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Prevention of acute vascular rejection in xenograft

异种移植物急性血管排斥反应的预防

基本信息

批准号：
12470239
负责人：
MIYAGAWA Shuji
金额：
$ 8.96万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470239/
关键词：
DAF C1-INH Complement regulatory protein pig endothelial cell CHO cell HSV-gC1 トランスジェニックマウス Factor I ブタ血管内皮細胞細胞傷害性試験

项目摘要

We constructed a cell surface-bound form of a fluid phase CRP. Factor I-PlThe cell membrane-bound form of factor I (fl-Pl) was constructed. An SEC line with a low expression of fl-Pl showed a weak inhibition of cell lysis in human serum, whereas a CHO cell transfectant with a high expression of fl-Pl showed over a 60% inhibition of cell lysis.C1-INHA surface-bound form of human C1-INH (C1-INH-Pl) was constructed Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-Pl showed a medium level of expression of these molecules. C1-INH-Pl block human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively.The codon-optimization DAFThe DAF gene was synthesized in order to adapt its codons for those which are more frequent in mammals, especially pigs.A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF. Consistent with the in vitro data, the expression of DAF in the mice which carry sDAF was higher than wild-type DAF in many organs.Assessment of the effect of various forms of C1-INH-Pl and DAFcDNAs of various deletion mutants of the C1-INH-Pl were established. While all deletion mutants of C1-INH-Pl except the delta-1-99AA were not expressed on the cell surface. Consequently, both the delta1-99AA C1-INH-Pl and delta-SCR1 DAF molecules are quite effective in downregulating the xenogeneic cell lysis, but accomplished this in different manners.HSV-gC1The complement regulatory function of a HSV-1 glycoprotein gC (HSV-gC1) molecule was investigated. The CHO cell transfectants showed a significant inhibition in cell lysis by the sera except for those that contained the preexisting anti-HSV-gC1 antibody. The SEC transfectants, however, showed a marked inhibition of cell lysis in all cases.

我们构建了细胞表面结合形式的液相 CRP。因子I-Pl构建了因子I(fl-Pl)的细胞膜结合形式。具有fl-Pl低表达的SEC系显示出对人血清中的细胞裂解的弱抑制，而具有fl-Pl高表达的CHO细胞转染子显示出超过60%的细胞裂解抑制。构建了人C1-INH (C1-INH-Pl)的C1-INHA表面结合形式的稳定CHO和具有C1-INH-Pl的SEC转染子的流式细胞术图谱显示这些细胞的中等水平的表达分子。 C1-INH-Pl 在 CHO 细胞上分别阻断人补体介导的细胞裂解约 75%，在 SEC 细胞上阻断约 60-65%。密码子优化 DAF 合成 DAF 基因是为了使其密码子适应哺乳动物（尤其是猪）中更常见的密码子。在合成 DAF 的转染子中观察到蛋白质产量显着增加，且未检测到 mRNA 升高 (sDAF)，与野生型DAF相比。与体外数据一致，携带sDAF的小鼠中许多器官中DAF的表达高于野生型DAF。建立了各种形式的C1-INH-Pl的作用评估和C1-INH-Pl的各种缺失突变体的DAFcDNA。而除delta-1-99AA外，C1-INH-P1的所有缺失突变体均不在细胞表面表达。因此，delta1-99AA C1-INH-Pl 和 delta-SCR1 DAF 分子在下调异种细胞裂解方面都非常有效，但以不同的方式实现这一点。 HSV-gC1 研究了 HSV-1 糖蛋白 gC (HSV-gC1) 分子的补体调节功能。除了含有预先存在的抗HSV-gC1抗体的那些之外，CHO细胞转染子显示出血清对细胞裂解的显着抑制。然而，SEC 转染子在所有情况下均显示出对细胞裂解的显着抑制。