혈청 선별 검사에서 개별 혈청과 혼합 혈청의 면역 반응성 비교 분석

이진영,이정옥,김지현,한영신,안강모 대한 소아알레르기 호흡기학회 2012 Allergy Asthma & Respiratory Disease Vol.22 No.4

Purpose:Serum screening test to detect specific immunoglobulin E (IgE) is an important step for the assessment of potential allergenicity of genetically modified (GM) food. The purpose of this study was to evaluate the usefulness of pooled serum for serum screening instead of individual serum. Methods:Children with allergic disease were recruited and those who were sensitized to peanut or egg white were selected to obtain their sera. Sensitization to these foods was determined when the level of specific IgE was over 0.35 kU/L by ImmunoCAP. The patients were divided into subgroups according to their level of specific IgE. Raw proteins were extracted and immunoblot analysis was performed to compare the immunoreactivity between individual serum and pooled serum. Results:Pooled serum from peanut-sensitized allergic children showed all the bands which were shown in immunoblot analysis by using individual serum and peanut protein extract. These findings were demonstrated both in pooled serum with low level of peanut-specific IgE and in those with high level of peanut-specific IgE. Likewise, there was no difference in the immunoreactivity between individual serum and pooled serum from egg white-sensitized allergic children. Conclusion:Pooled serum can be used as an alternative to individual serum for the serum screening in the allergenicity assessment of GM food. 목적:개별 혈청과 혼합 혈청을 이용한 immunoblotting을 비교함으로써 유전자 재조합 식품에 새로이 발현된 단백질에 대한 혈청 선별 검사에서 개별 혈청 대신 혼합 혈청을 사용할 수 있을지를 알아보고자 하였다. 방법:ImmunoCAP (Pharmacia, Uppsala, Sweden) 검사상 난백 특이 immunoglobulin E (IgE)에 양성을 보인 환아 15명과 땅콩 특이 IgE에 양성을 보인 환아 12명의 혈청을 확보하였다. 특이 IgE 수준에 따른 반응의 차이를 비교하기 위해 환자의 땅콩 특이 IgE 수준이 10 kU/L이하와 10 kU/L 이상, 계란은 5 kU/L 미만, 5–10 kU/L, 11kU/L 이상으로 나누어 땅콩 및 난백 단백질 추출물과 immunoblotting을 실시하였다. 결과:땅콩 특이 IgE 수준이 10 kU/L 이상이나 이하에 상관 없이 개별 혈청에서 반응하였던 단백질이 혼합 혈청과의 반응에서도 모두 선명하게 감지되었다. 난백 특이 IgE 양성인 혈청과 난백과의 반응에서도 특이 IgE 수준에 관계없이 모든 군에서 개별 혈청에 나타났던 양성 반응을 혼합 혈청에서도 확인할 수 있었다. 결론:혈청 내 특이 IgE와 특정 단백질 간의 반응성을 관찰하는 데에는 특이 IgE 값에 관계없이 개별 혈청 대신 혼합 혈청을 사용해도 될 것으로 판단된다.

Serum Resistance in Riemerella anatipestifer is Associated with Systemic Disease in Ducks

Bai Wei,Hye-Suk Seo,Ke Shang,Jong-Yeol Park,Yea-Jin Lee,Yu-ri Choi,Sang-Won Kim,Se-Yeoun Cha,Hyung-Kwan Jang,Min Kang 한국가금학회 2021 韓國家禽學會誌 Vol.48 No.4

