颌下腺细胞与丝素-壳聚糖在体内复合培养的动物实验

Experimental study on rad submandibular gland cell and silk fibroin-chitosan in vivo

目的探讨在丝素一壳聚糖（silk fibroin-chitosan，SFCS）支架上接种颌下腺细胞后，在大鼠体内构建组织工程化颌下腺的可行性及生物学特性。方法将36只8周龄SD大鼠分成实验组和对照组，每组各18只。取1～3d龄sD大鼠颌下腺原代细胞培养、传代、细胞来源鉴定后，将第二代细胞标记5-溴-2-脱氧尿嘧啶核苷（5’-BrdU），制备成5×10’个／L的颌下腺细胞悬液，将其接种于5mm×5mm×5mm的SFCS上，体外培养1周后将细胞一支架复合物植入实验组，将单纯SFCS支架植入对照组，分别于3、7、14d处死大鼠后取材，进行大体、组织学、免疫组织化学及扫描电镜观察。结果大体观察：植人物表面被膜覆盖，周围组织反应较轻。组织学观察：随着培养时间延长细胞数逐渐增多，细胞黏附于材料表面或分散在支架内，聚集成集落、呈岛状排列，分泌粉染的细胞外基质。BrdU免疫组织化学观察：术后3个时间点均存在阳性细胞。细胞角蛋白8（cytokeratin 8，CK-8）免疫组织化学观察：术后3个时间点均存在阳性细胞，随时间延长数目逐渐增多。扫描电镜观察：SFCS呈网状结构，植入初期的颌下腺细胞呈圆形、表面光滑、散在的黏附于SFCS表面或网孔内；随时间延长，颌下腺细胞表面凹凸不平，细胞数目增多，黏附于材料表面并伸出突起，细胞间相互接触。结论利用SFCS支架接种颌下腺细胞后，可在体内构建组织T程化涎腺样结构，为进一步进行组织工程化颌下腺的研究奠定理论基础。

Objective To evaluate the feasibility of the tissue engineered submandibular gland constructed in vivo based on submandibular gland cells and silk fibroin-chitosan （SFCS）. Methods Submandibular gland cells were obtained and purified. The second generation cells labeled by 5 ＇-BrdU were seeded on SFCS（5 mm ×5 mm× 5 mm）. Submandibular gland cells seeded on SFCS was implanted beneath the skin on the back. At 3, 7, 14 d post-implantation, implant sites were examined local and systemic responses. After paraffin embedding, serial sections 6 mm thick were cut and stained with either hematoxylin and eosin or brdu tissue stain for immunohistochemical studies and examined the responses of tissue. Scanning electron microscope was used to observe the growth behavior submandibular gland cells on SFCS scaffolds. Results General observation： at the 3, 7, 14 d after in vivo implantation, capsule formed in the surface of insert. Histological observation： in experimental group, submandibular gland cells proliferate on the SFCS scaffold fused to form unit 14 d after implantation. Brdu immunohistochemical observation： the resuhs of labelled cells were positive by immunohistochemical method at each time point. Cytokeratin-8 （ CK- 8） immunohistochemical observation： the results of labelled cells were positive by immunohistochemical method at each time point. With time, the positive cells gradually increased. Scanning electron microscope： the shape of the SFCS scaffold was mesh. At earlier, submandibular gland cells presence in disorder at attach to the SFCS. At the 14 d submandibular gland cells proliferate on the SFCS scaffold and form functional unit. Conclusions Such constructed tissue engineered submandibular gland based on submandibular gland cells and SFCS is promising.