日本人糖尿病母体から出生した児の糖代謝異常にHNF-1α遺伝子変異は関与しない

糖尿病母体から出生した児における糖代謝異常出現へのHNF(hepatocyte nuclear factor)-1α遺伝子,および環境因子の関与を検討した. 1989～1999年の追跡発育発達検診を受診し,遺伝子検索の同意が得られた134名(1型糖尿病母体出生児50名, 2型糖尿病母体出生児84名)を対象とした.対象を経口糖負荷試験で判定し,正常結果のみを正常群(N),糖尿病と診断した糖尿病群(D),その他を境界群(B)とした. HNF-1α遺伝子はPCR直接シークエンス法で変異を検索した.結果は, (1) 1型糖尿病母体出生児ではD1名(2%), B12名(24%), N37名(74%)であり, 2型糖尿病母体出生児ではD3名(3.8%), B31名(36.9%), N50名(59.5%)であり,糖尿病児の病型は全例が母の病型と一致していた. (2) 糖代謝異常は若年で出現していた. (3) 母体妊娠中平均血糖は, D>B>N群の順に高値であった. (4) 母の妊娠前のBMIと児の最大肥満度は正相関を示した. (5) 2型糖尿病母体出生児のうちの肥満児では,母が非肥満の群で糖代謝異常出現率が高値であった. (6) HNF-1α遺伝子変異は認めなかったが,既知の遺伝子多型が非糖尿病者における頻度に比し高率の傾向にあった.糖尿病母体出生児の糖代謝異常にHNF-1α遺伝子の関与は示されなかった.今後も対象を増やし,父方の関与についてもさらに検討する必要がある.A study on genetic and environmental factors has been carried out in the 134 offspring of diabetic mothers (50 from type 1 and 84 from type 2), who had been followed up for 1 to 10 years. Offspring were judged to be normal (N), diabetic (D), and borderline (B) by results of glucose torelance test. A search for mutations in the hepatocyte nuclear factor (HNF)-1α gene was conducted using the PCR direct sequencing method. (1) Diabetes and abnormal glucose tolerance was found 3.0% and 32.1% in offspring, respectively. (2) The mean age abnormal glucose tolerance discovered in the offspring was 11.4 to 14.4 years. (3) The rank of mean plasma glucose values during pregnancy in both mothers with type 1 and type 2 diabetes, in descending order, by offspring group was D>B >N. (4) There was a significant positive correlation between maternal body mass index in the pre-pregnant state and the maximum degree of obesity of the offspring. (5) Among the obese offspring, development of abnormal glucose tolerance was significantly more common in the offspring of non-obese mothers than in those of obese mothers. (6) No HNF-1α gene abnormalities were observed in any of the patients

