치주인대 세포의 교원질 생성에 대한 Substance P의 효과

전준영,최제용,경희문,성재현 대한치과교정학회 1996 대한치과교정학회지 Vol.26 No.1

Substance P는 교정력이 가해진 치아의 치주인대 중 인장력을 받는 부위에 많이 분포하는 neuropeptide 중의 하나이며, 또한 여러 조직에서 neurogenic inflammation을 야기하는 neuropeptide 중의 하나로도 알려져 있다. 그러나 중요한 세포의 단백기질인 교원질의 생성에 대한 Substance P의 효과는 잘 알려져 있지 않다. 따라서 이 연구의 목적은 배양 치주인대 세포에서 교원질 생성에 대한 Substance P의 효과를 평가하는 것이었다. collagenase-digestion method로 교원질 생성을 평가하였고 mRNA 수준에서 작용효과를 평가하기 위하여 Northern blot hybridization을 시행하였다. 이 연구는 또한 교원질 생성에 대한 prostaglandin과 gelatinase 생성도 포함하였으며 변성된 교원질의 분해를 평가하기 위하여 Zymography를 이용하였다. 비교원성 단백질, 교원성 단백질, 상대교원질에 대한 dose-dependent effect를 보면 Substance P는 비교원성 단백질 합성을 증가시켰으나 교원성 단백질 합성은 감소시켰다. 그리하여 총 단백합성에 대한 상대적인 교원질 생성을 나타내는 상대교원질은 7%에서 3.6%로 감소시켰다. 세포를 indomethacin과 동시에 처리할 때 substance P의 교원질 합성 억제효과는 나타나지 않았다. 이것은 Substance P의 교원질 합성 억제효과가 prostaglandin의 생성 때문이라는 것을 의미한다. Substance P의 교원질 합성 억제효과가 procollagen mRNA의 정상(steady-state)수준에 부합하는가를 평가하기 위하여 northern blot hybridization을 시행한 결과 Substance P는 α1(1) procollagen mRNA의 양적 변동을 일으키지 않았다. Substance P의 교원질 생성 억제효과는 전사이후의 어떤 단계에서 이루어지는 현상임을 나타낸다. 치주인대세포에서 gelatinase 생성에 대한 Substance P의 효과를 알아보기 위하여 zymography를 이용하였다. zymogram을 보면 Substance P는 치주인대세포에서 gelatinase 생성에는 아무 효과도 나타내지 않음을 알 수 있다. Substance P의 교원질 생성 억제효과가 치주인대세포에 대해 선택적인가 아닌가를 알아보기 위하여 MC3T3-E1세포를 이용하였는데 Substance P는 MC3T3-E1세포의 교원질 합성에는 영향을 미치지 않았다. 이상에서 Substance P는 인간의 치주인대세포에서 교원질 합성을 억제하였다. 이 효과는 procollagen mRNA와 gelatinase 생성의 정상(steady-state) 수준의 변화 때문이 아니라 prostaglandin 생성과 연관이 있음을 알았다. Substance P is one of the neuropeptide which presents highly in tension site of periodontal ligament during the orthodontic tooth movement. It has been also known as one of the neuropeptides which cause neurogenic inflammation in various tissues and organs. However, there is no report about the effect of substance P on major extracellualar matrix protein, collagen production. The purpose of this study was to evaluate the collagen production by substance P in human periodontal ligament cell, The collagenase-digestion method was used to evaluate collagen production and also used Northern blot hybridization for the evaluation of collagen mRNA level. This study also included in terms of prostanglandins and gelatinase production with respect to collagen production. For the collagen degradation, zymography was used to estimate denatured collagen degradation. Dose-dependent effect of substance P on noncollagen protein, collagen, and percent collagen was that substance P increased noncollagen protein synthesis, but decreased collagen systnisis. So the percent collagen, which determined by relative collagen production against total protein production, was decreased from 7% to 3.6%. This inhibitory effect of substance P on collagen production was disappeared when cells were treated concomitantly with indomethacin. It means that substance P-induced inhibitory effect on collagen production was due at least in part to the production of prostaglandins. To evaluate whether substance P-induced inhibitory effect on collagen production is correspond to the steady-state levels of procollagen mRNA, Northern blot hybridizartion was performed and it showed that substance P has no effect on the steady-state level of α1(1) procollagen mRNA. It means that the inhibitory effect of substance P on collagen production was due to the change of a certain mechanism after posttranscription. In this context, gelatinase production by substance P in periodontal ligament cells was evaluated by zymography. Zymogram showed that substance P has no effect on gelatinase production in periodontal ligament cells. To explore wheter substance P-induced inhibitory effect on collagen production is selevtive in periodontal ligament cells or not, MC3T3-31 cells which originated from mouse calvaria was used. It showed that substance P has no effect on collagen production in MCDTD-E1 cells. Taken together, substance P inhibits collagen production in human periodontal ligament cells. This effect was not due to the change of the steady-state level of procollagen mRNA and gelatinase production, but due at least in part to the change of prostaglandins production.

