혈관 생성 연구를 위한 세포 혼합 평가법 개발

강규태(Kyu Tae Kang) 대한약학회 2018 약학회지 Vol.62 No.5

Blood vessels are formed by two cell types: endothelial cells and vascular mural cells. However, most in vitro angiogenesis assays are performed by only endothelial cells. Here, we developed an in vitro angiogenesis assay system using two human vascular cell precursors: endothelial colony forming cells (ECFC) and mesenchymal stem cells (MSC). The progression of tube formation was monitored by a real-time cell recorder for 24 hours. We also utilized an image-stitching software to increase the analyzed image area up to 9-times larger than one image from 10X object lens. Tube number was higher in ECFC+MSC than ECFC at 6 hour time point, but became lower at 12 and 24 hours when compared with ECFC. The length per tube was higher in ECFC+MSC than ECFC at all-time points. VEGF treatment increased tube formation in both ECFC+MSC and ECFC. Vatalanib, VEGF inhibitor, inhibited tube formation of ECFC dose-dependently. Interestingly, ECFC+MSC-induced tube formation was not changed by vatalanib, suggesting that MSC may potentiate the stability and durability of tubular structures. These results indicate that two-cell tube formation assay system can mimic in vivo angiogenic process by two vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

Bone Formation of Embryonic Stem Cell-Derived Mesenchymal Stem Cells

정연태,유기연,이희수 한국조직공학과 재생의학회 2015 조직공학과 재생의학 Vol.12 No.4

Human embryonic stem cells are multipotent cells. In this study, We observed osteogenesis of human embryonic stem cell derived mesenchymal stem cells. mbryonic body formation method was used to derive mesenchymal stem cells from human embryonic stem cells. Embryonic stem cell derived mesenchymal stem cells were immunostained for CD 73 to make characterization of mesencymal stem cells. Osteogenesis of CD 73 positive mesencymal stem cells with media included ascorbic acid, dexamethasone and glycerophosphate was induced. After 10 days culture with osteogenesis media, the cells were immunostained with type I collagen, osteocalcin and Runx-2. After 21 days culture with osteogenesis media, the cells were stained with alizarin red to observe bone nodule formation. Mesenchymal stem cells were derived from human embryonic stem cells by embryonic body method. After these cells were cultured with osteogenesis media, the cells were differentiated into osteoblast and showed bone nodule formation.

Mxi1 influences cyst formation in three-dimensional cell culture

( Yeon Joo Yook ),( Kyung Hyun Yoo ),( Seon Ah Song ),( Min Ji Seo ),( Je Yeong Ko ),( Bo Hye Kim ),( Eun Ji Lee ),( Eunsun Chang ),( Yu Mi Woo ),( Jong Hoon Park ) 생화학분자생물학회 (구 한국생화학분자생물학회) 2012 BMB Reports Vol.45 No.3

Cyst formation is a major characteristic of ADPKD and is caused by the abnormal proliferation of epithelial cells. Renal cyst formation disrupts renal function and induces diverse complications. The mechanism of cyst formation is unclear. mIMCD-3 cells were established to develop simple epithelial cell cysts in 3-D culture. We confirmed previously that Mxi1 plays a role in cyst formation in Mxi1-deficient mice. Cysts in Mxi1 transfectanted cells were showed by collagen or mebiol gels in 3-D cell culture system. Causative genes of ADPKD were measured by q RT-PCR. Herein, Mxi1 transfectants rarely formed a simple epithelial cyst and induced cell death. Overexpression of Mxi1 resulted in a decrease in the PKD1, PKD2 and c-myc mRNA relating to the pathway of cyst formation. These data indicate that Mxi1 influences cyst formation of mIMCD-3 cells in 3-D culture and that Mxi1 may control the mechanism of renal cyst formation. [BMB reports 2012; 45(3): 189-193]

Bone Formation of Embryonic Stem Cell-Derived Mesenchymal Stem Cells

( Yeon Tae Jung ),( Ki Yeon Yoo ),( Hee Su Lee ) 한국조직공학·재생의학회 2015 조직공학과 재생의학 Vol.12 No.2s

