<原著>軟骨細胞の分化における HSP70 の発現

To know the role of HSP70 in chondrocytes, HSP70 expressions under heat stress or in a nonstress condition were examined by using electrophoresis, immunohistochemistry, and in situ hybridization. As a result, HSP70 was observed in proliferating chondrocytes in the micro-mass cultures without heat stress. In this culture, chondrocytes maintained the terminal differentiation potency. On the other hand, HSP70 did not appear in the chondrocytes in the logarithmic growth phase of the monolayer culture. In growth plates in vivo, HSP70 expressions in the chondrocytes located in the resting and hypertrophic zones were observed with immunohistochemistry. Appearance of HSP70 mRNA was also confirmed by in situ hybridization in the proliferating zone of growth plate. HSP70 can be expressed not only in chondrocytes under heat stress but also in the cells without stress, and the expression would be related to the terminal differentiation of chondrocytes. HSP70 is surmised to promote hypertrophy and calcification by stopping protein synthesis in chondrocytes which possess terminal differentiation potency.軟骨細胞との関連が注目されている熱ショック蛋白質の発現を, 遠沈管培養軟骨細胞と骨端軟骨板を用いて, in vitro および in vivo で検索した. 温熱ストレス下のラﾂト軟骨細胞では, 少なくとも3種類の熱ショック蛋白質の発現がみとめられ, そのうち HSP70 の誘導量が最大であった. 非ストレス下では, 遠沈管培養軟骨細胞のうち肥大軟骨細胞様細胞への分化能を有すると考えられる細胞に HSP70 の発現が認められた骨端軟骨板においては, 肥大軟骨細胞で HSP70 が誘導されていた. また, HSP70 mRNA は肥大層に隣接する増殖層および静止層の軟骨細胞で観察された. このことは, 肥大軟骨細胞への分化能を有する軟骨細胞では, すでに HSP70 mRNA が誘導されていることを示している. これらの結果から, HSP70 の発現が軟骨細胞の分化と関係していることが推察された

注射に起因する橈骨神経麻痺の2例

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<原著>培養軟骨細胞の分化機能発現と細胞増殖動態に関する実験的研究

