• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1304-1309
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• 2005

The production of therapeutic proteins for human diseases in plants results in many economic benefits, including reduced risk of animal virus contamination, high yields, and reduced production and storage costs. Human cytokines, interleukin-11 (hlL-11) and granulocyte-macrophage colony-stimulating factor (hGM-CSF), cDNAs were introduced into rice or tobacco, using either the maize ubiquitin promoter or the 35S promoter. The primary hIL-11 transgenic rice plants exhibited stunted growth and a sterile phenotype, whereas the hIL-11 transgenic tobacco plants did not. This suggests that hIL-11 expression in rice disrupts the normal growth and development of the plant. The regeneration efficiency of rice calli transformed with hGM-CSF was found to be approximately a quarter of that seen with the hIL-11, suggesting that hGM-CSF expression is more deleterious to the regeneration of rice calli than is hIL-11. However, the surviving hGM-CSF transgenic rice plants exhibited a normal phenotype of growth. Therefore, it appears that only those transgenic rice lines that expressed the human cytokines in small quantities were able to survive the selection process.

• Animal cells and systems
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• 제16권3호
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• pp.207-214
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• 2012

Burns are one of the most devastating forms of trauma and wound healing is a complex and multicellular process, which is executed and regulated by signaling networks involving numerous growth factors, cytokines, and chemokines. Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was specifically produced from rice cell culture through use of a recombinant technique in our laboratory. The effect of rhGM-CSF on promotion of deep second-degree burn wound healing on the back skin of a hamster model was evaluated through a randomized and double-blind trial. As macroscopic results, hamster skins of the experimental groups showed earlier recovery by new epidermis than the control groups. Immunohistochemical reactions of proliferating cell nuclear antigen and transforming growth factor-b1, which are indicators of cell proliferation, were more active in the experimental group, compared with the control group. On electron microscopy, basal cells in the epidermis of the experimental group showed oval nuclei, prominent nucleoli, numerous mitochondria and abundant free ribosomes. In addition, fibroblasts contained well-developed rough endoplasmic reticulum with dilated cisternae. Bundles of collagen fibrils filled the extracellular spaces. Particularly, ultrastructural features indicating active metabolism for regeneration of injured skin at 15 days after burn injury, including abundant euchromatin, plentiful free ribosomes, and numerous mitochondria, were observed. These findings suggest that use of rhGM-CSF could result in accelerated deep second-degree burn wound healing in animal models.

• Korean Journal of Acupuncture
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• 제27권3호
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• pp.109-118
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• 2010

Objectives : The purpose of this study is to investigate the effect of Bacillus-fermented Scutellariae Radix acupuncture solution (SB) on chemokine and growth factor production in RAW 264.7 mouse macrophages stimulated by lipopolysaccharide (LPS). Methods : Productions of chemokine and growth factor were measured by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on xMAP$^{(R)}$ technology. Firstly, cell culture supernatant was obtained after treatment with LPS (1 ${\mu}g$/mL) and SB for 24 hours. Then, it was incubated with the antibody-conjugated beads for 30 minutes. Detection antibody was then added and incubated for 30 minutes. After incubated for 30 minutes, strepavidin-conjugated phycoerythrin (SAPE) was added. After another 30 minutes incubation, the level of SAPE fluorescence was analyzed in Bio-plex Suspension Array System. Results : The results of the experiment are as follows. 1. SB significantly inhibited the LPS-induced production of vascular endothelial growth factor (VEGF), interferon-inducible protein (IP)-10, and granulocyte-colony stimulating factor (G-CSF) at the concentration of 25, 50, 100, and 200 ${\mu}g$/mL in RAW 264.7 cells (P < 0.05). 2. SB significantly inhibited the LPS-induced production of Eotaxin at the concentration of 25, 100, and 200 ${\mu}g$/mL in RAW 264.7 cells (P < 0.05). 3. SB significantly inhibited the LPS-induced production of MIP-$1\alpha$ at the concentration of 25 and 100 ${\mu}g$/mL in RAW 264.7 cells (P < 0.05). Conclusions : These results suggest that SB has immuno-modulatory property related with its inhibition of VEGF, IP-10, G-CSF, and Eotaxin production in macrophages.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.135-141
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• 2004

