• KSBB Journal
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• 제18권4호
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• pp.255-260
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• 2003

현재는 재조합 Erythropoietin의 생물학적 활성을 mouse를 이용한 in vivo bioassay로 실시하고 있으나, 이 방법의 여러가서 불편함으로 인하여, 이를 대체하기 위해 Ba/F3 세포주를 이용한 in vitro assay 방법을 확립하였으나, 위에 상술한 바와 같이 in vitro assay는 erythropoietin의 당분포에 의한 차이를 보이지 않게 때문에, 이를 보완하기 위해서는 in vivo bio-activity와 정량적인 상관관계가 있는 당단백질의 isoform 분석법인 Capillary zone electrophoresis (CZE) 와 sialic acid 함량 결과를 동시에 분석해야 했다. 위의 결과 sialic acid 함량은 erythropoietin 원액에서 10 mol/mol,EPO 이상의 sialic acid 함량을 가져야 되며, CZE 결과는 재조합 erythropoietin이 isoform 분포가 isoform 3의 경우 5.594～0.593％, isoform 4의 경우 31.598～11.704％, isoform 5의 경우 37.033～29.301％, isoform 6의 경우 27.837～18.807％, isoform 7의 경우 17.085～7.824％, isoform 8의 경우 7.642～1.964％을 보여줌과 동시에 isoform 4의 면적은 isoform 5의 면적보다 항상 작아야한다. 이상 두 가지 시험결과와 in vitro assay 결과를 combine해서 in vivo assay를 대체할 수 있다는 좋은 실험적 data를 얻었으므로 이상 위의 3가지 분석법을 활용한 combined in vitro bioassay 법의 기초를 확립하였다.

• BMB Reports
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• 제36권4호
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• pp.390-398
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• 2003

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that post-translational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.

• The Korean Journal of Physiology and Pharmacology
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• 제22권6호
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• pp.705-712
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• 2018

The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). In vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a $2{\times}2$ stitched area from the $4{\times}$ object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, $IC_{50}$ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.260-265
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• 1998

Angiogenic activity of Aloe vera gel was investigated by in vitro assay. We obtained the most active fraction from dichloromethane extract of Aloe vera gel by partitioning between hexane and 90% aqueous methanol. The most active fraction (F3) increased the proliferation of calf pulmonary artery endothelial (CPAE) cells. In addition, F3 fraction induced CPAE cells to invade type I collagen gel and form capillary-like tube through in vitro angiogenesis assay, and increased the invasion of CPAE cells into matrigel through in vitro invasion assay. Furthermore, the effect on the MRNA expression of proteolytic enzymes which are key participants in the regulation of extracellular matrix degradation was investigated by northern blot analysis. F3 fraction enhanced mRNA expression of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2), and membrane-type MMP (MT-MMP) in CPAE cells whereas the expression of plasminogen activator inhibitory (PAl-1) mRNA was not changed.

• 대한본초학회지
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• 제26권4호
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• pp.125-132
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• 2011

Objectives : An aim of study is to investigate effects of curculiginis rhizoma in vitro (factor Xa (FXa) inhibitor assay, prothrombinase assay, prothrombin time (PT) assay, activated partial thromboplastin time (aPTT) assay) and in vivo experiment (blood clotting time, thromboxane B2 content assay in serum and weight of thrombus by AV-shunt rat model). Methods : We gained a human serum and used serum in vitro study such as factor X activity (FXa) inhibition, prothrombinase inhibition, prothrombin time (PT) and activated partial thromboplastin time. Fifteen SD rats were divided into three groups (intact control group and two experimental group treated with extract of Curculiginis Rhizoma(ECR)). Rats were orally administrated DW (intact control group), 600 mg/kg concertration of ECR and 200 mg/kg concertration of ECR. After one hour, we anesthetized rats and made arteriovenous (AV) shunt rat models to study weights of thrombus, took a hole blood to study content of thromboxane B2 and blood clotting time. Results : In vitro, ECR increased a inhibitory activity of FXa, prothrombinase and aPTT compared than intact control group. Especially ECR made significant increase of FXa and prothrombinase inhibitory activity (p<0.05, p<0.01). And PT were increased in ECR control group compared with intact control group. In vivo, a blood clotting time of experiment group treated with ECR 600 mg/kg were significantly increased compared with that of intact control group (p<0.05) and content of thromboxane B2 was significantly decreased in group treated with ECR 600 mg/kg in seum. The weight of thrombus were significantly reduced in group treated with ECR 600 mg/kg compared with intact control group (p<0.05). But in vivo experiment study, those of group treated with ECR 200 mg/kg were reduced compared with those of intact control group without statistical significance. Conclusions : ECR has a antithromboic activity in internal course with inhibitory activity of FXa and prothrombinase in vitro, it required to research more study for effective compounds.

• Toxicological Research
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• 제25권1호
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• pp.51-58
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• 2009

Carrageenan is a naturally-occurring sulfated polygalactan which has been widely used in the dairy industry and a gelling agent in non-dairy products. In this study, four short-term in vitro genotoxicity assays were investigated to evaluate the potential genotoxic effects of carrageenan. The mutagenicity of carrageenan was evaluated up to a maximum dose of 5 mg/plate in Ames test. There was no increase in the number of revertant colonies compared to its negative control at any dose in all of strains tested. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. Carrageenan was not considered to be clastogenic in this assay at up to the highest feasible concentration which could be evaluated. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that carrageenan has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, our results indicate that carrageenan was not genotoxic based on four in vitro genotoxicity results.

