组织工程颌下腺细胞与胶原海绵复合培养的实验研究

目的研究大鼠颌下腺细胞在胶原海绵材料支架上的生长情况。方法取8d龄Wistar大鼠颌下腺细胞，传代培养至第2代细胞，按2×10^4／ml注射法接种于5mm×5mm×2mm胶原海绵表面进行培养。术后1、2、3周行组织学观察、扫描电镜观察胶原海绵上接种颌下腺细胞形态结构特点；取复合培养3周的颌下腺细胞行免疫组织化学细胞角蛋白（cytokratin8．13，CK8．13）、α-平滑肌肌动蛋白（α-smooth muscular actin，α-SMA）染色，透射电镜观察细胞超微结构，鉴定细胞来源。分别取复合培养1d，1、2、3周的培养上清，用Amano法测定淀粉酶含量，检测与胶原海绵复合培养的颌下腺细胞功能。结果细胞接种后第1周细胞分散于材料表面，无细胞突起形成；第2周细胞数量有所增加、细胞突起形成并锚定于胶原海绵表面；第3周时附着的细胞数目增多，可见细胞表层有丝状纤维形成。免疫组织化学染色观察复合培养的颌下腺细胞上皮细胞特异性抗体CK8。13呈强阳性，肌上皮细胞特异性抗体α-SMA染色呈阳性。透射电镜下颌下腺上皮细胞表面可见微绒毛、胞浆皱褶和细胞顶部的酶原颗粒，胞核大卵圆形，胞质内见线粒体和粗面内质网。随着接种后培养时间的延长，与胶原海绵复合培养的颌下腺细胞分泌的淀粉酶含量有不同程度的增加。结论胶原海绵具有良好的细胞相容性，颌下腺细胞与胶原海绵复合培养，细胞可保持增殖分化能力及分泌功能，有望成为颌下腺细胞的载体应用于组织工程支架材料。

AN EXPERIMENTAL RESEARCH OF TISSUE ENGINEERED SUBMANDIBULAR GLAND CELLS GROWING ON COLLAGEN SPONGE SCAFFOLD

OBJECTIVE: To make an experimental research of the tissue engineered rat submandibular glands (SMG) cells growing on a collagen sponge scaffold under an optimal culture condition. METHODS: The Wistar rat (8 days old) SMG cells of the second generation were seeded on the surface of the collagen sponge scaffold (5 mm X 5 mm x 2 mm) and were cultured under a physiologically optimal condition for 3 weeks. At 1, 2 and 3 weeks, the cultured cells were observed on their shapes and structures by the histological examination and the scanning electron microscopy. The cultured cells underwent the immunohistochemistry research (the cytokratin 8.13, CK8. 13; alpha-smooth muscular actin, alpha-SMA) staining performed at 3 weeks of the culture, and the amylase activity analysis (the Amano method) performed at 1 day, 1, 2 and 3 weeks of the culture for an evaluation on the secretion function of the cells; the ultrastructures of the cells were also observed by the transmission electron microscopy for an identification of their origins. RESULTS: The observation under the scanning electron microscope showed that at 1 week after the cell-seeding, the seeded cells were attached to the collagen sponge scaffold surface, with no cell process formed; at 2 weeks the cells increased, with formation of the cell process that was anchored on the collagen sponge scaffold surface; and at 3 weeks, the scaffold surface-attached cells increased, with formation of the filiform fibers in the surface layer of the cells. The immunohistochemistry staining showed that the cultured epithelial cells of SMG were strongly positive for the specific antibody of CK8. 13, and the myoepithelial cells were positive for the specific antibody of alpha-SMA. The transmission electron microscopy showed that in the surface layer of the cultured epithelial cells of SMG the microvilli, plasm crease, and zymogen granules were observed, with a big and oval-shaped nucleus in the cell, and mitochondria and rough endoplasmic reticulum in the cytoplasm of the cell. The amount of amylase secreted by the cells cultured with the collagen sponge scaffolds increased at a different degree with an extension of the culturing time. CONCLUSION: The collagen sponge has a satisfactory cell compatibility, and the SMG cells cultured with this kind of collagen sponge can keep their abilities of proliferation and differentiation and their function of secretion. Therefore, this kind of cultured SMG cells can be used as the tissue-engineered cells seeded in the scaffold.