The present study was undertaken to investigate the relationship among cell morphology, proliferation, and maturation of chondrocytes in primary cultures. Chondrocytes were isolated from the growth cartilages of the rat ribs and cultured for 6 days. In situ DNA cytofluorometry using an inverted epi-illumination cytofluorometer (Nikon P1-I) and 3H-thymidine autoradiography were carried out for the correlated analysis of cell morphology and proliferation. Cytoskeletal staining with fluorescent phalloidin and 35S-sulphate autoradiography were also performed. In addition, in situ hybridization to c-myc mRNA was carried out using DNA probe. According to the results obtained, the cultured chondrocytes were composed of mixed populations of large, polygonal cells and of small, round cells. The round cells showed a significantly higher 35S uptake than the polygonal cells. The cytoskeletal staining clearly revealed stress fibers in the cytoplasm of the polygonal cells, whereas only a fine filamentous structure was shown in the cytoplasm of the round cells. In situ DNA cytofluorometry clearly demonstrated that cell proliferative activity was high in the polygonal cells and low in the round cells. In addition, 3H-thymidine autoradiography with cumulative labeling method revealed that the polygonal cells were changing into the small, round cells. C myc mRNA signals were detected in the cytoplasm of over a half of the round cells, whereas no evidence of c-myc expression were found in the polygonal cells. From these results, it appears that as the shape of the cultured chondrocytes shifts from polygonal to round, the cell proliferative activity decreases in association with cell differentiation. It was also suggested that c-myc mRNA is amplified in the well differentiated round chondrocytes, and not in the proliferative polygonal cells.従来の培養軟骨細胞を用いた研究から, 軟骨細胞の形態と分化機能発現の聞には, 関連性のあることが示されている. 著者らは, 成長軟骨細胞の培養系において細胞形態, 機能が明らかに異なっている2種類の細胞が存在することを見いだした. 本研究では, この培養系を用い, 軟骨細胞の形態・細胞増殖動態・分化機能発現の3者の関連性を総合的に把握することを目的とした. このための方法論として, 細胞形態別増殖動態解析には, in situ DNA 顕微蛍光測光法と3_H-サイミジンオートラジオグラフィーを行い, 分化機能の検索には35_S オートラジオグラフィーを用いた. また, FITC-ファロイジン染色法により, 軟骨細胞の形態と細胞骨格の関係についても調べた. 更に, 本研究では, 悪性腫瘍以外に, 胎生期の細胞や分化途上の細胞にも出現し, 細胞の分化・増殖に深く関係があると考えられている c-myc 遺伝子の発現の有無を, in situ DNA- mRNA hybridization 法を用いて検索した. 実験には, ラット肋軟骨から分離・培養した成長軟骨細胞を用いた. 培養開始4 - 6日目頃の成長軟骨細胞は, 大型多角形の扁平な胞体を持ち, 大きな核を有する細胞(以下, 多角形細胞と略す)と, 比較的小型で類円形ないし球状の胞体と小さな核を有する細胞(以下, 円形細胞と略す)の2種類の細胞から構成されていた. in situ DNA 顕微蛍光測光法による細胞増殖動態解析の結果, 多角形細胞は, 活発な増殖性を示す2倍体細胞と少数の4倍体から構成されているのに対し, 円形細胞は, ほとんど増殖活性を持たない2倍体細胞から構成されていることが判った. 3_H-サイミジンの30分標識の結果から, 多角形細胞の標準率は11%, 円形細胞の標識率は0. 5%であり, その標識率の経時的変化はほとんど認められなかった. 3_H-サイミジンの持続標識実験の結果から, 多角形細胞が円形細胞に形態的に変化することが示唆された. また, 35_S オートラジオグラフィーより, 多角形細胞は, 軟骨基質の産生能が低く, 他方, 円形細胞では, 基質産生が亢進していることがわかった. FITC-ファロイジン染色によるアクチンの細胞内分布パタンを, 両細胞で比較したところ, 多角形細胞ではストレスファイバーがよく発達しているのに対し, 円形細胞には, 分断された線維性構造のみが観察された. 以上の結果をまとめると, 培養軟骨細胞の形態・増殖・分化の3者の間には, たがいに密接な関連が有り, 多角形細胞から円形細胞への形態変化に伴って, 増殖活性が低下し, 分化機能が発現されることが判明した. 次に, c-myc 遺伝子の発現の有無を in situ hybridization 法を用いて検索したところ, 円形細胞の過半数に, c-myc mRNA のシグナルが検出された. このことから, 軟骨細胞では, 分化機能発現と関連して c-myc 遺伝子が発現される可能性が示唆された

Analysis of heat shock proteins and cytokines expressed during early stages of osteoarthritis in a mouse model

SummaryObjective:Osteoarthritis (OA) is a debilitating disease of the joints. The joints of affected individuals are characterized by a progressive degeneration of articular cartilage leading to inflammation and pain. The expression of heat shock proteins (HSPs) is a ubiquitous self-protective mechanism of all cells under stress, furthermore, the synovium of osteoarthritic individuals contains high levels of cytokines. This study seeks to establish the role of HSPs and cytokines in OA.Methods:We have investigated the presence of HSPs and cytokines in articular cartilage during early stages of OA in a mouse that is known to develop spontaneous OA lesions (C57 black mouse). The articular cartilage from closely related mice (C57BL/6) was used as control. Messenger RNAs (mRNAs) for HSPs (HSP32, HSP47, HSP60, HSP70, HSP84 and HSP86) and cytokines [interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)] were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results:The mRNA levels of HSP47, HSP70, HSP86, IL-6, and IFN-γ were up-regulated in the cartilage of C57 black mice, whereas, the level of expression of HSP32, HSP60, HSP84 and IL-1β remained unchanged. Furthermore, the expression of IL-1β, IL-6, TNF-α and IFN-γ mRNA was associated with expression of HSP60, HSP47, HSP70 and HSP70/HSP86 mRNA, respectively.Conclusions:The findings in this study suggest that chondrocytes are conditioned under non-physiological stress during early stages of OA, In addition, among HSPs, HSP70 was associated with two different highly expressed cytokines in C57 black mice, indicating the possible role of HSP70 as a characteristic indicator of early stage of OA