Improvement of in vitro produced bovine embryo quality using Charcoal:Dextran Stripped Fetal Bovine Serum on culture media

Mesalam Ayman,Lianguang Xu,Byung-Hyun Choi,Beon-Young Park,Rami Kong,Mi-Ju Son,Jong-In Jin,Sung Woo Kim,Il-Keun Kong 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

Effect of charcoal:dextran stripped fetal bovine serum on <i>in vitro</i> development of bovine embryos

Mesalam, Ayman,Kong, Rami,Khan, Imran,Chowdhury, MMR,Choi, Byung-Hyun,Kim, Sung Woo,Cho, Kyu-Woan,Jin, Jong-In,Kong, Il-Keun Elsevier 2017 Reproductive biology Vol.17 No.4

<P><B>Abstract</B></P> <P>This study investigated the ability of charcoal:dextran stripped fetal bovine serum (CDS FBS) and heat-inactivated fetal bovine serum (HI FBS) to support <I>in vitro</I> development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentage of embryos that formed a blastocyst was significantly (<I>P<</I> 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84±0.78% vs. 36.85±0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly (<I>P<</I> 0.05) increased mitochondrial activity, as identified by MitoTracker Green, as well as a reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. Quantitative reverse transcription PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly (<I>P<</I> 0.05) increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and the anti-apoptotic gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (<I>P<</I> 0.05) higher in the CDS FBS-supplemented group than in the HI FBS-supplemented group, whereas that of the pro-apoptotic gene BCL2-associated X protein was significantly lower. Taken together, these data suggest that supplementation of medium with CDS FBS improves the <I>in vitro</I> developmental competence and cryo-tolerance of bovine embryos.</P>

Silk Protein as a Fetal Bovine Serum Substitute for Animal Cell Culture

조유영,권해영,지상덕,김종길,김기영 한국미생물·생명공학회 2019 한국미생물·생명공학회지 Vol.47 No.4

Fetal Bovine Serum (FBS) is an essential substance added to animal cell culture medium. However, its composition is unclear causing problems such as development of an immune response when cultured cells are transplanted into the human body. In this study, silk sericin, silk fibroin, and hemolymph obtained from silkworms were added to the cell culture medium in order to determine if it can replace FBS. After establishment of the cell culture, cell proliferation and expression levels of cell growth-related genes were compared with those of control cells (cells cultured in the medium with 10% FBS). Results showed that the test group treated with silk fibroin extracted from a Korean silkworm variety, Kumokjam could replace 10% FBS. In addition, expression levels of cell growth related genes such as Fibronectin and TGF-β1 increased significantly in cells cultured using silk fibroin, depending on the concentration used in cell adhesion and cell proliferation [24]. To date, no studies have been conducted to find a replacement for FBS. Thus, this study was carried out to develop a substitute for FBS by using silkworm-derived alternatives such as silkworm hemolymph, silk sericin, and silk fibroin, which are cheap and have various physiological effects, cell promoting effects, and can be mass produced.

SCFFBS1 Regulates Root Quiescent Center Cell Division via Protein Degradation of APC/CCCS52A2

Kyoung Rok Geem,Hyemin Kim,Hojin Ryu 한국분자세포생물학회 2022 Molecules and cells Vol.45 No.10

Homeostatic regulation of meristematic stem cells accomplished by maintaining a balance between stem cell self-renewal and differentiation is critical for proper plant growth and development. The quiescent center (QC) regulates root apical meristem homeostasis by maintaining stem cell fate during plant root development. Cell cycle checkpoints, such as anaphase promoting complex/cyclosome/CELL CYCLE SWITCH 52 A2 (APC/CCCS52A2), strictly control the low proliferation rate of QC cells. Although APC/CCCS52A2 plays a critical role in maintaining QC cell division, the molecular mechanism that regulates its activity remains largely unknown. Here, we identified SCFF-BOX STRESS INDUCED 1 (FBS1), a ubiquitin E3 ligase, as a key regulator of QC cell division through the direct proteolysis of CCS52A2. FBS1 activity is positively associated with QC cell division and CCS52A2 proteolysis. FBS1 overexpression or ccs52a2-1 knockout consistently resulted in abnormal root development, characterized by root growth inhibition and low mitotic activity in the meristematic zone. Loss-of-function mutation of FBS1, on the other hand, resulted in low QC cell division, extremely low WOX5 expression, and rapid root growth. The 26S proteasome-mediated degradation of CCS52A2 was facilitated by its direct interaction with F-box stress induced 1 (FBS1). The FBS1 genetically interacted with APC/CCCS52A2-ERF115-PSKR1 signaling module for QC division. Thus, our findings establish SCFFBS1-mediated CCS52A2 proteolysis as the molecular mechanism for controlling QC cell division in plants.