Optimization of Citric Acid Production by Immobilized Cells of Novel Yeast Isolates

( Abd El-latif Hesham ),( Yasser S. Mostafa ),( Laila Essa Omar Alsharqi ) 한국균학회 2020 Mycobiology Vol.48 No.2

Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30-40 g/L) was greater than that of freely suspended cells (8-19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ~52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 ℃, with NH₄Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 ℃ with (NH₄)₂ SO₄ as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.

Preparation of Corncob Grits as a Carrier for Immobilizing Yeast Cells for Ethanol Production

( Sang Eun Lee ),( Choon Geun Lee ),( Do Hyung Kang ),( Hyeon Yong Lee ),( Kyung Hwan Jung ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.12

In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized DEAE·HCl derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M DEAE·HCl, the yeast cell suspension (OD600 = 3.0) was adsorbed at >90% of the initial cell OD600. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The Qmax (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAEcorncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.

인체 세포 기반 융복합 의료제품의 최신 규제 동향

김미혜 ( Kim Mi-hye ),김주희 ( Kim Joo-hee ),김수동 ( Kim Su-dong ) 한남대학교 과학기술법연구원 2023 과학기술법연구 Vol.29 No.2

세포-지지체 복합제품은 최근 재생의료 수요의 증가, 세포 연구의 활용 분야의 확대로 인해 점점 다양한 형태로 개발되고 있다. 세포-지지체 복합제품들은 주로 인체 세포나 조직을 재생, 복구 또는 대체하기 위해 사용되며, 세포가 지지체와 결합을 통해 새로운 구조적 특성, 생물학적 활성 또는 생리적 기능을 획득하는 과정을 거쳐 제조되기 때문에 그 구조와 기능이 복잡한 특성을 갖는다. 최근에는 물리적·생물학적 특징이 다양한 지지체가 개발되고 있으며, 제품의 기능과 생산에 결정적인 영향을 주는 점을 감안하여 제품의 안전성과 유효성을 확인하는 것이 중요하다. 이러한 제품의 품질 최적화를 위해서는 구성부분들이 복합체로 제조되기 전에 세포와 지지체 각각에 대한 특성 분석뿐만 아니라 세포-지지체 간 상호작용, 완제품 조직의 기능성에 대한 제품별 검증이 필요하다. 인체 세포 기반 융복합 의료제품은 미국에서는 ‘인체 세포, 조직 혹은 세포·조직이용제품(HCT/Ps)’, 유럽에서는 첨단치료의약제품(ATMP), 일본에서는 재생의료 등 제품으로 불리고 있다. 이렇듯 국가별로 제품을 지칭하는 용어가 상이할 뿐만 아니라 해당 제품군을 규율하는 내용도 상이하다. 이에 본 논문은 우리나라의 인체 세포 기반 융복합 의료제품에 대한 규제현황과 선진국의 인체 세포 기반 융복합 의료제품의 규제 동향을 살펴본 후 인체 세포 기반 융복합 의료제품의 합리적 규제를 위한 입법적 제언을 제시하는 것으로 결론에 갈음하고자 한다. Cell-scaffold combination products are being developed in various forms due to the recent increase in demand for regenerative medicine and the expansion of the application field of cell research. Cell-scaffold combination products are mainly used to regenerate, repair or replace cells or tissues of the human body. Because cells are manufactured through the process of acquiring new structural properties, biological activities, or physiological functions through binding with scaffolds, Its structure and function are complex. Considering that the characteristics of a cell-scaffold combination products have a decisive influence on the function and production of the product, it is important to confirm the safety and effectiveness of the product. In order to optimize the quality of these products, it is necessary to verify the characteristics of each cell and scaffold as well as the cell-scaffold interaction and product-specific verification of the functionality of the finished product before components are manufactured as a composite. In the United States, cell-scaffold combination products are referred to as ‘human cells, tissues, or cell/tissue use products (HCT/PS)’, in Europe, they are referred to as advanced therapy medicinal products(ATMP), and in Japan, they are called products such as regenerative medicine. In this way, not only are the terms used to designate products different, but also the cont nt that regulates the product group in each country is different. Therefore, this paper examines the regulatory status of human cell-based combination products in Korea and the regulatory trend of human cell-based combination products in developed countries, and makes legislative suggestions for rational regulation of human cell-based combination products.

