Telocytes accompanying cardiomyocyte in primary culture: two‐ and three‐dimensional culture environment
Jin ZhouYe ZhangXinyu WenJunkai CaoDexue LiQiuxia LinHaibin WangZhiqiang LiuCuimi DuanKuiwu WuChangyong Wang
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Abstract:
Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra-digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two- and three-dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two-dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three-dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c-kit or CD34 immuno-positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three-dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non-muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.Keywords:
Desmin
Interstitial cell
뼈대근육은 손상의 요인에 따라 근섬유의 변성, 위축 및 재생시 조직화학적 변화가 다양하다. 뼈대근육에 물리적 손상을 주면 근섬유들은 변성과 동시에 재생이 일어난다. 근육의 재생과정에서 중요한 역할을 하는 것이 근육의 세포뼈대를 이루는 중간미세섬유이다. 이 중 desmin은 미성숙 근육섬유에서 더욱 강하게 발현되며 vimentin은 근육섬유의 재생과정이나 미성숙상태라는 것을 의미하므로 많은 연구에서 다루어져 왔다. 그러나 물리적 근손상후 재생과정일 때 운동이 미치는 영향을 desmin과 vimentin의 발현 여부로 추적한 연구는 흔치 않다. 따라서 이 연구에서는 생후 1개월 된 흰쥐 장딴지근에 30gauze needle로 200번을 찌르는 물리적 손상을 준 다음 운동을 시키지 않은 대조군과 트레드밀을 이용해 일정한 기간 (5, 10, 15, 20일)동안 운동을 시킨 실험군을 9,16, 23, 30일째에 근육 조직을 절취하여 면역조직화학적인 방법으로 중간미세섬유단백을 관찰하였다. 운동을 시킨 실험군이 대조군보다 근육재생이 빠르게 진행되었다. Desmin은 근섬유의 변성과 재생과정 모두에서 발현되었으며 vimentin은 재생과정에서만 발현되었다. 근손상후 근육조직을 절취한 9일의 대조군은 심하게 괴사된 근섬유를 보였고, 실험군에서는 재생이 진행되고 있는 근섬유가 관찰되었다. 16일의 대조군에서 근섬유가 재생됨이 관찰되었고, 실험군은 재생이 더욱 빨리 진행되고 있었다. 23일의 대조군에서 desmin과 vimentin 양성반응근섬유가 많이 관찰되나 실험군의 근섬유에서는 거의 관찰되지 않았다. 30일의 대조군은 형태적으로는 정상에 가까운 모습이나 vimentin이 발현되어 근육이 재생과정에 있는 것을 보여 주는 반면, 실험군 대부분은 vimentin 발현이 나타나지 않는 정상 근섬유로 회복되었다. 정상군에서 desmin은 매우 약한 양성반응이 나타났고, vimentin에 대한 양성 면역반응은 근섬유에서 나타나지 않았다. 근섬유의 형태학적인 변화는 30일에서 실험군의 변화는 정상에 가까운 형태를 띠었으나, 대조군은 정상치에 달하지 못하였다. 근내막의 형태학적인 변화 역시 실험군이 대조
군보다 비대현상도 작았으며 30일에 정상치에 달하였다. 이상의 결과로 근 손상 후 운동을 실시한 실험군과 대조군은 근육재생속도의 차이점을 보여 운동이 근육재생에 효과적임을 알 수 있었다. 따라서 이 연구결과는 근육의 손상 및 재생을 연구하는 분야나 다양한 뼈대근육 손상에 대한 임상적인 적용에 도움을 줄 것으로 생각된다.
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In order to study which classes of intermediate filaments (IF) comprise the IF of human trabecular cells, we undertook immunocytochemical investigations on cultured human trabecular cells using anti-keratin, anti-vimentin, anti-desmin and anti-glial fibrillar acidic protein (GFAP) rabbit sera. Immunostaining with the keratin antibody and the GFAP antibody only produced nonspecific staining. Immunostaining with the vimentin antibody and the desmin antibody produced intracytoplasmic filamentous staining. Pretreatment of the cells with colcemid to induce the perinuclear concentration of IF resulted in the demonstration of a characteristic perinuclear immunofluorescence, confirming the existence of vimentin and desmin. Some cells showed particularly strong positivity to desmin. The desmin antibody used in this study only faintly labeled the IF of cultured human skin fibroblasts, known to contain vimentin but not desmin. These results suggest that the IF of human trabecular cells are composed of vimentin and desmin, and that human trabecular cells possess muscle cell-like functions.
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The intermediate filament proteins desmin and vimentin from pregnant and non-pregnant uterine muscle and smooth-muscle cells in culture were analysed using SDS/PAGE. The desmin content in uterine muscle increases dramatically during pregnancy, whereas vimentin remains unchanged or changes very little. When muscle cells are kept in culture, a considerable increase in vimentin content is observed as compared with vimentin in freshly isolated non-pregnant uterine tissue. Our results strengthen the view that vimentin and desmin filaments have independent function and turnover, and point to a predominantly structural role for desmin filaments.
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To differentiate cultured rat liver myofibroblasts, fat-storing cells, aortic smooth muscle cells and skin fibroblasts from each other, desmin and vimentin stainings were undertaken by indirect immunofluorescence using monoclonal antibodies. In myofibroblasts, the reaction with antibodies to vimentin was positive but that with antibodies to desmin was virtually negative. In primary cultures as well as subsequent passage of fat-storing cells, reactions with antibodies to both desmin and vimentin were positive. In primary culture of smooth muscle cells, both reactions were positive, but in the first passage, smooth muscle cells lost the reactivity with antibodies to desmin. Fibroblasts showed a positive reaction with antibodies to vimentin and a negative one with antibodies to desmin. Thus, immunohistochemistry of intermediate filaments allows for the differentiation between fat-storing cells, which are desmin- and vimentin-positive, and myofibroblasts or fibroblasts, which are desmin-negative but vimentin-positive. Smooth muscle cells are also vimentin-positive and become desmin-negative after the first passage.
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Myofibroblastoma of the breast is a recently described entity. Since its first description in 1987, less than 50 cases have been reported. We present the first (reported) myofibroblastoma to be detected as a non-palpable mass on a routine screening mammogram and emphasize the importance of not mis-diagnosing this rare cellular lesion as malignant on frozen section. Review of the literature demonstrates changes in the clinical presentation of myofibroblastomas. Once considered more common in men than in women, myofibroblastomas are now being reported with increasing frequency in women. The age at presentation is a decade earlier, and not surprizingly, the size of the earlier detected lesion is smaller. Recently four different cytoskeletal phenotypes (V, VA, VAD and VD) of myofibroblastomas have been described, depending upon the vimentin (V), actin (A), and desmin (D) immunoreactivity. Whereas vimentin reactivity is universal, actin and desmin immunoreactivity is variable, desmin being more frequently positive than actin. As more is known about the clinical behavior of myofibroblastomas, their rate of recurrence and malignant potential, if any, the relationship of the cytoskeletal content to prognosis may become clearer. Currently, complete immunohistochemical analysis and electron microscopic examination of this interesting breast lesion is recommended. List of abbreviations-Vimentin (V), actin (A), and desmin (D), vimentin and actin (VA), vimentin and desmin (VD), vimentin, actin and desmin (VAD).
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