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Identification of Genes Differentially Regulated in Rat Alveolar Bone Wound Healing by Subtractive Hybridization

Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan;

View larger version (48K): [in a new window]   Figure 1. Histology of wound and detection of genes. (A) Histological findings of alveolar bone wound. Periodontium 1 wk after injury: Granulation tissues were observed in the defect (a,b). Two wks after injury: Granulation tissues and blood vessels were observed in the defect (c). Similar changes were observed at 2.5 wks (data not shown). Four wks after injury: Granulation tissues were contracted and connective tissue was partially remodeled (d). Bar equals 300 µm. After removal of the full-thickness flap, tissue proliferating in the bone cavity was recovered by dental curette (e). (B) General procedure of subtractive hybridization. The target c-sscDNA was synthesized from the target sscDNA-beads, and an auto-subtraction was performed. The target c-sscDNA was subtracted twice from the driver sscDNA-beads. The target c-sscDNA (up-regulated and down-regulated genes) was amplified by PCR. The PCR products were subjected to electrophoresis and used for cloning. (C) Representation of GAPDH cDNA after sequential subtraction. The amount of GAPDH cDNA in the sample was analyzed by PCR with primers designed for the 3' non-coding region of the rat GAPDH cDNA: sense, 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3'; antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3'. The following templates were used: cDNA from the one-week injured tissue (cDNA-injury), cDNA-injury subtracted once (cDNA-sub1), and cDNA-injury subtracted twice (cDNA-sub2). The amplification was performed for 20, 25, 30, and 35 cycles. (D) Display of amplified cDNAs followed by two-round subtraction. The PCR products underwent gel electrophoresis. Lane 1, one-week up-regulated genes; lane 2, two-week up-regulated genes; lane 3, 2.5-week up-regulated genes; lane 4, four-week up-regulated genes; lane 5, one-week down-regulated genes; lane 6, two-week down-regulated genes; lane 7, 2.5-week down-regulated genes; lane 8, four-week down-regulated genes; and lane M, 100-bp DNA ladder.

View larger version (36K): [in a new window]   Figure 2. Confirmation of quantitation by reverse Northern hybridization. (A) Detection of known cDNAs. Seven known cDNAs (BGP, Cbfa1, TGFß-1, ALK5, TGFß-RII, TGFß-RIII, and ß-actin) were amplified by PCR and cloned. Each clone (500 ng) was digested with EcoRI, subjected to gel electrophoresis (a), and then transferred to the membrane. Lanes: 1, 100-bp ladder; 2, BGP; 3, Cbfa1; 4, TGFß-a; 5, ALK5; 6, TGFß-RII; 7, TGFß-RIII; and 8, ß-actin. The membranes were hybridized with a mixture of probes at different concentrations as described in the lower table (b-f). To standardize the total amount of labeled probe, 5 x 105 cpm/mL of GAPDH probe was added to the mixture. The PCR primers used were: BGP sense, 5'-CTGAGTCTGACAAAGCCTTC-3', and BGP antisense, 5'-CCATAGAT GCGCTTGTAGGC-3'; Cbfa1 sense, 5'-ACCTCTGACTTCTGCCTCTG-3', Cbfa1 antisense, 5'-CGCCAAACAGACTCATCCAT-3'; TGFß-1 sense, 5'-CATGACATGAACCGGCCCTT-3', TGFß-1 antisense, 5'-AAATATA GGGGCAGGGTCCC-3'; ALK5 sense, 5'-GGACGCAGCTGTGGTTGGTG-3', ALK5 antisense, 5'-TTCCACCAATAGAACAGCGT-3'; TGFßRII sense 5'-CTTGACCTGTTGCCTGTGTG-3', TGFßRII antisense 5'-CATGCTCTCC ACACAGGGGT-3'; and TGFßRIII sense 5'-TACACCATCATCG AGAACAT-3', TGFßRIII antisense 5'-GAGTAG ATGTACCACAAGGC-3'. The ß-actin primers were purchased from Clontech (Rat Control Amplimer Set). Complementary DNA (1 ng) from injured rat tissue or mouse embryo was amplified by PCR according to the primers described above. After cDNAs were cloned, the nucleotide sequences were confirmed. (B) Quantitation of hybridization signals. The signal intensity of each cDNA was quantified with NIH Image and normalized against that of ß-actin. The mean value of 7 kinds of targets to the same probe concentration is plotted, and error bars indicate standard deviation.

View larger version (22K): [in a new window]   Figure 3. Expression patterns of genes and phases of wound repair. (A-E) Clustering of genes regulated in alveolar bone wound healing. On the basis of the changes in level of expression, 34 clones from injured tissues (Table) and 6 known genes were clustered into 5 groups: A, Cluster I (8 clones and ALK5); B, Cluster II (11 clones and BGP); C, Cluster III (3 clones, Cbfa1, and TGFß1); D, Cluster IV (9 clones and TGFßRIII); and E, Cluster V (3 clones and TGFßRII). (F) Phases of wound repair. The wound-healing process has been divided into three phases: (1) inflammation, (2) re-epithelialization, and granulation tissue formation, and (3) matrix formation and remodeling. This is modified from the figure described by Clark (1996).