줄기세포 기반 수혈용 적혈구 생산

김현옥 대한수혈학회 2016 大韓輸血學會誌 Vol.27 No.3

Blood transfusion is a well-established cell therapy. However, blood available for transfusion is a limited resource and is available only through donations by healthy volunteers. Moreover, the perpetual and widespread shortage of blood products, problems related to transfusion transmitted infections, and new emerging pathogens have elicited an increase in demand for artificial blood. Therefore, research for alternative RBC substitutes has begun in the 1960s. Hemoglobin-based oxygen carriers (HBOC) and perfluorocarbon-based oxygen carrier (PBOC) were two popular study subjects; however, research on these substitute candidates was halted due to unsatisfactory results and safety issues, including death, in the 1990s. Since then, worldwide efforts to produce RBC have shifted over to stem cell-derived RBC production using cord blood and G-CSF-mobilized peripheral blood stem cells, and some progress has been made. In terms of practical usefulness, however, large-scale production and cost effectiveness are still problematic. Recently, human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) have shown the potential to produce RBCs as unlimited cell sources. These two methods using hESCs and hiPSCs are also cost-effective since autologous and O, D negative blood RBCs will be used for alloimmunized patients with multiple alloantibodies or rare blood types (high incidence antigens) as well as universal blood production. We will review the current research on in vitro RBC production from hematopoietic stem cells and pluripotent stem cells and assess future directions in this field. 수혈은 적혈구, 백혈구, 혈소판 등 살아있는 세포가 환자에게 주입된다는 점에서 인류역사에서가장 오래된 세포치료제 중의 하나로 평가될 수있다. 그러나 혈액은 헌혈로서만 충당할 수 있는제제로 최근에는 혈액 부족현상이 발생하고 있으며, 특히 수혈로 인한 감염전파 문제는 새롭게 계속 나타나는 신종 바이러스 전염병이 보고될 때마다 수혈의 위험성이 강조되고 있다. 이런 문제를 극복하기 위한 인공혈액에 대한 연구는 1960 년대 중반부터 시작되었다. 산소를 운반하는 기능인 헤모글로빈으로 구성된 혈색소기반 산소운반체와 과불화탄소(perfluorocarbon)를 이용한 산소운반체가 개발되었으나 임상시험 진행 중 사망에 이르는 다양한 부작용이 보고되면서 개발이거의 중단되었다. 이후 줄기세포가 새로운 의료의 치료 패러다임으로 소개되면서 줄기세포를 이용하여 수혈용 적혈구를 생산하고자 하는 연구가진행되었고 제대혈과 G-CSF 가동 말초조혈모세포로부터 CD34+ 세포를 분리하여 적혈구를 생산하는 1단계 프로토콜은 완성되었다. 그러나 수혈용으로 사용하기 위해서는 대량생산 및 고가의생산비용이 필요한 점 등 모든 헌혈 혈액을 대체하기에는 역부족인 점이 지적되면서 만능줄기세포인 배야줄기세포나 역분화줄기세포로부터 적혈구를 생산하는 연구로 전환하게 되었다. 두 만능줄기세포로부터 만들어지는 적혈구의 수혈 대상은 첫째, 희귀혈액형 보유자나 복합 항체 보유로 적합한 혈액을 쉽게 찾을 수 없는 환자에 대한자가 혈액생산과 둘째 O형 D음성 혈액형을 가진만능 공혈자로부터 얻은 말초혈액에서 역분화줄기세포를 제작하고 여기서부터 적혈구로 분화시키는 기술 개발로 만능인공혈액을 생산하는 연구까지 진행되고 있다. 물론 아직까지 완벽한 수혈용 적혈구의 생산단계에는 이르지 못했으나 앞으로 역분화줄기세포로 확대된 줄기세포기반 적혈구 생산은 생각보다 빠르게 발전할 것이며, 안전성과 가격 문제가 해결된다면, 수년 내에 그 결과를 기대할 수 있을 것이다.