• Toxicological Research
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• 제24권1호
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• pp.37-44
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• 2008

The in vitro cell transformation assays (CTA) were performed using BALB/3T3 murine fibroblasts and HaCaT human keratinocytes in order to evaluate concordance between both in vitro CTAs and carcinogenicity with compounds differing in their genotoxic and carcinogenic potential. Six test articles were evaluated, two each from three classes of compounds: genotoxic carcinogens (2-amino-5-nitrophenol and 4-nitroquinoline-N-oxide), genotoxic noncarcinogens (8-hydroxyquinoline and benzyl alcohol), and nongenotoxic carcinogens (methyl carbamate and N-nitrosodiphenylamine). Any foci of size $\geq$2 mm regardless of invasiveness and piling was scored as positive in CTA with BALB/3T3. As expected, four carcinogens regardless of their genotoxicity had positive outcomes in two-stage CTA using BALB/3T3 cells. However, of the two genotoxic noncarcinogens, benzyl alcohol was positive CTA finding. We concluded that, of the 6 chemicals tested, the sensitivity for BALB/3T3 system was reasonably high, being 100%. The respective specificity for BALB/3T3 assay was 50%. We also investigated the correlation between results of BALB/3T3 assay and results from HaCaT assay in order to develop a reliable human cell transformation assay. However, evaluation of staining at later time points beyond the confluency stage did not yield further assessable data because most of HaCaT cells were detached after $2{\sim}3$ days of confluency. Thus, after test article treatment, HaCaT cells were split before massive cell death began. In this modified protocol for this HaCaT system, growing attached colonies were counted instead of transformed foci 3 weeks since last subculture. Compared to BALB/3T3 assay, HaCaT assay showed moderate low sensitivity and high specificity. Despite these differences in specificity and sensitivity, both cell systems did exhibit same good concordance between in vitro CTA and rodent carcinogenicity findings (overall 83% concordant results). At present the major weakness of these in vitro CTA is lack of validation for regulatory acceptance and use. Thus, more controlled studies will be needed in order to be better able to assess and quantitatively estimate in vitro CTA data.

• Toxicological Research
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• 제16권1호
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• pp.77-82
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• 2000

This study was conducted to assess a possible alternative method as replacements for in vivo test. The human fibroblasts were exposed to several photoxic chemicals (promethazine, neutral red, chlortetracyclone, amiodatone, bithional, 8-methyooxypsorale) and non-phototoxi substance, ammonium laureth sulfate and irradiatied with 5 J/$cm^2$ of UVA (3320~420nm). The cell viability was measured by NRU (neutral red uptake) assay. The photoxic potential of test chemicals in the NRU PT (phototoxicity test) was assessed by determining the PIF (photoirritancy Factor) by using a cut-off value of 5. The NRU PT responses of most chemicals showed a close agreement with in vivo response except bithinol. There was a relatively good agreement between in vitro NRU assay and in vivo data. These results suggest that NRU assay using fibroblast could be used to predict the phototoxicity.

• 한국환경성돌연변이발암원학회지
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• 제21권1호
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• pp.44-50
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• 2001

To identify nongenotoxic carcinogen determined as negative by ICH guideline-recommended standard genotoxicity test battery; Ames test, chromosome aberration assay, mouse lymphoma $tk^{+/-}$ assay, in vivo micronucleus assay, we picked bisphenol A as a model compound. In this study, we applied in vitro BalB/c 3T3 cell transformation assay and Syrian hamster embryo (SHE) cell transfarmation assay. Bisphenol A was treated upto $769.2 ug/m{\ell}$ in BalB/c 3T3 cells and upto $125 ug/m{\ell}$ in SHE cells. bisphenol A didn't induced morphological transformation both with one stage treatment protocol and with two stage treatment protocol. But, treated far 48 hr, Bisphenol A induced morphological transformation significantly in SHE cells.

• 환경생물
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• 제28권1호
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• pp.8-13
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• 2010

본 연구는 BRECs에서 AGEs로 유도된 세포의 이동 및 침윤에 있어서 AuNP의 역할에 관한 연구이다. 소 망막으로부터 내피세포를 분리하고, 세포 생존율은 MTT assay로 확인하였다. Wound migration assay는 BRECs의 이동력을 확인하기 위해 수행하였다. Tube formation은 on-gel system을 통해 확인하였다. AuNP의 apoptosis 유도는 caspase-3 assay를 통해 확인하였다. AGE-BSA은 세포증식 및 이동에 있어서 증가함을 보여주었다. 또한 AuNP는 AGE-BSA 존재 유무에 상관없이 세포의 증식, 이동, 신생혈관 형성을 억제하였고, caspase-3을 통해 apoptosis를 유도하였다. 이러한 결과, AuNP는 AGE로 유도된 신생혈관 형성 및 세포의 이동성을 억제하는 약재제로서, 당뇨성 합병증에 있어서 잠재적으로 중요한 분자가 될 것이다.