소배양법으로 배양한 생쥐췌장 베타세포의 형태학적 검정

유광성,전현우,박주홍,윤지원,고광삼 朝鮮大學校 附設 醫學硏究所 1986 The Medical Journal of Chosun University Vol.11 No.1

생쥐췌장 베타세포를 보다 순수하게 배양하기 위해 변형 고안한 소배양법을 검정하였다. 이 소배양법에 있어서는 96well plate에 islet cell을 이식하고 decantation한 다음 thimerosal에 노출시켜 많은 viable cell을 얻을 수 있었고, 생후 7일째의 생쥐의 췌장베타세포의 생존율이 가장 높았다. 또 이 소배양법으로 배양한 생쥐췌장 베타세포들은 위상차현미경, FITC-labelled anti-insulin antibody 염색 및 전자현미경으로 검정한 결과 형태학적으로 정상이었다. 기계적 선택방법으로서 생쥐췌장 베타세포의 decantation 최적시간은 세포를 이식한 후 12∼15시간 이었으며, 화학적 선택방법으로 0.6∼0.8㎍/㎖의 thimerosal에 노출시켜 fibroblastoid cell로 부터 생쥐췌장 베타세포의 분리를 증가시킬 수 있었으며, 두가지 방법을 동시에 응용하여 더 좋은 효율을 얻을 수 있었다. 또 이 소배양법에 있어서 cell duplication은 배양후 36시간후에 2배, 72시간후에 3배가 되었으며, 평균 generation시간은 46.8±7.0시간 이었다. 이들 결과로 미루어 보아 이소배양법은 생쥐췌장 베타세포 배양의 유용한 방법으로서, 췌장 베타세포에 연관된 각종 연구에 이용할 수 있을것으로 사료된다.

Low LET X-ray가 췌장β세포와 신경세포에 미치는 효과

박광훈(Kwang-hun Park),김구환(Kgu-hwan Kim) 대한방사선과학회(구 대한방사선기술학회) 2014 방사선기술과학 Vol.37 No.1

배양된 췌장베타세포와 신경세포를 10% FBS(fetal bovine serum), 11.1 mM glucose의 normal 조건과 1% FBS, 30 mM glucose의 hyperglycemia 조건으로 배양하고, 저 LET X-tjs을 0.5 Gy/hr의 선량률로 총 선량이 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 Gy가 되도록 저 LET 방사선을 조사한 후 MTT assay로 생존율을 측정하고 비교하였다. 분화된 췌장베타세포에 방사선을 조사하지 않았을 때는 normal 조건에 비해 hyperglycemia 조건의 생존율이 경미한 감소를 보였다. 그러나 X선의 총 선량이 점차 누적됨에 띠라 normal 조건에서 생존율이 조금씩 감소하는 것에 비해 hyperglycemia 조건에서는 급격히 감소하여 시너지 효과를 나타냈다. 미분화된 신경세포에서는 방사선을 조사하지 않은 때도 normal 조건에 비해 hyperglycemia 조건의 생존율이 뚜렷하게 감소하였다. X선의 총 선량이 점차 누적됨에 따라 normal 과 hyperglycemia 조건 모두에서 비교적 급격한 세포사멸이 나타났지만 그 생존율의 감소비율이 거의 평행하게 진행되어 시너지 효과를 보이지 않았다. Cultured pancreatic beta cells and nerve cells, it is given normal condition of 10% FBS (fetal bovine serum), 11.1 mM glucose and hyperglycemia codition of 1% FBS, 30 mM glucose. For low LET X-ray irradiated with 0.5 Gy/hr dose-rate(total dose: 0.5 to 5 Gy). Survival rates were measured by MIT assay. When non irradiated, differentiated in the pancreatic beta cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a small reduction. However increasing the total dose of X-ray, the survival rate of normal conditions decreased slightly compared to the survival rate of hyperglycemia conditions, the synergistic effect was drastically reduced. When non irradiated, undifferentiated in the nerve cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a large reduction. As the cumulative dose of X-ray normal conditions and hyperglycemia were all relatively rapid cell death. But the rate of decreased survivals by almost parallel to the reduction proceed and it didn't show synergistic effect.

