体外重建外泌汗腺的初步研究
发布时间：2019-03-29 08:42
【摘要】： 目的:1、体外培养的汗腺腺上皮细胞分别接种在Matrigel下表面、上表面、以及Matrigel中,探讨以Matrigel为主要基质构建汗腺样结构的方法。2、对已构建的汗腺样结构进行鉴定,探讨其在形态学、组织学上与正常汗腺的相似性。3、通过对钙离子激动剂和阻滞剂对体外重建的汗腺样结构钙通道的影响作用的研究,探讨其在功能上与正常汗腺的相似性。 方法:1、将酶消化法分离培养的原代汗腺腺上皮细胞分别接种在Matrigel下表面、上表面以及Matrigel中,倒置相差显微镜下观察细胞的生长情况。2、用倒置相差显微镜、激光扫描共聚焦显微镜、计算机三维重建、电镜、HE染色及免疫组织化学法观察汗腺样结构的形态学及组织学特性。3、钙荧光探针Fluo-3对汗腺样结构染色,分别在汗腺样结构中适时加入钙离子激动剂(氯化乙酰胆碱)和阻滞剂(阿托品),激光共聚焦显微镜观察其细胞内游离钙离子浓度变化情况。 结果:1、汗腺腺上皮细胞接种在Matrigel下表面,立体培养11d形成腔面结构,但细胞增殖不明显;当接种在Matrigel上表面时,立体培养8h,细胞胞浆伸长呈丝状,与邻近细胞相连呈腔面或半腔面形;而接种在Matrigel中,三维立体培养2~3d,细胞分裂增生,增生细胞彼此紧密排列成立体管腔结构,中间可见明显腔隙,随新生细胞增多,管腔结构逐渐演变为不规则球形结构。2、激光扫描共聚焦显微镜观察示细胞团呈球形结构,中央有管腔结构,细胞排列富有极性。计算机软件三维重建后从不同角度观察,细胞团呈不规则球形结构。透射电镜下可见含有大量高电子致密度颗粒的细胞,以及相对含有较少高电子致密度颗粒、但含有较多线粒体的细胞;细胞彼此排列形成管腔结构,其连接处可见指状突起以及类似分泌小管的腔隙。球形结构HE染色示其剖面为单层细胞相互连接形成环状结构,中间见明显的腔隙,细胞呈锥形,胞质嗜酸性,淡染,胞核蓝染,稍大,偏向基底部。免疫组织化学染色示CK18、CEA表达阳性。3、细胞外液钙浓度高时,加入氯化乙酰胆碱后,钙通道开放,细胞外钙内流,细胞内钙浓度明显增加;细胞外液钙浓度高时,先加入阿托品,5分钟后加入氯化乙酰胆碱,细胞内钙浓度无明显变化。 结论:体外培养的汗腺腺上皮细胞接种在Matrigel中,能形成汗腺样结构;所形成的汗腺样结构,在形态学、组织学及功能上都与在体汗腺相似,这为体外重建汗腺以及进一步研究汗腺的功能提供了重要实验依据。
[Abstract]:Objective: 1. The sweat gland epithelial cells cultured in vitro were inoculated into the lower surface, upper surface and Matrigel of Matrigel, respectively, to explore the method of constructing sweat gland-like structure with Matrigel as the main matrix. 2, to identify the constructed sweat gland-like structure. To investigate the morphological and histological similarities between the two groups and the normal sweat glands. 3. To explore the functional similarity between the calcium channel and the normal sweat gland by studying the effects of calcium agonists and blockers on the calcium channels of the reconstructed sweat gland-like structure in vitro. Methods: 1. The primary sweat gland epithelial cells isolated by enzyme digestion were inoculated into the lower surface of Matrigel, the upper surface of sweat gland and Matrigel respectively, and the growth of the cells was observed under inverted phase contrast microscope. 2, the growth of the cells was observed by inverted phase contrast microscope, and the growth of the cells was observed by inverted phase contrast microscope. Laser scanning confocal microscope, computer three-dimensional reconstruction, electron microscope, HE staining and immunohistochemistry were used to observe the morphological and histological characteristics of sweat gland-like structure. 3. The perspiration gland-like structure was stained by calcium fluorescence probe Fluo-3. Calcium agonist (acetylcholine chloride) and blocker (atropine) were added to the sweat gland-like structure respectively. The changes of intracellular free calcium concentration were observed by laser confocal microscope. Results: (1) the sweat gland epithelial cells were inoculated on the surface of Matrigel and cultured in three-dimensional culture for 11 days to form the cavity surface structure, but the proliferation of the cells was not obvious; When the cells were inoculated on the surface of Matrigel, the cytoplasmic elongation of the cells was filamentous and connected with the adjacent cells in the shape of cavity surface or semi-lumen surface after 8 hours of stereoscopic culture. On the other hand, when cultured in Matrigel for 2 ~ 3 days, the cells were divided and proliferated, and the hyperplastic cells were arranged closely into three-dimensional lumen structure with obvious lacunae in the middle, and the lumen structure gradually evolved into irregular spherical structure with the increase of the number of new-born cells. Laser scanning confocal microscope (LSCM) showed that the cells were spherical in shape, lumen in the center, and polar in the arrangement of cells. After three-dimensional reconstruction with computer software, the cell mass was observed from different angles, and the cell mass was irregular spherical structure. Cells containing a large number of high electron dense particles and relatively few high electron dense particles but more mitochondria were observed under transmission electron microscope. Cells arrange each other to form lumen structure, where finger-shaped processes and lacunae similar to secretory tubules can be seen. The HE staining of spherical structure showed that the single layer cells connected with each other to form a ring structure in the profile, and there were obvious lacunae in the middle. The cells were conical, eosinophilic, light stained, blue stained, slightly larger and inclined to the base of the base. The expression of CK18,CEA was positive by immunohistochemical staining. 3. When the concentration of calcium in extracellular fluid was high, calcium channel opened, extracellular calcium influx and intracellular calcium concentration increased significantly after adding acetylcholine chloride. When calcium concentration in extracellular fluid was high, atropine was added first, and acetylcholine chloride was added 5 minutes later, the intracellular calcium concentration had no obvious change. Conclusion: the eccrine gland epithelial cells cultured in vitro can form sweat gland-like structure when inoculated in Matrigel. The morphology, histology and function of the sweat glands are similar to those of the in vivo sweat glands, which provides an important experimental basis for the reconstruction of the sweat glands in vitro and the further study of the function of the sweat glands.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2449353