착상전 이배체 단위발생 돼지난자의 체외 배양에서 세포사멸과 세포사멸에 관여하는 유전자의 발현에 관한 연구

X. S. Cui,I. H. Kim,N. H. Kim 한국동물생명공학회(구 한국동물번식학회) 2003 Reproductive & developmental biology Vol.27 No.2

본 연구는 착상전 이배체 단위발생 돼지난자를 체외 배양시 우태아혈청 (FBS), 우혈청 알부민 (BSA) 및 상피세포성장인자 (EGF)를 배양액에 첨가하였을 때 배반포, 총 세포수, 세포사멸 및 세포사멸에 관여하는 유전자의 발현을 조사하고자 수행하였다. 0.4% BSA를 배양액에 첨가하였을 때 2세포기 단위발생 난자의 배반포까지의 발달율이 증가되었다(P<0.01). FBS는 배반포의 총세포수를 감소시 켰고 세포사멸을 증가하였다(P<0.01). 그리고 EGF는 BSA가 존재하는 조건하에서 배반포의 총세포수를 증가하였는데 EGF와 BSA가 각각 단독으로 존재할 때는 이런 작용이 없었다. 세포사멸도 이와 이슷한 경향을 보였는데 EGF와 BSA가 각각 존재할 때에는 비처리군과 차이가 없었지만 함께 존재할 때에는 세포사멸을 감소시켰다. RT-PCR의 결과에 의하면 EGF는 BSA가 존재하는 배양액에서 Bcl-xL 유전자의 상대적 발현량을 증가시키고 Bak 유전자의 상대적 발현량에는 영향을 주지 않는 과정을 통하여 세포사멸을 감소시키는것 같다. 반면에 FBS는 Bcl-xL의 발현량을 감소시키고 Bak 유전자의 상대적 발현량을 증가시킨다. 이러한 결과는 세포사멸에 관여하는 유전자의 발현은 배양액의 첨가물에 따라 유의적으로 영향을 받으며, 체외배양시 배아의 초기발달에 관여함을 시사한다. This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) bovine serum albumin (BSA) and epidermal growth factor (EGF) on blastocoel formation, total cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell stage parthenotes to the blastocysts stage (P<0.01). FBS reduced cell numbers of blastocysts (P<0.01) and increased percentage of apoptosis in the blastocysts (P<0.001). However, while BSA increased cell numbers, it did so only when EGF was present. Either agent on its own had no effect. Similarly, apoptosis in the blastocysts was not influenced by either agent on its own but was reduced when both BSA and EGF were present. Furthermore, semi-quantitative reverse-transcriptase polymerase chain reaction revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect, and EGF and/or BSA did not influence Bak gene expression in the blastocyst stage parthenotes. However FBS reduced Bcl-xL mRNA expression (P <0.05) and enhanced Bak expression. This result suggests that apoptosis related genes expression is significantly affected by supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

Production and Transfer of In Vitro fertilized Hanwoo Embryos with Serum-free Media

윤도중,김계웅,김곤중,박병권,Xiang-Shun Cui,김남형,이종완 사단법인 한국동물생명공학회 2009 한국동물생명공학회지 Vol.24 No.4

As a simple and economical method for in vitro produced embryos, we have used BSA instead of serum for the production and embryo transfer of Hanwoo in vitro fertilized (IVF) embryos and obtained the following results: 1) When using serum (FBS; fetal bovine serum) or BSA-containing culture media as the initial culture media for immature oocytes, it is regarded as inappropriate to add only BSA to the culture solutions from maturation of the immature oocytes to development stage culture, but serum still needs be added though there is no significant difference in the concentration, with a change from 5% to 10%. 2) The results of culturing IVF embryos after development (4 cell stage) in the Medium199 solutions containing BSA instead of serum (FBS) showed that 0.3% BSA concentration is not optimal and 0.5% or higher BSA concentration has no significant difference among 0.5%, 0.7%, 1% and 2% (p>0.05). 3) The post-freezing survival ratio after development in 5% FBS-Medium199 showed that 1% BSA concentration of the culture solution is the most suitable in the BSA concentrations of 0.3% (51%), 0.5% (67%), 0.7% (69%), 1% (77%) and 2% (75%). 4) The pregnancy rates of the transplanted fresh(not frozen) blastocyst had no significant concentration dependency (p>0.5), and the average pregnancy rate was 63.8%. 14% of overweight calves were found among the calves given birth to by the transfer of IVF blastocysts cultured in the serum-added culture solution, but none was found in the experimental groups in which BSA was added instead of serum.