인간 과립구 성장인자(hG-CSF)는 골수에서 생산되는 단백질로 호중구의 분화 및 생성을 촉진시키는 역할을 한다. 현재 재조합 hG-CSF는 암화학요법에 의한 호중구감소증, 골수이식시 호중구 감소증, 재생불량성 빈혈에 수반되는 호중구 감소증 등으로 적응증이 확대되고 있다. 본 연구에서는 OmpA signal sequence를 삽입하여 인간 과립구 성장인자(hG-CSF)가 분비발현되도록 고안된 T7 promoter 에 의하여 발현되는 pYRCl 발현백터를 제조하였다. E. coli BL2l (pYRCl) 발현시 $37^{\circ}C$에서 배양하는 경우 많은 양의 봉입체(aggregates)를 형성한다. 이에 비하여 $10\mu$M ucose를 포함하는 변형된 MBL배지에서 10 g/$\ell$IPTG를 유도물질로 7시간동안 $25^{\circ}C$에서 배양하였을 때 전체 periplasm단백질의 15%가 soluble rhG-CSF이었다. 또한, 유가식 배양방법을 사용하여 E. coli BL2l(pYRCl)에서 soluble rhG-CSF의 생산조건을 조사하였다. 유가식 배양에서 rhG-CSF의 발현량이 비증식속도를 $0.43 h^{-1}$ 에서 0.14 $h^{-1}$ 으로, 유도 배양시간을 최적화함으로써 rhG-CSF의 발현량이 4.4mg/$\ell$에서 24mg/$\ell$ 로 증가하였다.

• 생명과학회지
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• 제21권12호
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• pp.1778-1783
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• 2011

조혈에 관여하는 cytokine은 골수세포의 성장과 분화를 촉진시켜 혈구세포 생산을 조절한다. 이런 cytokine을 조혈성장인자(hematopoitic growth factor)이라고 하고, 그 중에서 호중구 세포(neutrophil) 성장에 관여하는 과립구 콜로니 자극 인자(granulocyte-colony stimulating factor, G-CSF)는 임상적 치료제로서 아주 중요하다. 왜냐하면 화학적 항암치료를 받는 환자들에게 심각한 호중구 세포가 감소하는 증세(neutropenia)가 발생하여 감염으로 인한 사망이 일어나기 때문이다. 두 종류의 G-CSF 재조합 단백질이 치료제로 승인 받아 사용되고 있으며, G-CSF 재조합 단백질 생산에 대한 연구가 지속적으로 이루어지고 있다. 선행연구에서 본 연구팀은 누에에서 유래된 Bm5 세포주에서 G-CSF의 생산을 증대하기 위해 누에 prophenoloxidase activating enzyme의 Endoplasmic reticulum targeting signal sequence유전자와 사람 G-CSF 유전자를 융합한 chimera 유전자를 제작하여 재조합 G-CSF 단백질을 생산하였다. 본 연구에서는 이 chimera 유전자가 생산하는 재조합 G-CSF 단백질의 N-말단에 3 개의 아미노산이 결여되는 3 종류의 돌연변이 유전자를 제작하여 G-CSF 단백질 생산에 미치는 영향을 조사하였다. 그 중 한 돌연변이 유전자에 의해 세포 밖으로 분비된 G-CSF 단백질의 생산이 현저히 감소하여, N-말단 부분이 이 단백질의 분비에 관여한다는 것을 알 수 있었다.

• 한방재활의학과학회지
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• 제18권2호
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• pp.97-109
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• 2008

Objectives : Effects of Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) on intellectual development and learning ability were investigated growth and intellectual impairment rat with insufficient nutrition diet. Methods : We divided male Sprague-Dawley rats into 4 groups(A, B, C, D). They were normal group(A), growth deficiency rat with insufficient nutrition diet group(B), growth deficiency rat with 0.1% Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) group(C) and growth deficiency rat with 0.2% Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) group(D). They were administered for 5 weeks. We measured body weight, serum growth hormone, insulin-like growth factor and thyroid stimulating hormone, RBC, concentration of Hb and PCV ratio, total WBC and its composition, the values of plasma glutamic oxaloacetic transaminase(GOT) and glutamic pyruvic transaminase(GPT) activities and morris water maze test in escape distance, escape time and escape speed. Results : 1. Body weight showed a tendency to increase in the Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) groups and 0.2% Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) group showed significantly different than control groups. 2. Serum growth hormone, insulin-like growth factor and thyroid stimulating hormone showed a tendency to increase in Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) groups, however these values showed no significantly different. 3. About the counts of RBC, 0.1% and 0.2% Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) groups showed significantly different than control groups. Concentration of Hb was higher than control group in Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) groups. And 0.2% Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) group showed significantly different than control groups in PCV ratio. 4. The counts of total WBC and its composition showed no significantly different in all treatment groups. 5. The values of plasma glutamic oxaloacetic transaminase(GOT) and glutamic pyruvic transaminase(GPT) activities showed no significantly different in all treatment groups. 6. In the morris water maze test, in escape distance and escape speed, Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) groups showed no significantly different than control group. But Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) groups showed the increasing tendency. Conclusions : So Kyungohkgo($Qi{\acute{o}}ng\;y{\grave{u}}\;g{\grave{a}}o$) have an effect of promoting growth of rats and might be effect to treat various kinds of growth delay in children.