Rhodopseudomonas sphaeroides의 고정화균체에 의한 수소생산의 효율적 기질공급

김진상,홍용기,신일식,조학래,장동석 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.1

Rhodopseudomonas sphaeroides B6 세포를 agar gel 중에 고정했을 때, 수소생산을 위한 최적 agar 농도는 2%(w/v)였다. B6의 2% agar gel 고정화균체(300㎖ gel: 2.85 ㎎ dry cells/㎖)와 비고정세포(1ℓ culture: 0.87㎎ dry cells/㎖)에 있어서 초기의 최고 수소 생산활성은 각각 47.5 및 48.O ㎖/hr/culture로 거의 같았다. 그러나, 수소생산이 거의 정지된 배양후기에 lactate의 제한공급(10mmole)에 의한 비고정세포의 활성회복은 50% 이하로 감소되었지만, 고정화세포의 활성은 거의 초기상태로 회복되었다. B6의 고정화균체를 이용하여 12시간 주기의 광조사 조건으로, 매 1ℓ의 수소생산시 마다 그에 소비된 만큼의 기질에 상당하는 9.3mmole의 DL-lactate와 1.86 mmole의 L-glutamate 함유 기본배지를 주기적으로 공급한 결과, 228시간의 배양기간 동안 명암의 반복 조건에도 불구하고 평균 510 ㎖/day/300㎖ ge1(2.9 ㎎ dry cells/㎖)의 속도로 수소생산이 지속되었다. 이 결과는 광합성세균에 의한 수소생산에 있어서, 보다 효율적 기질공급과 가변적인 태양광 이용 조건에 대응되는 자동배양 시스템의 구성 가능성을 시사한다. The Photosynthetic bacterium, Rhodopseudomonas sphaeroides strain B6 was immobilized on agar gel. The optimum concentration of agar for hydrogen production was 2% (w/v). Maximum rates of hydrogen production by immobilized (300㎖ of gel; 2.85㎎ dry cells/㎖) and free cells (1ℓ culture; 0.87㎎ dry cells/㎖) were 47.5 and 48.0 ㎖/hr/culture, respectively. However, when both cultures were fed by 10mmoles of lactate as limited electron donor at the later period of incubation, the activity of hydrogen production by free cells was significantly decreased but, immobilized cells continued hydrogen production with almost the same initial rate. We examined hydrogen production by immobilized cells of strain B6 under periodic illumination for 12 hr-intervals. When the culture was periodically fed by basal medium containing 9.3 mmoles of DL-lactate and 1.86 mmoles of L-glutamate as consumed electron donor and nitrogen source, respectively, for every one liter of hydrogen produced, hydrogen was evolved continuously with the average rate of 510 ㎖/day/300 ㎖ gel (2.9 ㎎ dry cells/㎖) during the incubation time for 228 hr.

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

Ahn, Woo Suk,Jeon, Jae-Jin,Jeong, Yeong-Ran,Lee, Seung Joo,Yoon, Sung Kwan Wiley Subscription Services, Inc., A Wiley Company 2008 Biotechnology and bioengineering Vol.101 No.6

<P>To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37°C until viable cell concentration reached 1 × 10<SUP>7</SUP> cells/mL, and then culture temperature was shifted to 25°C, 28°C, 30°C, 32°C, 37°C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37°C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q<SUB>EPO</SUB>, increased at low culture temperature and were the highest at 32°C and 30°C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32°C was not as high as the cumulative EPO production at 32°C although the q<SUB>EPO</SUB> at culture temperature below 32°C was comparable or even higher than the q<SUB>EPO</SUB> at 32°C. This implies that the beneficial effect of lowering culture temperature below 32°C on q<SUB>EPO</SUB> is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32°C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37°C. However, at culture temperatures below 32°C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32°C negatively affect the quality of EPO. Thus, taken together, cell culture at 32°C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32°C was not deteriorated as obtained at 37°C. Biotechnol. Bioeng. 2008;101: 1234–1244. © 2008 Wiley Periodicals, Inc.</P>