리메렐라 아나티페스티퍼 감염증은 오리와 거위에서 섬유소성 심막염, 간주위염증, 기낭염, 건락성난관염, 뇌막염을 특징으로 하는 급성 또는 만성 패혈증이다. 이 균은 혈청형 또는 분리주별로 병원성에 큰 차이가 나타나는 것으로 알려져 있다. 그럼에도 불구하고 지금까지 이러한 다양한 병원성과 그 이유에 대한 연구는 거의 이루어지지 않았다. 본 연구에서는 리메렐라 아나티페스티퍼의 병원성과 serum resistance 상관성을 구명하였다. 우리는 다양한 분리원으로부터 확보한 130주의 균주를 대상으로 serum resistance 특성을 분석하였다. 건강한 오리 인후두에서 분리된 균주들은 혈청에 대한 감수성이 높은 반면에 전신감염을 일으킨 균주들은 강한 serum resistance를 보였다. 또한 우리는 이러한 혈청의 살균효과가 혈청내 보체 성분에 의해 유도됨을 확인하였다. 강한 serum resistance를 유발하는 세균의 표면 유전자와의 관련성을 조사한 결과, 외막 단백질의 AS87_09335, AS87_00480, AS87_05195 유전자가 serum resistance와 관련 있음을 알 수 있었다. 본 연구 결과로 serum resistance 특성이 리메렐라 아나티페스티퍼의 병원성 결정 요소 중 하나라는 것을 확인하였다. Riemerella anatipestifer (RA) can cause septicemia, polyserositis, and ataxia in ducks. It can also colonize the upper respiratory tract of healthy ducks. These differences in pathogenicity are probably the result of diverse mechanisms of virulence in different strains. Since serum resistance is a feature frequently found in systemic pathogens, 130 RA strains having different clinical origins were tested. A variety of serum susceptibility levels were detected. Pharynx strains from healthy ducks were mainly susceptible to the bactericidal effect of the serum, while systemic strains were serum resistant. Heat-treatment of the sera abolished the bactericidal activity, indicating that complement is a key factor in this effect. In an attempt to associate serum-resistance to surface determinant genes of the bacteria, we screened for six genes involved in lipopolysaccharide synthesis and membrane proteins in RA. Of these, three genes (AS87_09335, AS87_00480, and AS87_05195) encoding outer membrane proteins might be implicated in serum resistance statistically. The results indicate that serum resistance is a virulence mechanism in RA.

횡문근육종 환아의 혈청 Neutral Ribonuclease의 분리와 성상에 관한 연구

박성원,이상훈,한중수,고재경 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.2

The activity of neutral ribonuclease (RNase) was determined in serum of patients with rhabdomyosarcoma to find out whether the serum enzyme could be used as a marker for rhabdomyosarcoma. Also analysed were isolation patterns of proteins and neutral RNases to investigate the persence of RNase specific to the rhabdomyosarcoma. In serum of patients with rhabdomyosarcoma, activities of amylase, alkaline phosphatase and 5'-nucleotidase were unchanged, but neutral RNase as a marker for the myosarcoma. Neutral RNases in serum of the rhabdomyosarcoma and in its control serum were separated by a DEAE-cellulose column chromatography into 5peaks each and the peak specific to the myosarcoma was not found. The peak I neutral RNase isozyme was, however, observed to be greater in rhabdomyosarcome serum than in the control serum and the peak Ⅴ neutral RNase isozyme was lesser in the myosarcoma than in the control. The peak I neutral RNase isozyme isolated from the rhabdomyosarcoma serum was active toward poly C and less active toward poly U and RNA, indicating the enzyme was the secretory type of RNase in nature. No activity was observed toward purine poly ribonucleotides, polyeosyribonucleotides and double stranded polyribonucleotides. Of the geteropolyribonucleotides used as substrate in the present study, the peak I neutral RNase isozyme from rhabdomyosarcoma serum was highly active toward poly AC and active toward poly ACU, AU, CI, CIU in the decreasing order. No or little activity was observed with poly AG, AGU and ACG. This would indicate that the RNase isozyme is active towasrd linkages made of cytosine and inactive toward linkages with guanine. Studies on the substrate specificity showed differences between the peak I neutral RNase isozymes from rhabdomyosarcoma and control serum, exhibiting decrease in the ratio of poly U/ poly C, RNA/ poly C and heteropolyribonucleotides/ poly C activities in the myosarcoma. This suggerts that the peak I neutral RNase isozyme minght be specific to the rhabdomyosarcoma. DEAE peak I proteins isolated from rhabomyosarcoma and control serum were further separated by a HPLC into 3 peaks each, the protein pattern was similar in DEAE peak I of both serum. The results obtained in the present study indicate that 1) neutral RNaes activity in serum of rhabdomyosarcoma was significantly increased suggesting the possible use of the RNase as a marker ofr the rhadbomyosarcoma and that 2) the peak I neutral RNase as a marker for the rhadbomyosarcoma and that cific to the myosarcome, exhibitin the difference of substrate specifity between the rhabdomyosarcoma and control.

