表面修饰化材料对颌下腺种子细胞生长影响的研究

Experiment research on reconstituting tissue engineering submandibular gland with surface modification technique

目的探讨应用表面修饰技术处理的生物支架材料与颌下腺种子细胞复合培养研究对种子细胞在支架材料上的附着、生长和分泌功能的影响。方法选用明胶海绵36块,均分为3组。A组:明胶海绵材料与鼠颌下腺细胞复合培养物;B组:层黏连蛋白(laminin,LN)表面修饰的明胶海绵材料与鼠颌下腺细胞复合培养物;C组:转化生长因子(TGF-β3)表面修饰的明胶海绵材料与鼠颌下腺细胞复合培养物。各组在体外进行复合培养,3、7和15 d各时间点行组织学观察和扫描电镜观察,15 d行免疫组织化学鉴定细胞来源,测定上清淀粉酶含量并进行比较。结果组织学和扫描电镜观察见培养第3天各组细胞分散于材料表面,无细胞突起形成;第7天见B组细胞数量明显多于A组和C组,细胞突起形成并锚定于胶原海绵表面;第15天,B组细胞数量仍多于A组和C组,并可见细胞之间有触突联系和腺管样结构形成。免疫组织化学染色观察复合培养的颌下腺细胞Brdu呈强阳性。随着接种后培养时间的延长,各组颌下腺细胞分泌的淀粉酶含量均有不同程度的增加。15 d,C组淀粉酶含量明显高于A组和B组(P<0.05)。结论应用细胞外基质和细胞因子成分对生物支架材料进行表面修饰,可促进颌下腺种子细胞在材料上的附着、增殖和分泌,有利于组织工程化颌下腺研究的进一步成功。

【Objective】 To study reconstitute tissue engineering submandibular gland by implant rat submandibular gland cells（RSGCs） combined with collagen sponge scaffold with surface modification tech-nique.【Methods】36 collagen sponges were divided into three groups according surface modify material（group A： blank control;group B： LN（laminin,LN） surface modify collagen sponges;group C： TGF-β3（Transforming growth factor-β3,TGF-β3） surface modify collagen sponges）.RSGCs labeled by Brdu were seeded on collagen sponges（1 cm×1 cm×1 cm）.At 3,7 and 15 days post-culture,HE and Brdu immuno-histochemical examined the cultured cells.Scanning electron microscope was used to observe the growth behavior of RSGCs on collagen sponge scaffolds.Supernatant anaylsis were examined by conventional clin-ical chemistry analyses.【Results】 The observation under histology and scanning electron microscope showed： at 7th day,RSGCs start to adhere on collagen fiber in group B.At the 15th day,RSGCs can keep proliferating and differentiating on collagen fiber and form functional lumen unit in group B.In group C,the number of RSGCs is similar to group A,but less than group B.The amount of amylase secreted increased at a different degree with an extension of the culturing time in all the groups.At the 15th day after culture,the amount of amylase in group C is higher than the other two groups.【Conclusions】Collagen sponge scaffold with LN and TGF-β3 surface modified can stimulate RSGCs adhere,proliferate and differentiate on collagen fiber in vivo.Surface modification technique can used to reconstitute tissue engineering submandibular gland.