• 생명과학회지
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• 제21권11호
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• pp.1652-1657
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• 2011

혈액세포의 분화와 성장은 20 여종 이상의 성장인자에 의해 조절된다. 혈액세포 생산에 관여하는 인자를 조혈 성장인자(hematopoitic growth factor)라고 한다. 조혈성장인자를 임상적으로 사용하기 위해 원핵생물 또는 진핵 생물 생산 시스템에서 재조합 단백질로 생산되고 있다. 그 중에서 Glranulocyte-Colony Stimulating Factor(G-CSF)는 호중구 세포 수가 감소된 암환자와 선천성 질병을 가진 환자에게 임상적 치료제로 아주 중요한 역할을 한다. 이 환자들은 충분하지 못한 호중구 세포로 말미암아 감염에 대한 위험이 아주 높으며 치사율 또한 높다. 두 종류의 재조합 G-CSF가 항암치료 후 발생하는 부작용으로 나타나는 호중구 세포 감소증 치료에 사용되고 있다. G-CSF의 중요성에 맞추어 G-CSF의 물리적 및 생물학적 기능에 대한 특성을 설명하였으며, 또한 항암치료와 G-CSF의 임상적 사용에 대한 연관성을 토론하였다. 마지막으로 두 종류의 재조합 G-CSF인 non-glycosylated G-CSF, filgrastim과 glycosylated G-CSF를 비교 설명하였으며, 이들 기존의 G-CSF에 비교되는 바이오시밀러에 대한 전망을 제시하였다.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.321-326
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• 2000

To examine the feasibility of the long-term production of the human granulocyte colony stimulating factor (hG-CSF) using the $_{L}-arabinose$ promoter system of Escherichia coli, flask relay culture and cyclic fed-batch culture were performed. In the flask relay culture, it was found that the pismid was maintained stably up to about 170 generations in an uninduced condition, whereby the cells could also maintain the capability of expressing hG-CSF expression were maintained stably up to at least 100 generations. In contrast, in the cyclid fed-batch culture, segregational plasmid instability was observed within about 4 generations after induction, even though the cell growth and hG-CSF production reached their maximum balues, 78.0 g/l of dry cell weight and 7.0 g/l of hG-CSF, respectively. It would appear that, when compared to the flask relay culture, the high-cell density and high-level expression of hG-CSF in the cyclic fed-batch cultrure led to the segregational plasmid instability; in other words, a severe metabolic burden existe on the cells due to the high-level expression of hG-CSF. Accordingly, based on these long-term cultures, the segregational and structural plasmid instability was observed and a strategy to overcome such problems could be designed.

• Nutrition Research and Practice
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• 제9권5호
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• pp.451-458
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• 2015

BACKGROUND/OBJECTIVES: We examined the chemical composition and the effect of fermented deer antler on hematopoietic factors in bone marrow cells. MATERIALS/METHODS: For the preparation of fermented deer antler extract (FAB), fermentation was carried out using Bacillus subtilis at $30^{\circ}C$ for 7 days. The hematopoietic effect of FAB was investigated hematopoietic factors in marrow cells. RESULTS: The contents of total sugar, sulfated glycosaminoglycans, and uronic acid and the dry weight gradually increased with fermentation time. The sialic acid content (from 0.14 mg/mL to 0.54 mg/mL) was the highest on the 4th day of fermentation after which it decreased. The proliferating activity of bone marrow cells increased with fermentation times. The levels of various hematopoietic growth factors were determined to verify the beneficial effect of deer antler extract fermented by B. subtilis on hematopoiesis. FAB increased the number of stem cell factors and granulocyte colony-stimulating factor in bone marrow cells. In addition, FAB augmented the burst-forming unit erythroid and total colonies in splenocyte-conditioned medium compared with non-fermented antler extract (NFA). However, FAB did not affect the mRNA levels of erythropoietin, an important factor for erythropoiesis. CONCLUSIONS: FAB, like NFA, did not directly affect hematopoiesis, but contributed to hematopoiesis by stimulating the production of hematopoietic factors.

• 대한의생명과학회지
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• 제13권2호
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• pp.157-160
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• 2007

Endoplasmic reticulum (ER) plays formation of disulfide bonds and proper folding of secretory proteins. Cellular responses to ER stress enhances the stress-activated kinase pathway and the induces a lot of immediate-early genes. Among of them, the early growth response-1 (Egr-1), a transcription factor, which plays an important role in cell growth, development, differentiation, apoptosis and various types of injury. For that reason, we have tested the expression of Egr-1 against ER stress inducible drugs (tunicamycin, DTT, A23187 and BFA) to understand what kind of aspect occurred by ER stresses.