Development of Cultured Epithelium Using β3 Integrin Subunit cDNA-Transduced Human Keratinocytes - To Improve the Take Rate -

用β3整合素亚基cDNA转导人角化细胞的体外细胞功能分析用逆转录病毒介导的方法转导人角化细胞在细胞表面表达β3和αvβ3。β3 cdna转导的角化细胞融合度为20-30%、50-60%、70-80%和100%时，β3阳性表达细胞的比例分别为69%、68%、65%和33%。另一方面，对照β-gal cdna转导的角质形成细胞β3或αvβ3均为阴性。β3整合素亚基cDNA的转导不影响其他αv整合素复合物如αvβ5或αvβ6的表达。1小时细胞粘附实验表明，与β-半乳糖苷酶（β-gal） cdna转导的人角质形成细胞（对照）相比，β3 cdna转导的人角质形成细胞与纤维蛋白（FB）、纤维蛋白原（FG）、玻璃体连接蛋白（VN）、纤维连接蛋白（FN）和变性胶原{明胶（GEL）}的粘附显著。用不同浓度αvβ3单克隆抗体（LM609）或正常小鼠IgG1或多肽GRGDSP或GRGESP在RT下孵育30分钟后，将细胞加入孔中，进行细胞对FB和GEL的粘附抑制实验。LM609和RGD肽以剂量依赖性的方式抑制β3 cdna转导的人角质形成细胞对FB和GEL的粘附，而正常小鼠IgG1和RGE肽则不受其影响。5天的细胞生长实验表明，β3整合素亚基介导的角质形成细胞在FB、FG、VN和变性胶原（GEL）上的生长显著高于β-gal介导的角质形成细胞（对照组）。β3 cdna转导的角化细胞在FG、FB、VN和GEL上的细胞生长速率（以细胞数增加速率计算）也明显高于对照组。两组间层粘连蛋白、IV型胶原和I型胶原的细胞生长均无差异。此外，14小时的趋向性迁移实验显示，β3 cdna转导的角质形成细胞向FG、FB、GEL、VN和FN的迁移明显多于对照细胞。两组细胞向I型胶原迁移的程度相同，未向牛血清白蛋白迁移。αvβ3单克隆抗体（LM609）显著抑制β3 cdna转导的角质形成细胞向FG、FB、GEL、VN和FN的迁移。因此，这些数据支持这样一种观点，即这些重组角化细胞在FB、FG和变性的富含胶原的皮肤伤口上应用时，提供了一种在移植手术中促进伤口愈合的方法。少

Analysis of in vitro cell functions with β3 cDNA-transduced human keratinocytesHuman keratinocytes transduced with β3 integrin subunit cDNA by a retrovirus-mediated transduction method expressed β3 and αvβ3 on the cell surface. The percentage of positive β3 expressed cells with the cultures of β3 cDNA-transduced keratinocytes having 20-30%, 50-60%, 70-80% and 100% confluence were 69%, 68%, 65% and 33%, respectively. On the other hand, the control β-gal cDNA-transduced keratinocytes were negative for β3 or αvβ3. The transduction of β3 integrin subunit cDNA did not affect the expression of other αv integrin complexes such as αvβ5 or αvβ6. One hour cell adhesion assays demonstrated that the β3 cDNA-transduced human keratinocytes adhered to fibrin (FB), fibrinogen (FG), vitronectin (VN), fibronectin (FN) and denatured collagen { gelatin (GEL) } significantly compared with β-galactosidase (β-gal) cDNA-transduced keratinocytes (control). Inhibition assay of the cell adhesion to FB and GEL wa … More s performed using cells which were incubated with various concentrations of monoclonal antibody to αvβ3 (LM609) or normal mouse IgG1 or alternatively with peptides GRGDSP or GRGESP for 30 min at RT before adding cells to the wells. The adhesion of the β3 cDNA-transduced human keratinocytes to FB and GEL was inhibited by the LM609 and RGD peptides in a dose-dependent fashion, but not by the normal mouse IgG1 nor RGE peptides. Five days cell growth assays showed that β3 integrin subunit cDNA-transduced keratinocytes grew on FB, FG, VN and denatured collagen (GEL) significantly more than the β-gal cDNA-transduced keratinocytes (control). The cell growth rate, calculated by the increased rate of cell numbers from one day to five days, of the β3 cDNA-transduced keratinocytes also increased on FG, FB, VN and GEL significantly more than the control. There were no differences in the cell growth on laminin, type IV collagen and type I collagen between the two groups. Furthermore, 14 hours haptotaxis migration assays revealed that the β3 cDNA-transduced keratinocytes migrated to FG, FB, GEL, VN and FN significantly more than the control cells did. Both groups of cells migrated to type I collagen to the same degree and did not migrate to BSA. A monoclonal antibody to αvβ3 (LM609) considerably inhibited the migration of the β3 cDNA-transduced keratinocytes to FG, FB, GEL, VN and FN.Thus, these data support the idea that these recombinant keratinocytes provide a method of enhancing wound healing in a graft procedure when they are applied to FB, FG and denatued collagen-rich cutanous wounds. Less