혈청 선별 검사에서 개별 혈청과 혼합 혈청의 면역 반응성 비교 분석

이진영 ( Jin Young Lee ),이정옥 ( Jeong Ok Lee ),김지현 ( Ji Hyun Kim ),한영신 ( Young Shin Han ),안강모 ( Kang Mo Ahn ) 대한소아알레르기호흡기학회(구 대한소아알레르기 및 호흡기학회) 2012 소아알레르기 및 호흡기학회지 Vol.22 No.4

목 적 : 개별 혈청과 혼합 혈청을 이용한 immunoblotting을 비교함으로써 유전자 재조합 식품에 새로이 발현된 단백질에 대한 혈청 선별 검사에서 개별 혈청 대신 혼합 혈청을 사용할 수 있을지를 알아보고자 하였다. 방 법 : ImmunoCAP (Pharmacia, Uppsala, Sweden) 검사상 난백 특이 immunoglobulin E (IgE)에 양성을 보인 환아 15명과 땅콩 특이 IgE에 양성을 보인 환아 12명의 혈청을 확보하였다. 특이 IgE 수준에 따른 반응의 차이를 비교하기 위해 환자의 땅콩 특이 IgE 수준이 10 kU/L이하와 10kU/L 이상, 계란은 5 kU/L 미만, 5-10 kU/L, 11kU/L 이상으로 나누어 땅콩 및 난백 단백질 추출물과 immuno-blotting을 실시하였다. 결 과: 땅콩 특이 IgE 수준이 10 kU/L 이상이나 이하에 상관 없이 개별 혈청에서 반응하였던 단백질이 혼합 혈청과의 반응에서도 모두 선명하게 감지되었다. 난백 특이 IgE 양성인 혈청과 난백과의 반응에서도 특이 IgE 수준에 관계없이 모든 군에서 개별 혈청에 나타났던 양성 반응을 혼합 혈청에서도 확인할 수 있었다. 결 론 : 혈청 내 특이 IgE와 특정 단백질 간의 반응성을 관찰하는 데에는 특이 IgE 값에 관계없이 개별 혈청 대신 혼합 혈청을 사용해도 될 것으로 판단된다. Purpose : Serum screening test to detect specific immunoglobulin E (IgE) is an important step for the assessment of potential allergenicity of genetically modified (GM) food. The purpose of this study was to evaluate the usefulness of pooled serum for serum screening instead of individual serum. Methods : Children with allergic disease were recruited and those who were sensitized to peanut or egg white were selected to obtain their sera. Sensitization to these foods was determined when the level of specific IgE was over 0.35 kU/L by ImmunoCAP. The patients were divided into subgroups according to their level of specific IgE. Raw proteins were extracted and immunoblot analysis was performed to compare the immunoreactivity between individual serum and pooled serum. Results : Pooled serum from peanut-sensitized allergic children showed all the bands which were shown in immunoblot analysis by using individual serum and peanut protein extract. These findings were demonstrated both in pooled serum with low level of peanut-specific IgE and in those with high level of peanut-specific IgE. Likewise, there was no difference in the immunoreactivity between individual serum and pooled serum from egg white-sensitized allergic children. Conclusion : Pooled serum can be used as an alternative to individual serum for the serum screening in the allergenicity assessment of GM food. [Pediatr Allergy Respir Dis(Korea) 2012;22:390-396]

호르몬 한정배지를 이용한 세포 초대배양계의 확립

한호재,강주원,박권무,이장헌,양일석,Han, Ho-jae,Kang, Ju-won,Park, Kwon-moo,Lee, Jang-hern,Yang, Il-suk 대한수의학회 1996 大韓獸醫學會誌 Vol.36 No.3

This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium. The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum- supplemented medium(p < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${\gamma}$-GTP) were increased(p < 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p < 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p < 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum-supplemented medium. The activities of $Na^+$glucose cotransporter($^{14}C$-a-MG uptake), $Na^+$phosphate cotransportere($^{32}P$ uptake) and $Na^+$ transporter($^{22}Na^+$ uptake) in the brush border membrane, and of $Na^+/K^+$-ATPase($^{86}Rb$ uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p < 0.05). In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.

