURPOSE. This study aimed to present the clinical applicability of restorations fabricated by a new method, by comparing the bond strength of between ceramic powder with different coefficient of thermal expansion and alloys fabricated by Selective laser sintering (SLS). MATERIALS AND METHODS. Fifty Co-Cr alloy specimens ($25.0{\times}3.0{\times}0.5mm$) were prepared by SLS and fired with the ceramic ($8.0{\times}3.0{\times}0.5mm$) (ISO 9693:1999). For comparison, ceramics with different coefficient of thermal expansion were used. The bond strength was measured by three-point bending testing and surfaces were observed with FE-SEM. Results were analyzed with a one-way ANOVA (${\alpha}$=.05). RESULTS. The mean values of Duceram Kiss ($61.18{\pm}6.86MPa$), Vita VM13 ($60.30{\pm}7.14MPa$), Ceramco 3 ($58.87{\pm}5.33MPa$), Noritake EX-3 ($55.86{\pm}7.53MPa$), and Vintage MP ($55.15{\pm}7.53MPa$) were found. No significant difference was observed between the bond strengths of the various metal-ceramics. The surfaces of the specimens possessed minute gaps between the additive manufactured layers. CONCLUSION. All the five powders have bond strengths higher than the required 25 MPa minimum (ISO 9693); therefore, various powders can be applied to metal structures fabricated by SLS.

• Clinical and Experimental Reproductive Medicine
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• 제17권2호
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• pp.185-196
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• 1990

These experiments were performed to know ultrastructural changes of the cumulus expansion in virot. SEM:In expanded oocyte-cumulus complex, the cell surface are characterized by the presence of many evaginations:they are relatively short and round shape. The mucous extracellular material were deposited between cumulus cells. TEM:In compact cumulus cells, golgi apparatus and rough endoplasmic reticulum developed. In expanded cumulus cells, rough endoplasmic reticulum decreased and the smooth endoplasmic reticulum increased. Also, there were numbers of mitochondria. Extracellular mucous material which is presumed to be hyaluronic acid appears when cumulus cell were expanded. In expanded cumulus cell, numbers of smooth endoplasmic reticulum help cumulus cell to develop in steroidogenic cell.

• Archives of Plastic Surgery
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• 제47권5호
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• pp.392-403
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• 2020

Severe cartilage defects and congenital anomalies affect millions of people and involve considerable medical expenses. Tissue engineering offers many advantages over conventional treatments, as therapy can be tailored to specific defects using abundant bioengineered resources. This article introduces the basic concepts of cartilage tissue engineering and reviews recent progress in the field, with a focus on craniofacial reconstruction and facial aesthetics. The basic concepts of tissue engineering consist of cells, scaffolds, and stimuli. Generally, the cartilage tissue engineering process includes the following steps: harvesting autologous chondrogenic cells, cell expansion, redifferentiation, in vitro incubation with a scaffold, and transfer to patients. Despite the promising prospects of cartilage tissue engineering, problems and challenges still exist due to certain limitations. The limited proliferation of chondrocytes and their tendency to dedifferentiate necessitate further developments in stem cell technology and chondrocyte molecular biology. Progress should be made in designing fully biocompatible scaffolds with a minimal immune response to regenerate tissue effectively

• 약학회지
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• 제49권1호
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• pp.103-108
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• 2005

Tacrine analogues for degenerative brain disease treatments have been designed. A series of diazaanthrine derivatives as novel analogues of tacrine has been prepared through the alkyl substitution and the ring expansion. They were expected to retain anti-inflammatory activity by inhibition of prostaglandin production with reduction of side effect as the selective prostaglandin synthase inhibitor. Prostaglandin synthase expression is associated with the deposition of beta-amyloid protein in neuritic plaques in brain inflammation. Therefore selective prostaglandin synthase blockade is important for the prevention and treatment of alzheimer's disease. To evaluate inhibitory effect of prostaglandin synthase, synthetic tacrine derivatives were screened with accumulation of prostaglandin biosynthesis by lipopolysaccharide in aspirin-treated murine macrophage cell. Most of synthetic compounds have shown significant prostaglandin synthase activities in vitro screening with $84.3{\sim}33.6\%$ inhibition of the prostaglandin $E_2$ production at $10\;{\mu}g/ml$.