Anti-cardiolipin 항체와 Cardiolipin의 결합에 미치는 $\beta_2$-GP1의 영향

강은영,장영주,Kang, Eun-Young,Jang, Young-Ju 대한면역학회 2004 Immune Network Vol.4 No.3

Background: Anti-cardiolipin antibody (Anti-CL Ab) is one of the various antiphospholipid antibodies (Anti-PL Abs) and found in the plasma of patients with systemic lupus erythematosus (SLE), atherosclerosis, and other infectious diseases. While anti-PL Abs found in the sera of patients with infectious diseases bind directly to CL, binding of anti-PL Abs to CL circulating in the sera of patients with autoimmune diseases is mediated by $\beta_2-$glycoprotein 1 ($\beta_2-GP1$). The purpose of this study is to investigate the effect of <$\beta_2-GP1$ on the antigen binding assay of anti-CL Abs present in the sera of patients with atherosclerosis, which has been known as one of autoimmune diseases. Methods: ELISA was performed with sera containing anti-CL Abs from three patients with atherosclerosis in the presence or absence of $\beta_2-GP1$ or FBS. Results: Reactivity of anti-CL Abs to CL was increased in the presence of $\beta_2-GP1$ or FBS in a dose dependent manner. Conclusion: <$\beta_2-GP1$ or FBS could be used as co-factor in CL ELISA with anti-CL Abs present in the sera of patients with atherosclerosis. It is suggested that anti-CL Abs found in atherosclerosis patients are similar in terms of antigen binding property to those circulating in the patients with autoimmune diseases, not to infectious diseases.

건강검진 수검자들의 대사증후군 위험인자별 유병률 및 비만지표와의 관련성

박규리(Park, Kyu-Ri),조영채(Cho, Young-Chae) 한국산학기술학회 2016 한국산학기술학회논문지 Vol.17 No.3

본 연구는 일반 성인을 대상으로 대사증후군 위험인자의 유병률을 파악하고, 각 대사증후군 위험인자들과 비만지표들과의 관련성을 파악하며, 비만지표들의 대사증후군을 예측하기 위한 관련성과 타당도를 검토하고자 시도하였다. 조사대상은 2014년 2월부터 11월까지의 기간에 한국건강관리협회 D지부 건강검진센타에서 종합건강검진을 받았던 20세 이상 1,051명을 대상으로 하였다. 연구결과, 조사대상자의 대사증후군 유병률은 21.5%이었으며, 각 위험인자의 유병률은 허리둘레의 경우 남자 31.8%, 여자 41.6%이었으며, TG는 남자 35.6%, 여자 17.3%이었고, HDL-C는 남자 17.6%, 여자 34.2%이었다. 혈압은 남자 53.5%, 여자 35.9%이었으며, FBS는 남자 14.9%, 여자 6.8%이었다. 이 같은 유병률은 연령이 높은 군일수록, BMI가 높은 군일수록, 음주횟수가 많은 군일수록, 유의하게 높았다. 비만지표별 대사증후군 위험요인 개수별 예측도를 보면 허리둘레/신장비가 높게 나타났다. 이상과 같은 결과를 볼 때, 다른 비만지표에 비해 허리둘레/신장비가 대사증후군 위험요인을 선별하는데 좀 더 예측력이 있다고 나타났다. 허리둘레/신장비는 간단하고 실용적인 복부비만의 지표가 될 수 있으며 대사증후군에 대한 선별검사로서 체질량지수와 허리둘레보다 우수한 것으로 나타났다. The aim of this study was to estimate the prevalence of the metabolic syndrome risk factors for adults in the general population, understand the relationship of each factor with the obesity indicators, and examine the relevance and validity for predicting the metabolic syndrome in obese indicators. The study subjects were 1,051 adults aged 20 years and over, who underwent a health package check-up at the Korea Association of Health Promotion, D-branch from Feb. to Nov. 2014. As a result, the prevalence rates of metabolic syndrome of the study subjects were 21.5%, and the prevalence rates of the metabolic risk factors were as follows: 31.8% of men and 41.6% of women for abdominal obesity; 35.6% of men and 17.3% of women for TG; 17.6% of men and 34.2% of women for HDL-C; 53.5% of men and 35.9% of women for blood pressure; and 14.9% of men and 6.8% of women for FBS. The prevalence rates of metabolic syndrome were significantly higher in the older age group, in the group with the higher BMI, and in the group of higher level of drinking frequency. In the predictive indicators of obesity on metabolic syndrome risk factors, the waist / height ratio was higher than other indicators. Conclusively, WHtR appeared to be a useful indicator to show abdominal obesity and is better in selecting metabolic syndrome patients compared to other obesity indicators.

