Method for treating autoimmune diseases and screening method for preventive or therapeutic agent for the same
a technology for autoimmune diseases and screening methods, applied in the direction of immunodeficiency syndrome, drug composition, sense disorder, etc., can solve the problems of no well-established therapeutic method, and nothing has been known in relation to the effect of rbap48 on improving the pathological condition of patients, and achieve excellent preventive or therapeutic effects
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example 1
Apoptosis of salivary Gland Cells of Normal Mice
[0028] C57BL / 6 mice and estrogen receptor knockout (ERαKO) mice were ovariectomized (Ovx), and salivary gland cells of each mouse were observed over time for six weeks after the ovariectomy in terms of apoptosis. Slices of the salivary gland cells were stained through TUNEL, and whether or not apoptosis occurred was evaluated from the stain index (TUNEL index). Sham operation groups (Sham), to which Ovx had not been performed, were used as controls. As a result, as shown in FIG. 1, transient apoptosis was observed from two to three weeks after Ovx in the salivary gland cells of the C57BL / 6 mice to which ovariectomy had been performed. In contrast, in the salivary gland cells of the ERαKO mice, increase in apoptotic cells was not observed. Thus, apoptosis of salivary gland cells was confirmed to involve estrogen deficiency.
example 2
Apoptosis Induction of Cultured Cells
[0029] Cells of each of the following cell lines were cultured through a routine method; mouse salivary gland cell lines (MSG:B6 and ERαKO); human salivary gland cell line (HSG); human breast cancer cultured-cell line (MCF-7); human acute T cell leukemia cell line (Jurkat); and human colon cancer cultured-cell lines (HT-29 and Colo201). The cultured cells were reacted with tamoxifen (Tam, final concentration: 10−7 M), an antagonist of estrogen. The resultant cells were observed in terms of Tam's effect on apoptosis and saliva-gland-specific antigens, and activity of apoptosis-related proteases (e.g., caspase) was investigated. Separately, the above cultured cells were reacted with staurosporin (an apoptosis inducer, final concentration: 10−6 M), and the resultant cells were used as a positive control group. Forty-eight hours after the addition of Tam, apoptosis of the cells was determined by means of a flow cytometer, by use of Annexin-V-FITC. A...
example 3
Identification of the Genes which Exhibit Enhanced Expression by Anti-Estrogen Effect
[0030] HSG cells were stimulated by Tam to thereby cause expression of mRNAs. mRNAs which exhibited enhanced expression as compared with those of non-stimulated HSG cells were extracted through a routine method and isolated through the differential display method (RNA image kit, Gene Hunter™, Toyobo). The thus-obtained DNA fragments were transferred into E. Coli for cloning. The thus-obtained mRNAs were investigated through northern blotting (FIG. 3) by use of HSG cells and the isolated DNA fragments serving as probes, to thereby determine mRNAs which exhibit enhanced expression by tamoxifen (final concentration: 10−7 M), and the expression was reduced by addition of estradiol (final concentration: 10−9 M). The amino acid sequences of the DNA fragments were determined through a routine sequencing method. The DNA fragments were identified to be the RBAp48 gene. Two hours after Tam stimulation, the H...
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