• 한국지역사회생활과학회지
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• 제28권3호
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• pp.365-375
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• 2017

The purpose of this study was to investigate the maximal elongation rate and area expansion ratio of human skin in various postures. Five males and five females (male: $23{\pm}2yr$ in age, $177.9{\pm}4.8cm$ in height, $76.7{\pm}8.8kg$ in body weight, $24.2{\pm}2.5$ in BMI, $16.2{\pm}3.4%$ in body fat; female: $22{\pm}1yr$, $163.2{\pm}3.6cm$, $51.4{\pm}2.7kg$, $19.3{\pm}1.6$, $27.4{\pm}6.7%BF$) participated in this study. Measurements were conducted using a pen and tape on the elbow, knee, wrist, shoulder, and neck. Subjects held postures so that each joint of the body regions was bent at its maximal level. The results were as follows: 1) The maximal elongation rate of skin showed a significant difference among the regions: $16.6{\pm}3.4%$ for the wrist, $22.4{\pm}5.5%$ for the neck (back), $37.6{\pm}11.3%$ for the shoulder, $42.6{\pm}10.0%$ for the knee, and $43.9{\pm}4.0%$ for the elbow (p<0.05). 2) The maximal expansion rate of the body surface area had the greatest values on the elbow ($93.7{\pm}6.4%$) and knee ($74.8{\pm}10.8%$). 3) No significant difference was found between males and females. In summary, maximal values of skin elongation and expansion rates in vivo were greater than in vitro values known from previous reports. These results can be applied to develop electronic fibers or textiles for wearable tight fit work clothing as well as fitness wear.

• 한국수정란이식학회지
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• 제32권3호
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• pp.139-146
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• 2017

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in vitro maturation of pig oocytes.

• 한국발생생물학회지:발생과생식
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• 제21권4호
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• pp.407-415
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• 2017

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)-related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: $32.5{\pm}10.1%$ vs control: $77.8{\pm}5.3%$). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, $200{\mu}M$) and melatonin ($0.1{\mu}M$), in BIP/GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, $p-eIF2{\alpha}$, $eIF2{\alpha}$, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.76-76
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• 2001

The objective of this study was to optimize the vitrification method of in vitro produced bovine embryos. Thus, in vitro produced embryos at 8 cell, morula and blastocyst stages were vitrified on electron microscope grids (EM grids) or in open pulled straws (OPS) with EG5.5 (5.5 M ethylene glycol, 1.0 M sucrose and 10% FBS in m-DPBS medium) freezing solution and their survival rates after thawing were compared. The embryos on EM grids or in OPS were briefly exposed to EG5.5 freezing solution and plunged directly into liquid nitrogen within 30 to 35 sec. Post-thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-DPBS, each for 1 min, and then cultured in CRI aa medium supplemented with 10% FBS. Embryonic survival rate was assessed as re-expanded and hatched rates of those embryos after warming. The rates of re-expansion embryos did not significantly different between EM grid (8 cell: 42.10%, morula: 66.66% and blastocyst: 77.08%) and OPS (8 cell: 47.36%, morula: 61.90% and blastocyst: 83.33%) methods. In addition, the hatched rates in EM grid (8 cell: 31.57%, morula: 57.14% and blastocyst: 72.91%) were similar to those in OPS (8 cell. 34.21%, morula: 50.00% and Blastocyst: 77.08%). Interestingly, even at the same blastocyst stage, the in vitro survival of day 7 embryos (EM grid: 79.48 and OPS: 87.18%) was higher than those of day 8 embryos (EM grid: 72.10 and OPS: 82.06%). The total cell number of blastocyst developed in vitro after vitrification was examined with Hoechst 33342 staining to compare the embryo quality among different treatment groups. The total cell number of blastocyst was not significantly different between vitrified groups (EM grid: 162.4$\pm$8.0 and OPS: 158.4$\pm$7.1) and unvitrified control (168.0$\pm$5.6). These results indicate that both vitrification containers can provide the high rate of embryo survival. Moreover, the OPS container may not need a cap to protect the container from floating after immersion in L$N_2$. Therefore, this study suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification method using EM grid or OPS with EG5.5 freezing solution. In the future, the Pregnancy rate would be investigated after transfer of our vitrified embryos into the appropriated recipients.