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1 Young Sook Choi, Kwang Soo Lee, M.D., Sang Ho Kim, M.D. Background: Complementary receptors have been suggested to play causative roles in the neuroinflammatory process of Alzheimer's disease (AD). The genetic expressions of the C3a receptor (C3aR), C5a receptor (C5aR) and the protein expressions of the C3aR and C5aR were examined in the human neuroblastoma cell line, SK-N-SH, after the administration of amyloid peptide (A1-42). Methods: SK-N-SH cells were incubated overnight with a single dose of 20 M of aggregated A (A1-42). An inhibition study was done with actinomycin D (ActD, 2.5 M) or with the administration of cycloheximide (CHX, 2.5 M) to the cell suspension. Messenger RNA expressions of C3aR and C5aR were detected by RT-PCR. The intensity of bands from 6% polyacrylamide electrophoretic gel was analyzed by a bioimage analyzer. The protein production of C3aR and C5aR in the A-treated cells was also measured by flow cytometry. NFB activation after treatment of A in the cells was detected by an electrophoretic mobility-shift assay. Results: A1-42 increased the expression of C3aR and C5aR. ActD inhibited the expression of both anaphylatoxin receptors but CHX only suppressed C5aR mrna expression. Activated NFB was demonstrated in the A-stimulated cells. Conclusions: C3aR and C5aR were constitutively expressed in the human neuroblastoma SK-N-SH cell. Expression of these anaphylatoxin receptors was upregulated after A1-42 stimulation, which as a result, may contribute to the complement-mediated neuroinflammation of AD. J Korean Neurol Assoc 22(1):52 58, 2004 Key Words: Complement C3a receptors, Complement C5a receptors, Alzheimer's disease, Actinomycin, Amyloid beta-protein 서론

2 대상과방법 22 권 1 호대한신경과학회지

결과 Figure 1. Messenger RNA expression of anaphylatoxin receptor genes. (A) Representative photograph shows RT-PCR products of C3a receptor and C5a receptor mrnas.

3 결과 Figure 1. Messenger RNA expression of anaphylatoxin receptor genes. (A) Representative photograph shows RT-PCR products of C3a receptor and C5a receptor mrnas. Neuroblastoma SK-N-SH cells are stimulated overnight by A 1-42 (20 M). The cdna has been obtained from a total RNA extract of cells. Electrophoretic separation is performed on a 6% polyacrylamide gel. Note the extraordinary increased mrna expression of C5a receptor after Astimulation. (B) Bar graphs show relative levels of anaphylatoxin receptor mrnas in cell extracts. Relative optical densities represent readings of gel band intensities as determined on a Fuji (BAS-2500) bioimage analyzer with quantitative analysis and use of Tina software 2.10 (Fuji). The mean±s.e. of 2~4 independent experiments are shown. * P<0.05 vs control(tnf-/a -). Student's t-test.

Figure 2. Flow cytometry analysis of the expression of anaphylatoxin receptors by SK-N-SH cells. (A) Dot plot of the C3aR and the C5aR from A 1-42 (20 M) stimulated cells.

4 Figure 2. Flow cytometry analysis of the expression of anaphylatoxin receptors by SK-N-SH cells. (A) Dot plot of the C3aR and the C5aR from A 1-42 (20 M) stimulated cells. Samples are prepared as described in Materials and Methods. The mean±s.e. of 3 independent experiments are shown. (B) Histograms of the C3aR and C5aR control; 20 M A Figure 3. Effect of actinomycin D (2.5 M) on the mrna expression of anaphylatoxin receptors from A 1-42 stimulated SK-N-SH cells. (A) The expression of mrnas for C3a receptor and C5a receptor is suppressed, respectively. (B) Optical density is measured with bioimage analyzer as in figure 1. The mean±s.e. of 2~4 independent experiments are shown. * P<0.05 vs A +/ActD-. Student's t-test. ActD; actinomycin D. 22 권 1 호대한신경과학회지

Figure 4. Effect of cycloheximide (2.5 M) on the mrna expression of anaphylatoxin receptor from A 1-42 stimulated SK-N-SH cells.

(B) Optical density is measured with a bioimage analyzer as in figure 1. The mean±s.e. of 2~4 independent experiments are shown.

(A) NF B activity is elevated in A 1-42 stimulated cells.

5 Figure 4. Effect of cycloheximide (2.5 M) on the mrna expression of anaphylatoxin receptor from A 1-42 stimulated SK-N-SH cells. (A) The expression of mrna for C5a receptor is obviously suppressed. (B) Optical density is measured with a bioimage analyzer as in figure 1. The mean±s.e. of 2~4 independent experiments are shown. * P<0.05 vs A +/CHX-. Student's t-test. CHX; cycloheximide. Figure 5. Effect of A 1-42 on NF B activity in SK-N-SH cells. (A) NF B activity is elevated in A 1-42 stimulated cells. (B) The nuclear protein-nf B complex (indicated by the arrow) is verified by suppression of the binding activity with an excess of cold NF B but not with cold AP-1 oligonucleotide. The supershift of the complex is seen by either anti-p50 and anti-p65 antibodies. 고찰

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Can032.hwp

Can032.hwp Chromosomal Alterations in Hepatocellular Carcinoma Cell Lines Detected by Comparative Genomic Hybridization Sang Jin Park 1, Mahn Joon Ha, Ph.D. 1, Hugh Chul Kim, M.D. 2 and Hyon Ju Kim, M.D. 1 1 Laboratory