In vitro biological activity of anti-C II TA hammerhead ribozyme--a novel approach for autoimmune diseases / 华中科技大学学报（医学）（英德文版）

This study investigated the feasibility of using an hammerhead ribozyme against C II TA, a major regulator of MHC II antigens, to repress the expression of MHC II molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C II TA and its target gene were transcribed, then mixed up and incubated in vitro. The cleavage products were analyzed by PAGE and silver-staining. Rz464 was then inserted into the pIRES2-EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class II MHC induction by recombinant human interferon-gamma (IFN-gamma). mRNA of C II TA was measured by RT-PCR. Our results showed that Rz464 could exclusively cleave C II TA RNA. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on pRz464+ Hela was induced, and the mRNA content of C II TA decreased too. It is concluded that Rz464 could inhibit C II TA and thus the family of genes was regulated by C II TA:MHC II molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto-immune diseases.

Inhibition of MHCⅡExpression on Human Dermal Fibroblast Through CⅡTA Anti-sense RNA / 中华皮肤科杂志

Objective To investigate the feasibility of using anti-sense RNA against classⅡmajor histocompatibility complex(MHCⅡ)transactivator(CⅡTA),which might regulate MHCⅡexpression,to suppress the relative immune response.Methods Stable transfectants of dermal fibroblasts with pDarⅡ(pDarⅡ-D)were tested for the expression of classic MHCⅡ(HLA-DR,-DP,-DQ)antigens induced with recombinant human interferon-gamma(IFN-?).mRNA abundance of CⅡTA,and classic MHCⅡwas mea-sured by RT-PCR.IL-2mRNA expressed in T cells,stimulated by transfected dermal fibroblasts,was de-termined by mixed lymphocyte reaction.Results When induced with IFN-?,the expression of HLA-DR and-DP antigens on pDarⅡ-D was reduced by95.63%and87.89%,respectively.Meanwhile,the mRNA contents of CⅡTA and classic MHCⅡwere decreased significantly(P

Experimental studies on hematopoiesis reconstitution by using of transplantation of gene-modified bone marrow cells / 中华器官移植杂志

Objective To explore the biological features of hematopoiesis reconstitution by using genetic marking.Methods NeoR gene was transduced into bone marrow(BM)cells of mouse mediated by liposome.Then these cells were transplanted into lethally irradiated recipients.The hematopoiesis reconstitution was observed and the marker gene in spleen and BM cells of recipients after hematopoiesis reconstitution was examined.Results The transplanted recipients remained alive and healthy.But the irradiated mice with no transplantation died from BM aplasia soon.Meanwhile,the cells from spleen and BM of transplanted mice could be alive in G418 system,and contained the DNA fragments of NeoR gene by PCR.Conclusion Gene-modified BM cells could be used to reconstitute hematopoiesis successfully and express the foreign gene to some extent stably.

In vitro induction of p210~(Bcr-Abl) protein-specific cytotoxic T cell responses using 562 cell total RNA transfected human dendritic cells / 中国输血杂志

Objective To investigate whether human monocyte-derived dendritic cells (DCs) can express p210 Bcr-Abl protein and induce antigen-specific CTL responses in vitro after transfection with total RNA of K562 cells (K562-RNA).Methods Immature DCs were derived from human peripheral blood monocytes after 5 day incubation in the presence of GM-CSF and IL-4. The cells were then transfected with K562-RNA using electroporation or DOTAP lipofection. To verify the successful transfection of DCs with K562-RNA, Bcr-Abl fusion gene expression was determined by RT-PCR and Western blot. The immune phenotypes of the DCs were analyzed by flow cytometry. CTL cytotoxicity was assayed by propidium iodide (PI) stain and flow cytometry. The amount of DCs, CD1a expression and purity of DCs were measured by FACS.Results Bcr-Abl fusion gene appeared in the DCs after transfection with K562 cell total RNA. But 24 hours later, the Bcr-Abl mRNA from the K562-RNA transfected DCs disappeared, while the cells were expressing p210 Bcr-Abl protein. The transfected DCs could significantly promote T lymphocytes to kill the target K562 cells. We found that PBMC can be induced to DC in culture medium containing human plasma, suggesting a potential for clinical application.Conclusion Human dendritic cells transfected by K562 total RNA can induce effective p210 Bcr-Abl protein-specific immune responses, which might broaden the spectrum of possible DC-based clinical applications.