體外重建外泌汗腺的初步研究
發(fā)布時(shí)間：2019-03-29 08:42
【摘要】： 目的:1、體外培養(yǎng)的汗腺腺上皮細(xì)胞分別接種在Matrigel下表面、上表面、以及Matrigel中,探討以Matrigel為主要基質(zhì)構(gòu)建汗腺樣結(jié)構(gòu)的方法。2、對(duì)已構(gòu)建的汗腺樣結(jié)構(gòu)進(jìn)行鑒定,探討其在形態(tài)學(xué)、組織學(xué)上與正常汗腺的相似性。3、通過(guò)對(duì)鈣離子激動(dòng)劑和阻滯劑對(duì)體外重建的汗腺樣結(jié)構(gòu)鈣通道的影響作用的研究,探討其在功能上與正常汗腺的相似性。 方法:1、將酶消化法分離培養(yǎng)的原代汗腺腺上皮細(xì)胞分別接種在Matrigel下表面、上表面以及Matrigel中,倒置相差顯微鏡下觀察細(xì)胞的生長(zhǎng)情況。2、用倒置相差顯微鏡、激光掃描共聚焦顯微鏡、計(jì)算機(jī)三維重建、電鏡、HE染色及免疫組織化學(xué)法觀察汗腺樣結(jié)構(gòu)的形態(tài)學(xué)及組織學(xué)特性。3、鈣熒光探針Fluo-3對(duì)汗腺樣結(jié)構(gòu)染色,分別在汗腺樣結(jié)構(gòu)中適時(shí)加入鈣離子激動(dòng)劑(氯化乙酰膽堿)和阻滯劑(阿托品),激光共聚焦顯微鏡觀察其細(xì)胞內(nèi)游離鈣離子濃度變化情況。 結(jié)果:1、汗腺腺上皮細(xì)胞接種在Matrigel下表面,立體培養(yǎng)11d形成腔面結(jié)構(gòu),但細(xì)胞增殖不明顯;當(dāng)接種在Matrigel上表面時(shí),立體培養(yǎng)8h,細(xì)胞胞漿伸長(zhǎng)呈絲狀,與鄰近細(xì)胞相連呈腔面或半腔面形;而接種在Matrigel中,三維立體培養(yǎng)2~3d,細(xì)胞分裂增生,增生細(xì)胞彼此緊密排列成立體管腔結(jié)構(gòu),中間可見(jiàn)明顯腔隙,隨新生細(xì)胞增多,管腔結(jié)構(gòu)逐漸演變?yōu)椴灰?guī)則球形結(jié)構(gòu)。2、激光掃描共聚焦顯微鏡觀察示細(xì)胞團(tuán)呈球形結(jié)構(gòu),中央有管腔結(jié)構(gòu),細(xì)胞排列富有極性。計(jì)算機(jī)軟件三維重建后從不同角度觀察,細(xì)胞團(tuán)呈不規(guī)則球形結(jié)構(gòu)。透射電鏡下可見(jiàn)含有大量高電子致密度顆粒的細(xì)胞,以及相對(duì)含有較少高電子致密度顆粒、但含有較多線粒體的細(xì)胞;細(xì)胞彼此排列形成管腔結(jié)構(gòu),其連接處可見(jiàn)指狀突起以及類似分泌小管的腔隙。球形結(jié)構(gòu)HE染色示其剖面為單層細(xì)胞相互連接形成環(huán)狀結(jié)構(gòu),中間見(jiàn)明顯的腔隙,細(xì)胞呈錐形,胞質(zhì)嗜酸性,淡染,胞核藍(lán)染,稍大,偏向基底部。免疫組織化學(xué)染色示CK18、CEA表達(dá)陽(yáng)性。3、細(xì)胞外液鈣濃度高時(shí),加入氯化乙酰膽堿后,鈣通道開(kāi)放,細(xì)胞外鈣內(nèi)流,細(xì)胞內(nèi)鈣濃度明顯增加;細(xì)胞外液鈣濃度高時(shí),先加入阿托品,5分鐘后加入氯化乙酰膽堿,細(xì)胞內(nèi)鈣濃度無(wú)明顯變化。 結(jié)論:體外培養(yǎng)的汗腺腺上皮細(xì)胞接種在Matrigel中,能形成汗腺樣結(jié)構(gòu);所形成的汗腺樣結(jié)構(gòu),在形態(tài)學(xué)、組織學(xué)及功能上都與在體汗腺相似,這為體外重建汗腺以及進(jìn)一步研究汗腺的功能提供了重要實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
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