동맥혈관 평활근세포 증식에 대한 오보바톨 유도체(A-8)의 억제효과

임용(Yong Lim),이미애(Mi-Yea Lee),투데브멍흐(Munkhtsetseg Tudev),박은석(Eun-Seok Park),정재경(Jae-Kyung Jung),윤여표(Yeo-Pyo Yun) 대한약학회 2011 약학회지 Vol.55 No.2

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of proliferative cardiovascular diseases, including hypertension and atherosclerosis. To find antiproliferative agent (A)-8 had inhibitory effect on VSMCs proliferation. Therefore, we examined the antiproliferative effect of A-8, a newly synthesized obovatol derivative. To investigate the antiproliferative effect of A-8, we examined cell counting and [3H]-thymidine incorporation assays. The pre-incubation of A-8 (1~4 μM) significantly inhibited proliferation and DNA synthesis of 5% fetal bovine serum (FBS)-stimulated rat aortic VSMCs in concentration-dependent manner. Whereas, A-8 did not show any cytotoxicity in rat aortic VSMCs in this experimental condition by WST-1 assay. In addition, A-8 significantly inhibited 5% FBS-induced cell cycle progression in rat aortic VSMCs. These results show that A-8 may be developed as a potential antiproliferative agent for treatment of angioplasty restenosis and atherosclerosis. Furthermore, A-8 should be examined for further clinical application either as a single agent or in combination with other angioplasty restenosis or atherosclerosis agents.

절피원심법으로 대규모 채취한 누에체액의 곤충세포 증식효과

최지영 ( Ji Young Choi ),김종길 ( Jong Gill Kim ),최영철 ( Young Cheol Choi ),김삼은 ( Sam Eun Kim ),한명세 ( Myung Sae Han ) 한국잠사학회 2007 한국잠사곤충학회지 Vol.49 No.1

누에체액이 동물세포 배양의 필수첨가물인 FBS를 대체할 수 있는 세포증식 효과가 있는 것으로 확인되었으나, 누에의 배발에 상처를 내어 채혈하는 방법으로는 대량 채취가 어렵다. 본 연구에서는 이러한 문제점을 해결코자 대량의 체액을 한꺼번에 얻을 수 있는 대규모 채혈법을 검토하게 되었다. 1. 누에 머리부분과 꼬리부분 (배발 포함)에 상처를 낸 후 원심하여 체액을 채취하는 방법 (절피원심법)이 기존의 소규모 채취한 체액과 유사한 세포증식 촉진효과를 보였다. 2. 절피원심한 체액의 원심속도에 따른 세포증식 촉진 효과는 500 rpm과 1000 rpm에서 양호한 값을 보였다. 3. 원심속도를 500 rpm으로 고정한 경우 원심시간을 5분에서 30분까지 달리하여도 세포중식 효과는 유사하였고, 체액 회수율은 원심시간이 길어질수록 증가하였으나 일정시간 이상부터는 증가하지 않았다. 4. 절피원심법으로 대규모 채취한 체액의 액상회수율은 누에(백옥잠) 생체중의 19.5%이었고, 분말회수율은 1.4%이었으며, 분말체액에서도 세포증식 촉진 효과가 인정되었다. This study was carried out to investigate the utilization of hemolymph of silkworm, Bombyx mori, as a substitute for fetal bovine serum (FBS) in the insect cell culture. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate for large scale collection. The hemolymph was collected from 5 th instar larva by centrifugation after cutting of the abdominal legs was more appropriate procedure for large scale collection. The cell growth in the medium supplemented with hemolymph (Baekokjam) collected in large scale was almost same as that in the medium hemolymph supplemented with hemolymph collected in small scale. However, the mutant (wEb) hemolymph collected in large scale was still less effective in the cell growth, as compared to the Baekokjam hemolymph collected in large scale. The optimum centrifugation condition for large-scale bleeding was 500 rpm and 15 mm.