• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.3
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• pp.543-548
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• 2000

EC-2B와 EC-2C 획분은 정향(Eugenia caryophyllata)의 알칼리 추출물로부터 에탄올 침전, cetavlon 처리 및 한외여과를 거쳐 분획하였다. EC-2B 획분은 APTT에서 항응고 활성을 가지는 반면, EC-2C 획분은 APTT와 TT 모두에서 항응고 활성을 가지고 있으며 EC-2B와 EC-2C에서 모두 혈소판 응집억제능을 관찰할 수 있었다. EC-2B와 EC-2C 획분의 경구투여에서 두 획분 모두 독성이 없었으며 EC-2B 획문은 1,000 mg/kg (mouse, intravenours)에서도 독성이 없었으나, EC-2C 획분은 LD50 322 mg/kg 정도의 독성을 가지고 있었다. 두 획분의 in vivo 성에서의 항응고 활성을 60% 생존율을 갖는 dose로 표시한 결과 EC-2B는 131 mg/kgdlsep 비해 EC-2C는 58 mg/kg로 활성의 차이가 in vitro에서 보다 크게 나타났다. 이 두획분을 sulfation시킨 후 활성의 변화를 ex vivo를 통해 확인한 결과 두 획분 모두 활성이 증가하였으며 특히 EC-2B 획분의 활성이 급격히 증가하였다.

• The Korea Journal of Herbology
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• v.39 no.3
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• pp.57-67
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• 2024

Objectives : The aim of this study is to evaluate the potential biological activity of Veronica incana extracts (VIE) through in vitro, ex vivo, and in vivo experiments. Methods : In vitro, we conducted analyses on the total polyphenol (TP) and total flavonoid (TF) levels, alongside DPPHand ABTS radical scavenging activities. Ex vivo evaluations on adipose tissue measured glycerol release as a marker of lipolysis. In LPS-induced RAW 264.7 cells, we quantified nitric oxide (NO) production. Following H₂O₂ induction in U2OS cells, we performed mitochondrial assays such as MitoSox and MitoTracker. Moreover, Bodipy assays were conducted in 3T3-L1 cells. In vivo, we performed anti-osteoarthritis effect of VIE against monosodium iodoacetate (MIA)-induced osteoarthritis in rats. Results : The results presented encompass a myriad of models, from cell culture to animal experiments as well as ex vivo studies. VIE demonstrated high TP and TF contents, potent DPPH and ABTS scavenging activities, and regulated glycerol release. Moreover, the inhibition of NO production in LPS-induced inflammation was notably confirmed and the reduction of lipid droplets was distinctly shown. Furthermore, in H₂O₂-induced U2OS cells, MitoSox was effectively reduced while MitoTracker noticeably increased. In vivo assays confirmed a significant increase in hindpaw weight distribution (HWD) decreased by MIA after VIE treatment. Additionally, VIE inhibited serum inflammatory cytokines (TNF-𝛼, IL-6, and IL-1𝛽) and MDA levels in joint tissue. Conclusion : In conclusion, Veronica incana exhibited various pharmacological effects including antioxidant, anti-obesity, and anti-inflammatory properties.

• International Journal of Stem Cells
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• v.16 no.4
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• pp.438-447
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• 2023

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.77-77
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• 2003

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. $CD34^+/ $ cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 $\times$ $10^5$ cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 $\times$ $10^5$ cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was $26.6 \pm 8.4.$ In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 $\pm$ 0.5 and that of HSCs cultured onto an insert was $46.9 \pm 8.4.$ The percentage of BM-MSCs cells remained being fluorescent was $97.9 \pm 0.3%$ after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.

• Journal of Life Science
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• v.27 no.1
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• pp.78-88
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• 2017

Angiogenesis, the formation of new blood vessels, plays an important role in tumor growth and metastasis; therefore, it has become an important target in cancer therapy. Novel anticancer pharmaceutical products that have relatively few side effects or are non-cytotoxic must be developed, and such products may be obtained from traditional herbal medicines. Coptis chinensis Franch. is an herb used in traditional medicine for the treatment of inflammatory diseases and diabetes. However, potential antiangiogenic effects of C. chinensis water extract (CCFWE) have not yet been studied. The purpose of this study was to determine the antiangiogenic effect of CCFWE in order to evaluate its potential for an anticancer drug. We found that the treatment with CCFWE inhibited the major steps of the angiogenesis process, such as the endothelial cell proliferation, migration, invasion, and capillary-like tube formation in response to vascular endothelial growth factor (VEGF), and also resulted in the growth inhibition of new blood vessels in an ex vivo rat aortic ring assay. We also observed that CCFWE treatment arrested the cell cycle at the G0/G1 phase, preventing the G0/G1 to S phase cell cycle progression in response to VEGF. In addition, the treatment reduced the VEGF-induced activation of matrix metalloproteinases 2 and 9. Taken together, these findings indicate that CCFWE should be considered a potential anticancer therapy against pathological conditions where angiogenesis is stimulated during tumor development.

• Clinical and Experimental Pediatrics
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• v.48 no.8
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• pp.894-900
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• 2005

Purpose : Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. Methods : The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. Results : The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. Conclusion : The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.