Comparative Analysis of MSC-Derived Exosomes Depending on Cell Culture Media for Regenerative Bioactivity

Kim Jun Yong,임원규,Seo Hyo Jeong,Lee Joo Youn,박천권,Han Dong Keun 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.3

Background: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. Methods: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor™ CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. Results: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. Conclusion: CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serum-derived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM. Background: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. Methods: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor™ CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. Results: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. Conclusion: CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serum-derived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.

복수의 감별에 있어 혈청과 복수내 CEA 및 CA19-9의 가치에 관한 연구

김선민 ( Kim Seon Min ),박경식 ( Park Gyeong Sig ),마상인 ( Ma Sang In ),김휘정 ( Kim Hwi Jeong ),유승박 ( Yu Seung Bag ),조준환 ( Jo Jun Hwan ),이재동 ( Lee Jae Dong ),이중건 ( Lee Jung Geon ) 대한내과학회 1992 대한내과학회지 Vol.42 No.4

1989년 4월 1990년 12월까지 본원 내과에서 입원하였던 복수 환자 81예를 대상을 그 원인 악성 질환에 의한 것인지 양성 질환에 의한 것인지를 감별하기 위해 혈청 및 복수내의 CEA, CA19-9의 농도를 측정 비교하여 그 임상적 의의에 대해 다음과 같은 결과를 얻었다. 1) 악성 질환에서의 CEA의 혈청내 평균치는 양성질환보다 의의있는 상승을 보였으며(p<0.05), 복수내 평균치 또한 유의한 상승을 보였다(p<0.01)> 2) CA19-9의 혈청과 복수에서의 평균치는 악성 복수군에서 현저한 상승을 보였으나 통계적 유의성은 없었다.(p>0.05). 3) 복수의 감별진다에 있어 CEA의 복수내의 민감도는 84%였으며 혈청에서는 71%이였으며 CA19-9는 복수내의 민감도가 68% 혈청에서는 60%로 CEA보다는 전체적으로 민감도가 낮았다. 4) 혈청과 복수에서 CEA 병합 검사시 민감도는 86%로 상승하였으며 CA19-9병합 검사시 민감도 또한 73%로 상승 하였으며 모두 병합 검사시 민감도는 92%로 의의있는 상승을 보였다. 5) 혈청내 CEA 복수내 CEA 혈청내 CA19-9 및 복수내 CA19-9 검사간에는 유의한 상관관계가 있었다(p<0.005, p<0.001). 이상과 같이 혈청과 복수에서의 CEA와 CA19-9 농도 측정은 복수의 원인이 악성 질환에 의한 것인지를 아는데 도움이 될 것으로 사료된다. Ascites may be caused by various benign and malignant disease, but it is sometimes difficult to determine whether it is caused by benign disease or malignant diseases. In order to determine whether CEA, CA 19-9 in ascitic fluid and serum assist in the diagnosis of malignant disease that causing ascites, we studied 38 cases of malignant ascites and 43 cases of benign ascites patients who were admitted to Seoul Adventist Hospital from April 1989 to December 1990. The results were obtained as follows: 1) The mean value of CEA in serum and ascitic fluid of the malignant ascites group were significantly higher than that of the benign ascites group in serum (p<0.05), in ascites (p<0.01). 2) The mean value of CA 19-9 in serum and ascitic fluid of the malignant ascites group was signficantly higher than that of the benign ascites group was significantly higher than that of the benign ascites group, there was no significance in statistical aspects(p>0.05). 3) The sensitivity of CEA was 81% in ascites, 78% in serum, the sensitivity of CA 19-9 was 63% in ascites, 65% in serum. 4) The sensitivity of CEA was increased to 87% in serum and ascites combinations, the sensitivity of CA 19-9 was also increased to 81% in serum and ascites combinations. The sensitivity was significantly elevated to 92% in case of all tumor marker combinations. 5) There was a significant correlations between serum CEA and ascites CEA, serum CA 19-9 and ascites CA 19-9. (p<0.01, in all cases) It was concluded that the measurement of serum and ascites CEA, CA 19-9 levels is useful lin differention between ascites caused by benign and malignant diseases. Combinantions of each tumor markers in serum and ascites were more accurate than single test.

