Preliminary Study On Three-dimensional Reconstruction Of Eccrine Sweat Glands In Vitro

Objective:1. The sweat glands epithelial cells were inoculated respectively on the upper surface,inside and under the lower surface of Matrigel in order to explore a method to reconstruct eccrine sweat gland-like structure in Matrige. 2. to identify the sweat gland-like structure restructured in vitro in order to explain its similarity with the normal sweat gland on biology. 3. To study the effects of agonists and antagonists of calcium ion on calcium channel of eccrine sweat gland-like structure in order to prove its function.Methods:1. The sweat glands epithelial cells isolated and cultured by enzymatic digestion were inoculated respectively on the upper surface,inside,and under the lower surface of Matrigel and then were observed with inverted phase contrast microscope. 2. the biological characteristics of the sweat gland-like structures were observed with laser scanning confocal microscopy,computer software,electron microscopy,HE staining and immunohistochemistry. 3. The agonists (Ach) and antagonists (Atropine) of calcium ion were added to the eccrine sweat gland-like structure timely.[Ca2+ ]i was examined by confocal laser scanning microscopy (SISM)with the Ca2+ sensitive dye Fluo-3.Results:1.when sweat gland epithelial cells had been cultured under the lower surface of Matrigel,the tubular structure formed after 11 days;but no significant proliferation could be observed.when sweat gland epithelial cells had been cultured on the upper surface of Matrigel,cytoplasm strethed to be filamentous shape and the adjacent cells were connected to show lumen or half-lumen after 8 hours.when sweat gland epithelial cells had been cultured in the three-dimensional Matrigel,cells divided and hyperplasied after 2~3 days,Proliferative cells were closely arranged in tubular structures, lacunar can be seen clearly in the middle of tubular structures,With the number of newborn cells,the tubular structure gradually developed into a irregular spherical structure.2.the center of cell mass existed lumen structure,the lumen structure was observed in the middle of spherical structure by CLSM, electron microscopy and HE staining and the CK18,CEA of spherical structure were positive expression with immunohistochemistry,the spherical structure were very similar to the Lumen structure of secretory portion of sweat gland.3.After Ach was added, opening of the calcium channel and significant[Ca2+ ]i increase were observed when the similarity eccrine sweat gland structure incubated with high concentration of calcium (2mmol/L),no significant [Ca2+ ]i increase was observed,Atropine hand been added before Ach was added.Conclusion:The sweat glands epithelial cells cultured in vitro and inoculated in Matrigel can form eccrine sweat gland-like structure and eccrine sweat gland-like structure restructured was very similar to the normal sweat gland on the morphology, histology, biology and function. The reconstruction in vitro and further study the function of sweat glands may provide an important experimental basis.