• Journal of Nutrition and Health
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• v.42 no.3
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• pp.207-212
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• 2009

Codonopsis lanceolatae has been used as one of the traditional remedies as well as food source. However, few studies on their immunomodulating effects have been reported. We previously reported that ex vivo supplementation of Codonopsis lanceolatae water extracts enhanced splenocyte proliferation compared to the control group. In order to elucidate its ex vivo effect, six to seven week old balb/c mice were fed ad libitum on a chow diet and water extracts of Codonopsis lanceolatae were orally administrated every other day for four weeks at two different concentrations (50 and 500 mg/kg B.W). After preparing the single cell suspension, the proliferation of splenocytes was determined by MTT (3- [4,5-dimethylthiazol-2-y] -2,5-diphenyl terazolium bromide) assay. The production of cytokine (IL-1${\beta}$, IL-6, TNF-${\alpha}$), secreted by macrophages stimulated with LPS or not, was detected by ELISA assay using a cytokine kit. After 48 hrs of incubation with the mitogen (ConA or LPS) stimulation, the mice splenocyte proliferation in experimental group was statistically increased at two different concentrations than that in control group. The cytokines production was more significantly enhanced at the lower supplementation (500 mg/kg B.W.) group rather than higher concentration (500 mg/kg B.W.) compared to the control group. The results of this study may suggest that the supplementation of water extract of plant mixture could regulate the immune function by increasing the splenocyte proliferation and enhance the immune function through regulating cytokine production capacity by activated macrophages in mice.

• Journal of Yeungnam Medical Science
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• v.24 no.2
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• pp.262-274
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• 2007

Purpose : Bone morphogenetic proteins (BMPs) play an important role in the formation of cartilage and bone, as well as regulating the growth of chondroblasts and osteoblasts. In this study, we investigated whether recombinant human BMP adenoviruses are available for ex vivo gene therapy, using human fibroblasts and human bone marrow stromal cells in an animal spinal fusion model. Materials and Methods : Human fibroblasts and human bone marrow stromal cells were transduced with recombinant BMP-2 adenovirus (AdBMP-2) or recombinant BMP-7 adenovirus (AdBMP-7), referred to as AdBMP-7/BMSC, AdBMP-2/BMSC, AdBMP-7/HuFb, and AdBMP-2/HuFb. We showed that each cell secreted active BMPs by alkaline phosphatase staining. Since AdBMP-2 or AdBMP-7 tranducing cells were injected into the paravertebral muscle of athymic nude mice, at 4 weeks and 7 weeks, we confirmed that new bone formation occurred by induction of spinal fusion on radiographs and histochemical staining. Results : In the region where the AdBMP-7/BMSC was injected, new bone formation was observed in all cases and spinal fusion was induced in two of these. AdBMP-2/BMSC induced bone formation and spinal fusion occurred among one of five. However, in the region where AdBMP/HuFb was injected, neither bone formation nor spinal fusion was observed. Conclusion : The osteoinductivity of AdBMP-7 was superior to that of AdBMP-2. In addition, the human bone marrow stromal cells were more efficient than the human fibroblasts for bone formation and spinal fusion. Therefore, the results of this study suggest that AdBMP-7/BMSC would be the most useful approach to ex vivo gene therapy for an animal spinal fusion model.

• Korean Chemical Engineering Research
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• v.44 no.1
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• pp.73-80
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• 2006

This study compared cell expansion and colony forming ability in human cord blood stem cells cultured ex vivo with two kinds of cytokine combinations, two kinds of media, presence or absence of fetal bovine serum (FBS) and two or three dimensional (2D or 3D) culture environments. Purified $CD34^+$ cells were cultured in the IMDM (Iscove's Modified Dulbecco's Medium) and SFM (Serum Free Medium) containing a cytokine cocktail-I (coc-I) (EPO, GMCSF, SCF, and IL-3) or a cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher cellular and clonogenic expansion were observed in the coc-I cytokine condition, compared to coc-II cytokine condition. 3D (Methocult) and 2D (IMDM + coc-I + FBS) conditions gave the greatest cell ($2,258{\pm}456$ fold) and CFU (BFU-E: $652{\pm}19$, CFU-GM: $520{\pm}58$, CFU-GEMM: $339{\pm}100$ fold) expansions, respectively. In aspect of medium, IMDM was better than SFM, except for coc-II condition without FBS. In conclusion, 'IMDM + coc-I + FBS' and 'IMDM + coc-I' were the best CFU expansions on the occasion of all culture conditions. FBS and 2D conditions had affirmative effect on CFU expansion, generally. These data might provide a variety of notions about ex vivo expansion of hematopoietic stem cells.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.31-38
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• 2007

Infection with virulent or attenuated Salmonella typhimuriumhas known to induce reduction in proliferative responses of spleen cells. We investigated a role of lipid A from S. typhimurium, a B cell mitogen, on proliferation of spleen cells by T cell mitogens such as concanavaline A and phytohemagglutinin under in vitro and ex vivo conditions. Lipid A alone induced proliferation of spleen cells in vitroin a dose-dependent manner. However, subsequent treatment of concanavaline A or phytohemagglutin in after lipid A treatment induced proliferation suppression of murine spleen cells in vitro and ex vivo. Removal of macrophages from spleen cells, which were obtained from a lipid A-injected mouse, restored proliferation by concanavaline A and phytohemagglutinin, indicating that macrophages appeared to play a role in lipid A-induced suppression. Secreted molecules from macrophages did not accounted for the suppression because suppressive effect was not achieved when the supernatant from macrophage-containing spleen cell culture was conditoned to macrophage-depleted spleen cell culture. Co-culture of spleen cells from lipid A-treated and - untreated mice showed proliferation suppression as increasing cell numbers of lipid A-treated mouse. These data suggested that the cell-to-cell contact of macrophage with splenic lymphocyte cells is responsible for immune responses against lipid A, which is applicable to the case of human S. typhi infection.