쥐췌장 베타세포 소배양법의 생리적 검정

고광삼,김성식,김양균,이근배 朝鮮大學校 附設 醫學硏究所 1984 The Medical Journal of Chosun University Vol.9 No.1

쥐 췌장 베타세포의 소 배양법에 대한 생리적 반응을 알아보기 위하여 cell density를 측정하는 방법으로 DNA 농도를 측정하였고, cell density가 intracellular와 extracellular immunoreactive insulin (IRI) 농도에 미치는 영향을 검토하였으며, glucose, arginine, isoleucine 및 leucine이 insulin 분비에 미치는 영향을 알아보았다. 그 결과 DNA 농도는 배양후 2일째에 가장 높았으며, intracellular IRI은 배양후 6일째까지 점차적으로 증가되었고, extracellular IRI은 배양시작후 8일째까지 증가되었으나 그후 감소되었다. Cell density는 intracellular IRI와 extracellular IRI 농도에 영향을 주지 않았으며, glucose는 0~300㎎/100㎖ 농도에서 점차적으로 IRI 분비를 증가시켰으나 300~500㎎/100㎖ 농도에서는 변화가 없었다. Leucine은 IRI 분비를 3배증가시켰으며, arginine과 isoleucine은 2배 증가시켰다. 이로 미루어 보아 본 연구에 이용한 쥐 췌장 베타세포와 소 배양법으로 배양한 쥐 췌장 베타세포는 적절한 생리적 반응을 나타내고 있음을 알 수 있었다. Physiological responses of monolayered mouse beta cell vary according to culture conditons. These cells maintained normal morphological characteristics when cultured in a microculture system. Following determination of DNA concentration as a means of measuring cell density, the effect of cell density on intracellular and extracellular immunoreactive insulin (IRI) conccntration was determmed. Then the effect of glucose, arginine, isoleucine and lencine on insuline secretions was evaluated. When 2 davs old cultures with cell densities between 10^(3) and 10^(6) were compared. the increase of DNA concentration was highest in the 2 days old cultures and no further change by the 14th days. The intracellular IRI through the increase of cell population increased progressively up to culture day 6, and then slowly decreased. Extracellular IRI increased until day 8 and then decreased thereafter regardless cell density. The production of either intracellular or extracellular IRI was therefore independent from the density of cultured cells, Exposure to glucose in the range of 0 to 300㎎/100㎖ resulted in a progressive increase of IRI secretions, but from 300 to 500㎎/100㎖ there was no change. Exposure to leucine resulted in a three-fold increase of IRI secretions, whereas arginine and isoleucine caused these microcultured mouse beta cells to double their IRI secretions. This invetigation, therefore, not only demonstrated that cultured cells in the microculture system were morphologically normal, but also the system was suitable for studies of physiological responses to various culture conditions of murine pancreatic beta cells.

제1형 당뇨병

이명식,김경아 대한의사협회 2009 대한의사협회지 Vol.52 No.7

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by selective autoimmune- mediated destruction of pancreatic islet beta- cells leading gradually to absolute insulin deficiency. T1D is under polygenic control. The HLA complex attributes 50% of the genetic risk for T1D while as many as 20 genes influence susceptibility to T1D. The autoimmune beta-cell destruction could be triggered by environmental factors. While the exact trigger of anti-islet autoimmunity remains elusive, it can lead to an imbalance between regulatory T cells and autoimmune effector T cells. During the initiation of insulitis, emerging evidences suggest that the infiltrating macrophages via toll-like receptor 2 (TLR2) activation lead to induction and amplification of insulitis. Following the priming of diabetogenic T-cells, autoreactive T effector cells destroy the beta cells by direct contact- dependent cytolysis or by soluble mediators secreted from macrophages or CD4 T effector cells. The hyperglycemia occurs late in its course after 80% of the beta cells have been destroyed. Although no current cure exists, refinement of genetic studies and islet autoantibodies has improved the ability to predict the risk of T1D and aid the establishment of rationally designed preventive therapies. Other strategies involve beta-cell replacement by islet transplantation. Extensive and long-term research on the efficacy of islet transplantation and preservation of beta-cell function is keenly needed.

Retinoic Acid-induced Differentiation of Rat Mesenchymal Stem Cells into β-Cell Lineage

김재형,김경식,이상우,김현우,주동진,김유선,서활 대한이식학회 2015 Korean Journal of Transplantation Vol.29 No.3

Backgrounds: Type I diabetes mellitus (T1DM), an autoimmune disease, is associated with insulin deficiency due to the death of β-cells. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are capable of tissue repair and thus are a promising source of β-cell surrogates. Methods: In this study, the therapeutic potential of BM-MSCs as β-cell replacements was analyzed both in vitro and in vivo. First, we used retinoic acid (RA) to induce rat BM-MSCs to differentiate into cells of endodermal/pancreatic lineage. Then, differentiated rat BM-MSCs were syngeneically injected under the renal capsule of rats. Results: Analysis of gene expression revealed that rat BM-MSCs showed signs of early pancreatic development, and differentiated cells were qualitatively and quantitatively confirmed to produce insulin in vitro. In vivo study was performed for short-term (3 weeks) and long-term (8 weeks) period of time. Rats that were injected with differentiated MSCs exhibited a reduction in blood glucose levels throughout 8 weeks, and grafted cells survived in vivo for at least 3 weeks. Conclusions: These findings show that RA can induce differentiation of MSCs into the β-cell lineage and demonstrate the potential of BM-MSCs to serve as therapeutic tools for T1DM.