• Journal of Acupuncture Research
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• 제23권5호
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• pp.69-77
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• 2006

Background : Mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in human subchondral bone and the capacity of these cells to differentiate to osteoblast. Methods : Human subchondral bone were digested with collagenase. Isolated cells were cultured with a-MEM, 15% FBS, 10-8M dexamethasone and 50 ng/mL ascoric acid. Cells from 0 day(isolated cells), 7 day (first subculture) and 14 days (third subculture) were used to carry out phenotypic characterization experiments flowcytometry analysis with 11 monoclonal antibodies) and osteogenic differentiation experiments. Osteogenic differentiation of cells was assessment by quantification of bone extracellular matrix components by following analysis: alkaline phosphatase(ALP) stains to detect ALP activity, RT-PCR and western blot to detect osteocalcin (OCN), osteopontin (OPN) and type I collagen(Col I), and Alizarin red stains to detect calcium deposition. Results : Flowcytometry analyses showed that in our population more than 98% of cells were positive for MSC markers: SH-2(CD105, 99%), CD29 (95%), CD73 (95%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CDl17 (c-kit) (15.1%), and CD166 (74.9%), and cell adhesion molecules such as CD54 (78.1%) and CD106 (63.5%). The osteogenic specific marker analyses showed that the culture of these cells for 7 and 14 days stimulates ALP, OCN, OPN and Col I synthesis by RT-PCR and Western blot analysis. Also, after 14 days in the culture of MSCs induces mineralization by Arizarin red stain. Conclusion : In this work, we demonstrated a new and efficient method for osteoblastic differentiation of human subchondral bone stem cells. As MSCs takes part in reparative processes of adult tissues, these cells could play an important role in osteogenesis.

• Journal of Animal Science and Technology
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• 제51권1호
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• pp.25-32
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• 2009

본 실험은 이유자돈 120두를 이용하여 생균제의 첨가가 이유자돈의 성장 능력, 영양소 소화율, 혈중 요소태 질소, 설사 빈도, 면역 반응에 미치는 영향에 대해 조사하였다. 이유자돈 사료에 항생제 대체제인 생균제의 첨가는 전 사양 기간에 걸쳐 사양 성적이 항생제 첨가구(PC)에 비해 유의적으로 낮게 나타났다. 항생제 첨가구(PC)를 제외하고 비교하였을 때에는 비록 유의적인 차이는 발생하지 않았지만 생균제 첨가구가 항생제 무첨가구(NC)에 비해 일당증체량, 사료효율에서 높은 경향을 보였으며, 이는 항생제 첨가 효과에는 미치지 못하지만 항생제 대체제로서 그 가능성을 보인 것이라고 하겠다. 특히 생균제 A 첨가구는 0~5 주차 사료 효율(P<0.05)에서 항생제 첨가구(PC)와 비슷한 성적을 나타내어, 항생제 대체제로서 이유자돈의 성장을 개선 시킬 가능성이 가장 높다고 하겠다. 본 실험에서는 5주동안 이유자돈의 사료에 생균제를 첨가하여 사양성적을 보았는데, 생균제의 급여기간을 좀 더 늘리면 생균제의 처리효과가 더욱 뚜렷하게 나타날 수도 있으리라 생각된다. 이유자돈 사료에 생균제의 첨가가 건물, 조단백질, 조지방, 조회분 소화율에 어떠한 영향도 미치지 않았으며(P>0.10), 질소 축적율은 생균제 첨가구에서 항생제 첨가구(PC)에 비해 높게 나타났으나 일관된 효과를 보이지는 않았다. 혈중 요소태 질소 농도에서는 2주차와 4주차에서 유의적인 차이가 나타났다(P<0.05). 또한 생균제 B 첨가구가 다른 처리구에 비해 혈중 요소태 질소 농도가 가장 낮게 나타났다. 설사 발생 빈도 측정 결과, 항생제 첨가구(PC)에서 설사 발생 빈도가 유의적으로 낮게 나타났으며, 생균제 C 첨가구에서 다음으로 낮게 나타났다(P=0.18). 면역 반응 실험 결과, $CD^{4+}$ T-cell 수의 경우 생균제 C 첨가구에서 다른 처리구에 비해 가장 낮게 나타났으며(P<0.01), $CD^{8+}$ T-cell 수와 $CD^{4+}:CD^{8+}$ 비율의 경우에는 모든 처리구에서 유의적인 차이가 발생하지 않았다(P>0.10). 결론적으로 이유자돈사료 내 생균제의 첨가가 이유자돈의 성장 능력, 질소축적율, 혈중 요소태 질소, 설사 빈도, 및 면역 능력에 일정한 개선 효과를 갖고 있지만, 항생제 첨가 효과에는 미치지 못한다고 하겠다. 본 실험을 통해 이유자돈 사료에 생균제의 첨가는 항생제 대체제로서 일정 부분 개선효과가 있는 것으로 사료된다.