簡便な成熟ブタ膵内分泌細胞分離法の検討

膵細胞移植に際しては,大量かつ高純度の膵内分泌細胞を得ることが重要である.本研究では,リンパ球分離溶液を用いた簡便な成熟ブタ膵内分泌細胞の分離法を検討した.屠殺場より入手した成熟ブタ膵を細切・攪拌した後,細胞懸濁液を遠心分離し単離肝細胞を収集した.これをリンパ球分離溶液であモノポリ分離溶液(mono-poly resolving medium: MPRM)を用いて,膵内分泌細胞を外分泌細胞および血管内皮細胞,血液細胞などより分離・精製した.得られた膵分泌細胞数を計測し,形態学的・機能的観察を行った.その結果,(1)得られた膵内分泌細胞数:3.40±1.32×10^5/gのうち,ジチゾン染色陽性細胞数は2.81±1.09×10^5/gで,純度は82.6±2.5%であった.(2)免疫組織化学染色では60%がB細胞であった.(3)電顕では,典型的な分泌顆粒を有するB細胞,A細胞が認められた.(4)グルコース負荷試験では,分離直後のインスリン分泌能低下は1週間の培養で改善し,また40日間の培養中インスリンの分泌が保たれた.成熟ブタ膵内分泌細胞径は,ヒトリンパ球径に類似しており,MPRMを用いた1回の遠心操作で,膵分泌細胞は明瞭に分離され安定した細胞数が得られた.また,膵内分泌細胞の構成は膵島におけるそれと類似しており,超微細構造も保たれていた.以上より,MPRMを用いた膵内分泌細胞の分離法は,簡便で安定した細胞数が得られ,その形態・機能とも良好で有用な方法であると考えられた.Adult pig pancreatic endocrine cells were harvested by auto-digestion without added enzymes. The isolated, crude cells were purified by Mono-poly resolving medium (MPRM). The purity of the harvested cells was determined by dithizone staining and the number of pancreatic endocrine cells was counted. A large number of the cells were stained red with dithizone and showed high viability and a good insulin secretory response to glucose stimulation. The average number of cells purified by MPRM was 3.40 ± 1.32 × 10^5 cells/g pancreas and the number of dithizone-stained cells was 2.81 ± 1.09 × 10^5 cells/g pancreas. The insulin secretion from the pancreatic endocrine cells was maintained throughout a 40-day observation period and high glucose stimulation induced an increase in insulin secretion from the cultured cells. In the cells purified by MPRM, light and electron microscopic studies showed the cells to be typical pancreatic endocrine cells. The present purification method using MPRM allowed us to obtain quickly a large amount of adult pig pancreatic endocrine cells from the unpurified preparations. This is useful for transplantation and biochemical or biological studies of adult pig pancreatic endocrine cells

簡便な成熟ブタ膵内分泌細胞分離法の検討

膵細胞移植に際しては,大量かつ高純度の膵内分泌細胞を得ることが重要である.本研究では,リンパ球分離溶液を用いた簡便な成熟ブタ膵内分泌細胞の分離法を検討した.屠殺場より入手した成熟ブタ膵を細切・攪拌した後,細胞懸濁液を遠心分離し単離肝細胞を収集した.これをリンパ球分離溶液であモノポリ分離溶液(mono-poly resolving medium: MPRM)を用いて,膵内分泌細胞を外分泌細胞および血管内皮細胞,血液細胞などより分離・精製した.得られた膵分泌細胞数を計測し,形態学的・機能的観察を行った.その結果,(1)得られた膵内分泌細胞数:3.40±1.32×10^5/gのうち,ジチゾン染色陽性細胞数は2.81±1.09×10^5/gで,純度は82.6±2.5%であった.(2)免疫組織化学染色では60%がB細胞であった.(3)電顕では,典型的な分泌顆粒を有するB細胞,A細胞が認められた.(4)グルコース負荷試験では,分離直後のインスリン分泌能低下は1週間の培養で改善し,また40日間の培養中インスリンの分泌が保たれた.成熟ブタ膵内分泌細胞径は,ヒトリンパ球径に類似しており,MPRMを用いた1回の遠心操作で,膵分泌細胞は明瞭に分離され安定した細胞数が得られた.また,膵内分泌細胞の構成は膵島におけるそれと類似しており,超微細構造も保たれていた.以上より,MPRMを用いた膵内分泌細胞の分離法は,簡便で安定した細胞数が得られ,その形態・機能とも良好で有用な方法であると考えられた.Adult pig pancreatic endocrine cells were harvested by auto-digestion without added enzymes. The isolated, crude cells were purified by Mono-poly resolving medium (MPRM). The purity of the harvested cells was determined by dithizone staining and the number of pancreatic endocrine cells was counted. A large number of the cells were stained red with dithizone and showed high viability and a good insulin secretory response to glucose stimulation. The average number of cells purified by MPRM was 3.40 ± 1.32 × 10^5 cells/g pancreas and the number of dithizone-stained cells was 2.81 ± 1.09 × 10^5 cells/g pancreas. The insulin secretion from the pancreatic endocrine cells was maintained throughout a 40-day observation period and high glucose stimulation induced an increase in insulin secretion from the cultured cells. In the cells purified by MPRM, light and electron microscopic studies showed the cells to be typical pancreatic endocrine cells. The present purification method using MPRM allowed us to obtain quickly a large amount of adult pig pancreatic endocrine cells from the unpurified preparations. This is useful for transplantation and biochemical or biological studies of adult pig pancreatic endocrine cells