줄기세포 유래 세포치료제의 개발 전략 및 임상화 과정

조명수 ( Myung Soo Cho ),유대훈 ( Dae Hoon Yoo ),송슬애 ( Seul Ae Song ),최영민 ( Young Min Choi ) 서울대학교 인구의학연구소 2012 人口醫學硏究論集 Vol.25 No.-

Stem cells are good cell sources for cell replacement therapy of degenerative or incurable disorders. However, there are some limitations such as immune response, differentiation efficiency, safety and ethical issues for clinical application. These barriers must be considered thoroughly from starting point of the investigational study. For clinical application and commercialization of stem cell-derived cell therapy products, strict preparation is required for formal approval of regulatory body at each stage of the clinical process. This preparation process can be another barrier and challenge to the researcher or developer related to stem cell study. However, These challenges will become increasingly important to stem cell research as clinical translation progresses and helpful for birth of authentic novel cell therapy products. In this brief review, we will suggest more effective strategy of the stem cell research and development and introduce the clinical process for cell therapy products briefly.

Inflammatory Mediators Modulate NK Cell-stimulating Activity of Dendritic Cells by Inducing Development of Polarized Effector Function

Kim, Kwang-Dong,Choi, Seung-Chul,Lee, Eun-Sil,Kim, Ae-Yung,Lim, Jong-Seok The Korean Association of Immunobiologists 2007 Immune Network Vol.7 No.3

Background: It is well established that cross talk between natural killer (NK) cells and myeloid dendritic cells (DC) leads to NK cell activation and DC maturation. In the present study, we investigated whether type 1-polarized DC (DC1) matured in the presence of IFN-${\gamma}$ and type 2-polarized DC (DC2) matured in the presence of PGE2 can differentially activate NK cells. Methods: In order to generate DC, plastic adherent monocytes were cultured in RPMI 1640 containing GM-CSF and IL-4. At day 6, maturation was induced by culturing the cells for 2 days with cytokines or PGE2 in the presence or absence of LPS. Each population of DC was cocultured with NK cells for 24 h. The antigen expression on DC was analyzed by flow cytometry and cytokine production in culture supernatant was measured by ELISA or a bioassay for TNF-${\alpha}$ determination. NK cell-mediated lysis was determined using a standard 4h chromium release assay. Results: DC2, unlike DC1, had weak, if any, ability to induce NK cell activation as measured by IFN-${\gamma}$ production and cytolytic activity. DC2 were weakly stimulated by activated NK cells compared to DC1. In addition, IFN-${\gamma}$-primed mature DC appeared to be most resistant to active NK cell-mediated lysis even at a high NK cell/DC ratio. On the other hand, PGE2-primed DC were less resistant to feedback regulation by NK cells than IFN-${\gamma}$-primed mature DC. Finally, we showed that the differential effect of two types of DC population on NK cell activity is not due to differences in their ability to form conjugates with NK cells. Conclusion: These results suggest that different combinations of inflammatory mediators differentially affect the effector function of DC and, as a result, the function of NK cells, eventually leading to distinct levels of activation in adaptive immunity.

AHP기반 에어백 조립 생산 배치 개선

최진영 ( Jin Young Choi ),최윤규 ( Yun Kyu Choi ) 대한설비관리학회 2020 대한설비관리학회지 Vol.25 No.3

The automobile airbag assembly production processes in company A can be characterized as multi-product middle-quantity continuous assembly production. Therefore, production cells were arranged in a grid shape and production processes in a cell were placed in a U shape so that various products were produced in one cell. However, recently it has become an urgent task to make feeding materials, withdrawing finished products, and saving production space efficient. Based on this motivation, in this paper, we propose a new improved assembly production layout by using Analytic Hierarchy Process (AHP) to evaluate layout alternatives. Specifically, we extract four decision criteria, generate three production layout alternatives by brainstorming, and choose the best one by using AHP. The selected layout, with U shape of cells and two parallel lines of processes in a cell, could reduce 77.5% of cell feeder’s moving distance and 59.8% of production space area compared to the current one.