Human embryonic stem cells are multipotent cells. In this study, We observed osteogenesis of human embryonic stem cell derived mesenchymal stem cells. mbryonic body formation method was used to derive mesenchymal stem cells from human embryonic stem cells. Embryonic stem cell derived mesenchymal stem cells were immunostained for CD 73 to make characterization of mesencymal stem cells. Osteogenesis of CD 73 positive mesencymal stem cells with media included ascorbic acid, dexamethasone and glycerophosphate was induced. After 10 days culture with osteogenesis media, the cells were immunostained with type I collagen, osteocalcin and Runx-2. After 21 days culture with osteogenesis media, the cells were stained with alizarin red to observe bone nodule formation. Mesenchymal stem cells were derived from human embryonic stem cells by embryonic body method. After these cells were cultured with osteogenesis media, the cells were differentiated into osteoblast and showed bone nodule formation. Tissue Eng Regen Med 2015;12(Suppl 2):132-137

Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration

Noh, Mi Ra,Jang, Hee-Seong,Song, Dae-Kyu,Lee, Seong-Ryong,Lipschutz, Joshua H.,Park, Kwon Moo,Kim, Jee In Elsevier 2018 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Exocyst Sec10 decreases by I/R injury and restores with functional recovery. </LI> <LI> Sec10 inversely regulates kidney tubule cell wound healing after scratch injury. </LI> <LI> Sec10 inversely regulates ruffle formation at the leading edge during recovery. </LI> <LI> Upregulation of Sec10 increases DGKγ expression at the leading edge during recovery. </LI> <LI> DGK inhibitor reversed Sec10-mediated ruffle formation and wound healing defect. </LI> </UL> </P>

Cell death induction and intracellular vesicle formation in human colorectal cancer cells treated with Δ9-Tetrahydrocannabinol

Hwang Yu-Na,Kwon In-Seo,Park Ju-Hee,Na Han-Heom,Kwon Tae-Hyung,Park Jin-Sung,Kim Keun-Cheol 한국유전학회 2023 Genes & Genomics Vol.45 No.12

Background Δ9-Tetrahydrocannabinol (Δ9-THC) is a principal psychoactive extract of Cannabis sativa and has been traditionally used as palliative medicine for neuropathic pain. Cannabidiol (CBD), an extract of hemp species, has recently attracted increased attention as a cancer treatment, but Δ9-THC is also requiring explored pharmacological application. Objective This study evaluated the pharmacological effects of Δ9-THC in two human colorectal cancer cell lines. We investigated whether Δ9-THC treatment induces cell death in human colorectal cancer cells. Methods We performed an MTT assay to determine the pharmacological concentration of Δ9-THC. Annxein V and Western blot analysis confirmed that Δ9-THC induced apoptosis in colorectal cancer cells. Metabolic activity was evaluated using MitoTracker staining and ATP determination. We investigated vesicle formation by Δ9-THC treatment using GW9662, known as a PPARγ inhibitor. Results The MTT assay showed that treatment with 40 μM Δ9-THC and above inhibited the proliferation of colorectal cancer cells. Multiple intracytoplasmic vesicles were detected upon microscopic observation, and fluorescence-activated cell sorting analysis showed cell death via G1 arrest. Δ9-THC treatment increased the expression of cell death marker proteins, including p53, cleaved PARP-1, RIP1, and RIP3, suggesting that Δ9-THC induced the death of colorectal cancer cells. Δ9-THC treatment also reduced ATP production via changes in Bax and Bcl-2. Δ9-THC regulated intracytoplasmic vesicle formation by modulating the expression of PPARγ and clathrin, adding that antiproliferative activity of Δ9-THC was also affected. Conclusion In conclusion, Δ9-THC regulated two functional mechanisms, intracellular vesicle formation and cell death. These findings can help to determine how cannabinoids can be used most effectively to improve the efficacy of cancer treatment.

자기조직화 신경망을 이용한 셀 형성 문제의 기계 배치순서 결정 알고리듬

전용덕(Yong-Deok Jeon) 한국산업경영시스템학회 2019 한국산업경영시스템학회지 Vol.42 No.2

Self Organizing Map (SOM) is a neural network that is effective in classifying patterns that form the feature map by extracting characteristics of the input data. In this study, we propose an algorithm to determine the cell formation and the machine layout within the cell for the cell formation problem with operation sequence using the SOM. In the proposed algorithm, the output layer of the SOM is a one-dimensional structure, and the SOM is applied to the parts and the machine in two steps. The initial cell is formed when the formed clusters is grouped largely by the utilization of the machine within the cell. At this stage, machine cell are formed. The next step is to create a flow matrix of the all machine that calculates the frequency of consecutive forward movement for the machine. The machine layout order in each machine cell is determined based on this flow matrix so that the machine operation sequence is most reflected. The final step is to optimize the overall machine and parts to increase machine layout efficiency. As a result, the final cell is formed and the machine layout within the cell is determined. The proposed algorithm was tested on well-known cell formation problems with operation sequence shown in previous papers. The proposed algorithm has better performance than the other algorithms.

