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

使用 β3 整合素亚基 cDNA 转导的人角质形成细胞开发培养上皮 - 提高摄取率 -

基本信息

批准号：
15390543
负责人：
KUBO Miyoko
金额：
$ 9.47万
依托单位：
Kawasaki Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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

用逆转录病毒介导的方法将β3整合素亚基cDNA导入人角质形成细胞，使其在细胞表面表达β3和αvβ3。在20- 30%、50- 60%、70-80%和100%汇合时，β 3基因转染的角质形成细胞的阳性表达率分别为69%、68%、65%和33%。另一方面，对照β-gal cDNA转导的角质形成细胞对β3或αvβ3呈阴性。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小时细胞粘附试验表明，与β-半乳糖苷酶（β-gal）cDNA转导的角质形成细胞（对照）相比，β3 cDNA转导的角质形成细胞显著粘附于纤维蛋白（FB）、纤维蛋白原（FG）、玻连蛋白（VN）、纤连蛋白（FN）和变性胶原{明胶（GEL）}。细胞对FB和GEL的粘附抑制试验用MTT法进行。 ...更多信息使用在室温下与不同浓度的抗αvβ3单克隆抗体（LM 609）或正常小鼠IgG 1或肽GRGDSP或GRGESP孵育30 min的细胞进行，然后将细胞加入威尔斯孔中。LM 609和RGD肽以剂量依赖性方式抑制β3 cDNA转导的人角质形成细胞与FB和GEL的粘附，但不被正常小鼠IgG 1和RGE肽抑制。5天细胞生长试验显示，β3整合素亚基cDNA转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 cDNA转导的角质形成细胞（对照）。FG、FB、VN和GEL上β3 cDNA转导的角质形成细胞的细胞生长率（通过1天至5天的细胞数量增加率计算）也明显高于对照组。两组细胞在层粘连蛋白、IV型胶原和I型胶原上的生长无差异。14 h的趋触性迁移实验显示，转染β3 cDNA的角质形成细胞向FG、FB、GEL、VN和FN的迁移率明显高于对照细胞。两组细胞迁移到I型胶原蛋白的程度相同，并没有迁移到BSA。抗αvβ3单克隆抗体（LM 609）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，这些数据支持了这样的观点，即这些重组角质形成细胞在应用于FB、FG和变性的富含胶原蛋白的皮肤伤口时，提供了一种在移植手术中促进伤口愈合的方法。少