• 대한본초학회지
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• 제23권2호
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• pp.87-97
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• 2008

Objectives : The present study was carried out to investigate the effect of Herba Polygalae Japonica on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mice by ovalbumin (OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dessiciated with collagenase (1 ${\mu}g$/ml). Eosinophils were activated by rIL-3/rmIL-5 co-treatments. The lung cells were treated with extract of Herba Polygalae Japonica (EPJ), incubated for 48 hr at $37^{\circ}C$, and analyzed by flow cytometer, ELISA, RT-PCR and immunocytochemistry stain. Results : A significant cytotoxicity by drug treatment was not observed. The cell number ratio of granulocyte, CD3e-/CCR3+, CD3e+CD69+, CD4+, CD23+/B220+ cells was increased in rmIL-5/rIL-3 treated control group compared to the normal group. Cells numbers in the experimental animal group treated with EPJ was all decreased. In ELISA analysis, IL-4, IL-5, IL-13 levels and histamine release level were increased in the control group compared to the normal animal group, then significantly decreased in the experimental group with 100 ${\mu}g$/ml of EPJ treatment. In RT-PCR analysis, mRNA expressions of IL-4, IL-5, IL-13, CCR3 and eotaxin were increased in the control group compared to the normal animal group, then decreased in the experimental group with 100 ${\mu}g$/ml of EPJ treatment. And eosinophil proliferation levels were 18847${\pm}$,1527 (cpm) in the control group, 4676${\pm}$972 (cpm) in the positive control group, and 7709 ${\pm}$ 549 (cpm), 16839 ${\pm}$ 1403 (cpm), 16385 ${\pm}$ 1723 (cpm) in the experimental group with 100 ${\mu}g$/ml, 10 ${\mu}g$/ml, 1 ${\mu}g$/ml of EPJ treatment. Conclusions : The present data suggested that Herba polygalae japonica may have an effects on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of EPJ.

• 대한본초학회지
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• 제23권1호
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• pp.75-83
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• 2008

Objectives : The present study was carried out to investigate the effect of Hyperici Japonici Herba on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mice by ovalbumin(OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dessiciated with collagenase(1${\mu}g$/ml). Eosinophils were activated by rIL-3/rmIL-5 co-treatments. The lung cells were treated with extract of Hyperici Japonici Herba(EHH), incubated for 48 hr at $37^{\circ}C$, and analyzed by flow cytometer. ELBA, RT-PCR, immunocytochemistry stain. Results : The cell number ratio of granulocyte, $CD3e^-$/$CCR3^+$, $CD3e^+$/$CD69^+$, $CD4^+$, $CD23^+$/$B220^+$ cells was increased in rmIL-5/rIL-3 treated control group compared to the normal group. Cells numbers in the experimental animal group treated with EHH was all decreased. In ELISA analysis, IL-4, IL-5, IL-13 protein levels and histamine release level were greatly increased in the control group compared to the normal animal group, then significantly decreased in the experimental group with 100 ${\mu}g$/ml of EHH treatment. In RT-PCR analysis, the HT value of IL-4, IL-5, IL-13, CCR3, Eotaxin were increased in the control group compared to the normal animal group, then decreased in the experimental group with 100 ${\mu}g$/ml of EHH treatment. And eosinophil proliferation levels were 18847${\pm}$1527(cpm) in the control group, 4676${\pm}$972(cpm) in the positive control group, and 8675${\pm}$159(cpm), 11352${\pm}$1005(cpm), 14325${\pm}$677(cpm) in the experimental group with 100 ${\mu}g$/ml, 10 ${\mu}g$/ml, 1 ${\mu}g$/ml of EHH treatment. Conclusions : The present data suggested that Hyperici Japonici Herba may have an effects on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of EHH.

