Steroid sulfataseの腎細胞癌とその非腫瘍部組織における発現 : 免疫組織化学・酵素組織化学・in situ hybridizationによる検討

[目的]腎近位尿細管は,ホルモンの標的器官として注目されている.性ステロイドホルモンがrenal cell carcinoma (RCC)の発症原因になるか否かについての詳細はいまだ不明である.循環血液中に存在する性ステロイドホルモンは糸球体で濾過されないが,例えばestrogen sulfate (EIS)は糸球体で濾過され,近位尿細管で再吸収される.そこで,EISのステロール環3β硫酸基を,水酸基に置き換えるsteroid sulfatase (STS)の腎近位尿細管における局在についてRCC腎,健常腎において検討した.[方法]STS-peptideを合成し,polyclonal抗体を作製し,免疫組織化学法を行った.STS mRNAをdigoxigenin標識法で観察した.STS活性は4-methyl-umbelliferyl sulfateを基質とした組織化学的方法で検出した.[結果]免疫組織化学およびmRNAは近位尿細管を中心にRCC腎で陽性所見が見られた.STS活性は核膜,小胞体に局在した.活性は管腔側に強く発現した.[結論]硫酸基をもつEISは近位尿細管でSTSにより活性化され,受容体と結合してその作用を発現すると考えられた.性ステロイドホルモンは細胞の増殖分化発生に影響するため,RCC腎の近位尿細管におけるこのようなSTSの局在は興味深い.Purpose: Steroid hormones are known to be potential endogenous carcinogens. Estrogen receptors have been identified in the kidney, raising unresolved question about involvement of steroid sulfatase (STS), an enzyme that converts soluble estrogen (E1S) to active estrogen (E2), in induction of renal cell carcinoma (RCC). We studied the renal distribution of STS activity, proteins and mRNA in surgical and autopsy specimens with or without renal cell carcinoma. Materials and methods: Immunohistochemical staining for STS and in situ hybridization for the corresponding mRNA were performed. The STS enzyme activity was assessed by ultrastructural histochemistry after incubation in 4-methylumbelliferyl sulfate. Results: Immunoreactivity for STS protein was seen in nontumor tissue adjacent RCC and showed some weak staining in tumor tissue. The ultrastructural reaction product which indicates enzyme activity was not present in tumors but was seen in adjacent tissue mainly in the endoplasmic reticulum of proximal tubule cells. In situ hybridization demonstrated STS mRNA primarily in proximal tubules of renal tissue adjacent to tumors but not in tumor tissue. Conclusion: STS is synthesized in the proximal tubules of the human kidney, and augmented STS activity may be related to induction of RCC

Steroid sulfataseの腎細胞癌とその非腫瘍部組織における発現 : 免疫組織化学・酵素組織化学・in situ hybridizationによる検討

[目的]腎近位尿細管は,ホルモンの標的器官として注目されている.性ステロイドホルモンがrenal cell carcinoma (RCC)の発症原因になるか否かについての詳細はいまだ不明である.循環血液中に存在する性ステロイドホルモンは糸球体で濾過されないが,例えばestrogen sulfate (EIS)は糸球体で濾過され,近位尿細管で再吸収される.そこで,EISのステロール環3β硫酸基を,水酸基に置き換えるsteroid sulfatase (STS)の腎近位尿細管における局在についてRCC腎,健常腎において検討した.[方法]STS-peptideを合成し,polyclonal抗体を作製し,免疫組織化学法を行った.STS mRNAをdigoxigenin標識法で観察した.STS活性は4-methyl-umbelliferyl sulfateを基質とした組織化学的方法で検出した.[結果]免疫組織化学およびmRNAは近位尿細管を中心にRCC腎で陽性所見が見られた.STS活性は核膜,小胞体に局在した.活性は管腔側に強く発現した.[結論]硫酸基をもつEISは近位尿細管でSTSにより活性化され,受容体と結合してその作用を発現すると考えられた.性ステロイドホルモンは細胞の増殖分化発生に影響するため,RCC腎の近位尿細管におけるこのようなSTSの局在は興味深い.Purpose: Steroid hormones are known to be potential endogenous carcinogens. Estrogen receptors have been identified in the kidney, raising unresolved question about involvement of steroid sulfatase (STS), an enzyme that converts soluble estrogen (E1S) to active estrogen (E2), in induction of renal cell carcinoma (RCC). We studied the renal distribution of STS activity, proteins and mRNA in surgical and autopsy specimens with or without renal cell carcinoma. Materials and methods: Immunohistochemical staining for STS and in situ hybridization for the corresponding mRNA were performed. The STS enzyme activity was assessed by ultrastructural histochemistry after incubation in 4-methylumbelliferyl sulfate. Results: Immunoreactivity for STS protein was seen in nontumor tissue adjacent RCC and showed some weak staining in tumor tissue. The ultrastructural reaction product which indicates enzyme activity was not present in tumors but was seen in adjacent tissue mainly in the endoplasmic reticulum of proximal tubule cells. In situ hybridization demonstrated STS mRNA primarily in proximal tubules of renal tissue adjacent to tumors but not in tumor tissue. Conclusion: STS is synthesized in the proximal tubules of the human kidney, and augmented STS activity may be related to induction of RCC