Apoptotic Cell Death in TrkA-overexpressing Cells: Kinetic Regulation of ERK Phosphorylation and Caspase-7 Activation

Jung, Eun Joo,Kim, Deok Ryong Korean Society for Molecular Biology 2008 Molecules and cells Vol.26 No.1

The TrkA tyrosine kinase is activated by autophosphorylation in response to NGF, and plays an important role in cell survival, differentiation, and apoptosis. To investigate its role in cell fate determination, we produced stable TrkA-inducible SK-N-MC and U2OS cell lines using the Tet-On system. Interestingly, TrkA overexpression induced substantial cell death even in the absence of NGF, by stimulating ERK phosphorylation and caspase-7 activation leading to PARP cleavage. TrkA-mediated cell death was shown by the annexin-V binding assay to be, at least in part, apoptotic in both SK-N-MC and U2OS cells. Furthermore, the truncated form (p18) of Bax accumulated in the TrkA-induced cells, suggesting that TrkA induces mitochondria-mediated apoptosis. NGF treatment augmented the cell death induced by TrkA overexpression. This TrkA-induced cell death was blocked by the tyrosine kinase inhibitors, K-252a and GW441756. Moreover, TrkA overexpression inhibited long-term proliferation of both the neuronal SK-N-MC cells and the non-neuronal U2OS cells, suggesting a potential role of TrkA as a tumor suppressor.

Apoptotic Cell Death in TrkA-overexpressing Cells: Kinetic Regulation of ERK Phosphorylation and Caspase-7 Activation

정은주,김덕룡 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.1

The TrkA tyrosine kinase is activated by autophosphorylation in response to NGF, and plays an important role in cell survival, differentiation, and apoptosis. To investigate its role in cell fate determination, we produced stable TrkA-inducible SK-N-MC and U2OS cell lines using the Tet-On system. Interestingly, TrkA overexpression induced substantial cell death even in the absence of NGF, by stimulating ERK phosphorylation and caspase-7 activation leading to PARP cleavage. TrkA-mediated cell death was shown by the annexin-V binding assay to be, at least in part, apoptotic in both SK-N-MC and U2OS cells. Furthermore, the truncated form (p18) of Bax accumulated in the TrkA-induced cells, suggesting that TrkA induces mitochondria-mediated apoptosis. NGF treatment augmented the cell death induced by TrkA overexpression. This TrkA-induced cell death was blocked by the tyrosine kinase inhibitors, K-252a and GW441756. Moreover, TrkA overexpression inhibited long-term proliferation of both the neuronal SK-N-MC cells and the non-neuronal U2OS cells, suggesting a potential role of TrkA as a tumor suppressor.

Hesperidin Inhibits Vascular Formation by Blocking the AKT/mTOR Signaling Pathways.

김기대 한국식품영양과학회 2015 Preventive Nutrition and Food Science Vol.20 No.4

Hesperidin has been shown to possess a potential inhibitory effect on vascular formation in endothelial cells. However, the fundamental mechanism for the anti-angiogenic activity of hesperidin is not fully understood. In the present study, we evaluated whether hesperidin has anti-angiogenic effects in mouse embryonic stem cell (mES)-derived endothelial-like cells, and human umbilical vascular endothelial cells (HUVECs), and evaluated their mechanism via the AKT/mammalian target of rapamycin (mTOR) signaling pathway. The endothelial cells were treated with several doses of hesperidin (12.5, 25, 50, and 100 μM) for 24 h. Cell viability and vascular formation were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and tube formation assay, respectively. Alteration of the AKT/mTOR signaling in vascular formation was analyzed by western blot. In addition, a mouse aortic ring assay was used to determine the effect of hesperidin on vascular formation. There were no differences between the viability of mES-derived endothelial-like cells and HUVECs after hesperidin treatment. However, hesperidin significantly inhibited cell migration and tube formation of HUVECs (P<0.05) and suppressed sprouting of microvessels in the mouse aortic ring assay. Moreover, hesperidin suppressed the expression of AKT and mTOR in HUVECs. Taken together, these findings suggest that hesperidin inhibits vascular formation by blocking the AKT/mTOR signaling pathways.