Stein Elisabeth, Blaimauer Karin, Bauer Sieglinde, Erovic Boban M, Turhani Dritan, Thurnher Dietmar
Department of Otorhinolaryngology, Head and Neck Surgery, Medical University of Vienna, Vienna, Austria.
Wien Klin Wochenschr. 2007;119(9-10):318-22. doi: 10.1007/s00508-007-0783-6.
Large defects of the oral mucosa are still a major challenge in reconstructive surgery. For the development of oral mucosal grafts, successful enrichment of cells with a high proliferative potential is highly desirable. Therefore, the aim of this study was to separate two fractions of oral keratinocytes based on their affinity to collagen type IV.
Oral keratinocytes were isolated from oral mucosa and separated into two fractions with different affinities to collagen type IV. Growth curves, Western blot analysis and immunohistochemical staining were used to detect differences between the two cell fractions.
The cell fraction (RAC-IV) that adhered to collagen type IV within 20 min showed higher proliferative potential, significantly higher (P < 0.05) expression of integrin beta1 and fewer apoptotic cells. In particular, this fraction included small proliferating cells with the typical polygonal shape of oral keratinocytes, whereas the non-proliferating cells (RAC-IV-D) were irregularly shaped. Immunohistochemical staining showed only some apoptotic RAC-IV cells, whereas RAC-IV-D cells showed a significant increase of M30-positive cells. In addition, Western blotting revealed significantly higher (P < 0.05) expression of integrin beta1 in the RAC-IV fraction than in the RAC-IV-D fraction.
Our results show that it is possible to enrich a fraction of highly proliferative oral keratinocytes by means of their high affinity to collagen type IV.
口腔黏膜大面积缺损仍是重建手术中的一项重大挑战。对于口腔黏膜移植物的研发而言,成功富集具有高增殖潜能的细胞是非常必要的。因此,本研究的目的是根据口腔角质形成细胞对IV型胶原的亲和力分离出两个细胞组分。
从口腔黏膜中分离出口腔角质形成细胞,并将其分离为对IV型胶原具有不同亲和力的两个组分。采用生长曲线、蛋白质免疫印迹分析和免疫组织化学染色来检测这两个细胞组分之间的差异。
在20分钟内黏附于IV型胶原的细胞组分(RAC-IV)显示出更高的增殖潜能、整合素β1的表达显著更高(P < 0.05)且凋亡细胞更少。特别地,该组分包含具有典型口腔角质形成细胞多边形形状的小增殖细胞,而非增殖细胞(RAC-IV-D)则形状不规则。免疫组织化学染色显示只有一些RAC-IV凋亡细胞,而RAC-IV-D细胞显示M30阳性细胞显著增加。此外,蛋白质免疫印迹显示RAC-IV组分中整合素β1的表达显著高于RAC-IV-D组分(P < 0.05)。
我们的结果表明,通过利用口腔角质形成细胞对IV型胶原的高亲和力,可以富集出一部分高增殖性的口腔角质形成细胞。