老化促進マウス(SAM)の血管周囲マクロファージ内顆粒における加齢変化の促進

老化促進マウス(SAMP)の加齢変化は,対照群となるSAMRよりも早く現れることが知られている.著者らは,これまでラット脳血管周囲マクロファージが加齢とともに,その数や細胞形態において変化することを報告してきた.そこで,この研究では. SAMP8と対照群のSAMR1を用いて,脳の血管周囲マクロファージの電子密度の高い顆粒(dense granule)と泡沫状顆粒(foamy granule)について,それぞれの形態と数の変化について年齢を追って観察した.類粒は加齢とともにelectron-denseからfoamyに変わりdense granuleは減少する.逆にfoamy granuleは4～6ヵ月頃から急速に増え,さらに加齢とともに増える.以上の結果より,マクロファージ内のfoamy granuleへの変化は,老化の初期段階の兆候の一つであり,この兆候は,SAMP8においてSAMR1より,促進されていた.Both morphological and quantitative changes in electron-dense granules and foamy granules of the brain perivascular macrophages were investigated using senescence accelerated prone mice (SAMP) at different ages. Perivascular macrophages scavenge high molecular weight substances, such as low-density lipoprotein, incorporating them into granules in the cytoplasm. In this study, SAMPS and control SAMR1, 2-19 months of age, were examined to characterize these granules in perivascular macrophages of the brain. The number of granules were counted in animals of each age studied and were observed to change with aging from dense to foamy granules in perivascular macrophages. Dense granules gradually diminished starting after 2 months until 10 months postnatally, but foamy granules increased after 4-6 postnatal months. Foamy granules showed a quicker increase in SAMPS than in SAMR1 around 6 months of age. These results suggested that the morphological changes in granules of perivascular macrophages are characteristics of aging and more accelerated in senescence accelerated SAMPS, than in control SAMR1

老化促進マウス(SAM)の血管周囲マクロファージ内顆粒における加齢変化の促進

老化促進マウス(SAMP)の加齢変化は,対照群となるSAMRよりも早く現れることが知られている.著者らは,これまでラット脳血管周囲マクロファージが加齢とともに,その数や細胞形態において変化することを報告してきた.そこで,この研究では. SAMP8と対照群のSAMR1を用いて,脳の血管周囲マクロファージの電子密度の高い顆粒(dense granule)と泡沫状顆粒(foamy granule)について,それぞれの形態と数の変化について年齢を追って観察した.類粒は加齢とともにelectron-denseからfoamyに変わりdense granuleは減少する.逆にfoamy granuleは4～6ヵ月頃から急速に増え,さらに加齢とともに増える.以上の結果より,マクロファージ内のfoamy granuleへの変化は,老化の初期段階の兆候の一つであり,この兆候は,SAMP8においてSAMR1より,促進されていた.Both morphological and quantitative changes in electron-dense granules and foamy granules of the brain perivascular macrophages were investigated using senescence accelerated prone mice (SAMP) at different ages. Perivascular macrophages scavenge high molecular weight substances, such as low-density lipoprotein, incorporating them into granules in the cytoplasm. In this study, SAMPS and control SAMR1, 2-19 months of age, were examined to characterize these granules in perivascular macrophages of the brain. The number of granules were counted in animals of each age studied and were observed to change with aging from dense to foamy granules in perivascular macrophages. Dense granules gradually diminished starting after 2 months until 10 months postnatally, but foamy granules increased after 4-6 postnatal months. Foamy granules showed a quicker increase in SAMPS than in SAMR1 around 6 months of age. These results suggested that the morphological changes in granules of perivascular macrophages are characteristics of aging and more accelerated in senescence accelerated SAMPS, than in control SAMR1

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