[Abstract]:Objective: 1. The sweat gland epithelial cells cultured in vitro were inoculated into the lower surface, upper surface and Matrigel of Matrigel, respectively, to explore the method of constructing sweat gland-like structure with Matrigel as the main matrix. 2, to identify the constructed sweat gland-like structure. To investigate the morphological and histological similarities between the two groups and the normal sweat glands. 3. To explore the functional similarity between the calcium channel and the normal sweat gland by studying the effects of calcium agonists and blockers on the calcium channels of the reconstructed sweat gland-like structure in vitro. Methods: 1. The primary sweat gland epithelial cells isolated by enzyme digestion were inoculated into the lower surface of Matrigel, the upper surface of sweat gland and Matrigel respectively, and the growth of the cells was observed under inverted phase contrast microscope. 2, the growth of the cells was observed by inverted phase contrast microscope, and the growth of the cells was observed by inverted phase contrast microscope. Laser scanning confocal microscope, computer three-dimensional reconstruction, electron microscope, HE staining and immunohistochemistry were used to observe the morphological and histological characteristics of sweat gland-like structure. 3. The perspiration gland-like structure was stained by calcium fluorescence probe Fluo-3. Calcium agonist (acetylcholine chloride) and blocker (atropine) were added to the sweat gland-like structure respectively. The changes of intracellular free calcium concentration were observed by laser confocal microscope. Results: (1) the sweat gland epithelial cells were inoculated on the surface of Matrigel and cultured in three-dimensional culture for 11 days to form the cavity surface structure, but the proliferation of the cells was not obvious; When the cells were inoculated on the surface of Matrigel, the cytoplasmic elongation of the cells was filamentous and connected with the adjacent cells in the shape of cavity surface or semi-lumen surface after 8 hours of stereoscopic culture. On the other hand, when cultured in Matrigel for 2 ~ 3 days, the cells were divided and proliferated, and the hyperplastic cells were arranged closely into three-dimensional lumen structure with obvious lacunae in the middle, and the lumen structure gradually evolved into irregular spherical structure with the increase of the number of new-born cells. Laser scanning confocal microscope (LSCM) showed that the cells were spherical in shape, lumen in the center, and polar in the arrangement of cells. After three-dimensional reconstruction with computer software, the cell mass was observed from different angles, and the cell mass was irregular spherical structure. Cells containing a large number of high electron dense particles and relatively few high electron dense particles but more mitochondria were observed under transmission electron microscope. Cells arrange each other to form lumen structure, where finger-shaped processes and lacunae similar to secretory tubules can be seen. The HE staining of spherical structure showed that the single layer cells connected with each other to form a ring structure in the profile, and there were obvious lacunae in the middle. The cells were conical, eosinophilic, light stained, blue stained, slightly larger and inclined to the base of the base. The expression of CK18,CEA was positive by immunohistochemical staining. 3. When the concentration of calcium in extracellular fluid was high, calcium channel opened, extracellular calcium influx and intracellular calcium concentration increased significantly after adding acetylcholine chloride. When calcium concentration in extracellular fluid was high, atropine was added first, and acetylcholine chloride was added 5 minutes later, the intracellular calcium concentration had no obvious change. Conclusion: the eccrine gland epithelial cells cultured in vitro can form sweat gland-like structure when inoculated in Matrigel. The morphology, histology and function of the sweat glands are similar to those of the in vivo sweat glands, which provides an important experimental basis for the reconstruction of the sweat glands in vitro and the further study of the function of the sweat glands.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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