Evaluation and Optimization of a Serum-based Minimum Inhibitory Concentration Assay to Caspofungin in Candida albicans Clinical Isolates

Young Bin Yoo,Sung-Soon Kim,Young Kwon Kim,Sunghyun Kim 대한의생명과학회 2016 Biomedical Science Letters Vol.22 No.4

In the present study, a serum-based minimum inhibitory concentration (MIC) testing to caspofungin was optimized and evaluated to solve the limitations of the conventional Clinical and Laboratory Standards Institute (CLSI) guideline-based antifungal agent MIC test and the usefulness of this testing for clinical application was determined. A total of 105 Candida albicans clinical isolates were used for measuring MIC to caspofungin. Results showed that growth characteristics were different according to types of serum and the mouse serum was the most suitable for this assay. In order to measure the optimal concentration of mouse serum, 0 to 100% mouse serum were added to the media during fungal culture. The optimal concentration of serum was 50% when consideration of antifungal agent administration and inoculum size, serum components and ease of hyphae separated, and the consideration of the degree of growth. In comparison of the usefulness between the conventional Alamar-modified broth microdilution MIC assay and 50% mouse serum-based MIC testing, the range of MIC80 of the Alamar-modified broth microdilution MIC assay was 0.13~2.0 μg/mL (SD ±0.42 μg/mL) and that of the 50% mouse serum-based MIC assay was 2.0~32.0 μg/mL (SD ±9.01 μg/mL). The range of MIC50 of the Alamar-modified broth microdilution MIC assay was 0.13~2.0 μg/mL (SD ±0.40 μg/mL) and that of the 50% mouse serum-based MIC assay was 1.0~16.0 μg/mL (SD ±2.36 μg/mL). The MICs of 50% mouse serum-based MIC testing was increased by up to 4 to 64 times than Alamar-modified broth microdilution MIC assay. In conclusion, a 50% mouse serum-based MIC assay was more useful for measuring MIC in Candida albicans clinical isolates than conventional colorimetric broth microdilution MIC testing.

Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media

Yun-Song Lee,Huong Thi Lan Tran,Quang Van Ta 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.4

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/ chemokines. Whereas the MEK-ERK and PI3KAkt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-α, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125- mediated MMP-9 induction, similar to IFN-γ. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-γ. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression. Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/ chemokines. Whereas the MEK-ERK and PI3KAkt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-α, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125- mediated MMP-9 induction, similar to IFN-γ. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-γ. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.

Effect of Serum on Nitric Oxide Synthesis in Mouse Embryonic Liver Cells

Chung,Hun Taeg,Pae, Hyun-Ock,Yoo,Ji Chang 啓明大學校 醫科大學 1998 계명의대학술지 Vol.17 No.4

Serum that is commonly used as a culture medium supplement contains biologically active factors, including growth factors and cytokines. Nitric oxide (NO) is an important mediator of various biochemical and physiological processes. Its synthesis by various cells is controlled by various stimuli including cytokines or growth factors. To study the control of NO synthesis in the liver cells, the effects of serum on NO synthesis were investigated in this study. Mouse embryonic liver cell line BNL CL.2 cells were cultured 6hr in 20%, 10%, 5%, 2% serum-supplied or serum-free DMEM, respectively, and stimulated with interferon-Υ (IFN-Υ), lipopolysaccharide (LPS), or combination of two stimuli. Cells cultured in 2% serum-supplied or serum-free DMED synthesized NO in response to IFN-Υ alone in a dose- and time-dependent manner, while cells cultured in 20%, 10% or 5% serum-supplied DMEM did not. When cells were stimulated with IFN-Υ plus LPS in 2% serum-supplied or serum-free DMEM, a synergistic increase in NO synthesis was observed, whereas decrease in NO synthesis was observed in uncreasing serum concentration. These findings evidence that in vitro NO synthesis by mouse liver cells may be dependent on concentrations of serum in culture.