• Childhood Kidney Diseases
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• 제22권1호
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• pp.1-6
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• 2018

Rituximab (RTX) is a chimeric monoclonal antibody that inhibits CD20-mediated B-cell proliferation and differentiation. Several studies have examined its use in intractable nephrotic syndrome (NS) with some positive results. However, those studies examined such effects for a short-term period of 1 year, and some patients continued to relapse after a lapse in RTX treatment. Our use of RTX as a maintenance therapy (RTX injection when the CD19 cell count exceeded $100-200/{\mu}L$ before relapse) showed some noticeable efficacy. We used RTX in 19 patients with steroid-dependent NS (SDNS). In 12 patients treated with RTX maintenance therapy, only one relapse occurred. The mean treatment period was $23.4{\pm}12.7months$, and the mean number of RTX administrations was $3.9{\pm}1.6$. The relapse rates were decreased (from 2.68/year to 0.04/year), and the drug-free period also increased (from 22.5 days/year to 357.1 days/year) during maintenance therapy. The other seven patients were treated with one cycle of RTX or additional cycles in case of relapse (non-maintenance therapy). Relapse rates were significantly decreased after RTX treatment (from 1.76/year to 0.96/year, P=0.017). The relapse-free period was $15.55{\pm}7.38$ (range, 5.3-30.7) months. No severe side effects of RTX were found except for a hypersensitivity reaction such as fever and chills during its infusion. In conclusion, RTX is considered an effective and safe option to reduce the relapse rate by a single- or maintenance-interval therapy in SDNS.

• 대한예방한의학회지
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• 제27권2호
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• pp.23-33
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• 2023

Objectives : Sojadodamgangki-tang and its main components are traditional korean medicinal methods for treatment of cough, sputum and dyspnea. Using a respiratory inflammatory model, we intend to reveal the anti-inflammatory effect and its immune mechanism of Sojadodamgangki-tang. Methods : We used a papain-induced respiratory inflammatory mouse model. 8-week-old female BALB/C mice were divided into 3 groups as follows: the following groups: saline control group, papain treated group (vehicle), papain and Sojadodamgangki-tang(200 mg/kg) treated group (n=4). To evaluate the anti-inflammatory effect of Sojadodamgangki-tang extracts, inflammatory cell infiltration was measured in bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF). In addition, the effects of Sojadodamgangki-tang extracts on Th2 cell population in lung were determined by using flow cytometry. Results : Sojadodamgangki-tang extracts administration reduced inflammatory cell infiltration in BALF and NALF, especially of eosinophils. Furthermore, total immunogloblin (Ig)-E levels was reduced in BALF and serum by drug administration. Interestingly, Sojadodamgangki-tang extracts treatment also decreased the Th2 cell (CD4⁺GATA3⁺) population in lung. Conclusions : Our findings indicate Sojadodamgangki-tang extracts have anti-inflammatory effects by mediating Th2 cell and B cell activation.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.106-115
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• 2010

Phosphorylated dextran (P-Dex) is an acidic polysaccharide that functions as an immune adjuvant. P-Dex is known to regulate immune response by maintaining a balance between Th1 and Th2 cells in vitro, and thus may also be important in the control of allergic reactions. In the current study, we report the optimum conditions required for the efficient phosphorylation of dextran without toxicity. We found that when dextran was heated at 160${^{\circ}C}$ for 24 h in phosphate buffer (pH 5.0), the resulting P-Dex demonstrated the highest phosphorus content (6.8%). We also report that P-Dex enhances mitogenic activity in mouse splenocytes and induces expression of CD69 and CD86 on the surface of B cells and dendritic cells (DC) in vitro. Oral administration of P-Dex to ovalubmin (OVA)-immunized mice was found to reduce antigen-induced cell proliferation and suppress the expression of CD86 on Th2-inducing DC via exogenous OVA stimulation. P-Dex was also found to increase IL-10 expression in the splenocytes of treated mice. These findings suggest that oral administration of P-Dex increases immunological tolerance and improves the specificity of immunological response to specific antigens.

• IMMUNE NETWORK
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• 제23권4호
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• pp.33.1-33.13
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• 2023

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been acknowledged as an effective mean of preventing infection and hospitalization. However, the emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) has led to substantial increase in infections among children and adolescents. Vaccine-induced immunity and longevity have not been well defined in this population. Therefore, we aimed to analyze humoral and cellular immune responses against ancestral and SARS-CoV-2 variants after two shots of the BNT162b2 vaccine in healthy adolescents. Although vaccination induced a robust increase of spike-specific binding Abs and neutralizing Abs against the ancestral and SARS-CoV-2 variants, the neutralizing activity against the Omicron variant was significantly low. On the contrary, vaccine-induced memory CD4⁺ T cells exhibited substantial responses against both ancestral and Omicron spike proteins. Notably, CD4⁺ T cell responses against both ancestral and Omicron strains were preserved at 3 months after two shots of the BNT162b2 vaccine without waning. Polyfunctionality of vaccine-induced memory T cells was also preserved in response to Omicron spike protein. The present findings characterize the protective immunity of vaccination for adolescents in the era of continuous emergence of variants/subvariants.

• 동의생리병리학회지
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• 제20권1호
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• pp.163-170
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• 2006

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .

• 동의생리병리학회지
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• 제23권5호
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• pp.1106-1115
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• 2009

Mori Ramulus has multiple applications in Korean traditional medicine prescription because it has antioxidant and anti-inflammatory effects by reducing macrophage activities. Yet, no studies on the anti-arthritic activity of EMR (extract of Mori Ramulus) have been reported in vitro and in vivo. Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic inflammation characterized by hyperplasia of synovial cells in affected joints, which ultimately leads to the destruction of cartilage and bone. Because collagen-induced arthritis (CIA) is similar to RA in pathological symptoms and immune reactions, there have been several reports concerning RA using CIA mouse model. Here, we investigated the effects of Mori Ramulus on RA using CIA mice. The importance of CD4+ Th1 cells in RA progress was previously indicated and studies further showed that Th17 cells play a prime role in severity of disease. Accordingly, the present study was focused on CIA associated with CD4+ Th1 cells and Th1 7 cells. DBA/1OlaHsd mice were immunized with bovine type II collagen (CII). After a second collagen immunization, mice were treated with EMR once a day for 4 weeks. The severity of arthritis within the paw joints was evaluated by histological assessment of cartilage destruction and pannus formation. Immune cells in peripheral blood mononuclear cells (PBMC), draining lymph node (DLN) and paw joints, cytokine production and gene expression were assessed from CIA mouse using ELISA, FACS and real-time PCR analysis. Administration of EMR significantly suppressed the progression of CIA and inhibited the production of TNF-$\alpha$, IL-6 and IL-17 in the serum. The erosion of cartilage was dramatically reduced in mouse knees after treatment with EMR. In conclusion, our results demonstrate that EMR significantly suppressed the progression of CIA and that this action was mediated by the decreased production of TNF-$\alpha$, IL-6, IL-17 and collagen II-specific antibody in the serum. EMR suppressed Th17 cells and reduced level of IL-6 via B cell suppression, and thus, the levels of autoantibodies produced from B cells were decreased. Furthermore, EMR suppressed NKT cells which directly stimulate B cells and develop imbalance of Th1/Th2 cell. Oral administration of EMR (100 mg/kg or 200 mg/kg) significantly suppressed the progression of CIA, which is comparable to that of methotrexate (MTX, 0.3 mg/kg) used as a positive control. We are currently studying the mechanism underlying the therapeutic role for EMR in CIA mice.