カヘンガタ イデンシ トラップ ベクター pU-17 オ モチイタ トラップ ライン ノ ジュリツ ト カイセキ

We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-βgeo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of β galactosidase/neomycin-resistancefusion gene(β geo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre-mutant lox system, we successfully replaced the β geo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp-deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the β geo gene. Thus, using this pU-17 trap vector, we can initially carry out random mutagenesis, and then replace the β geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination

FGF-2 Stimulates the Growth of Tenogenic Progenitor Cells to Facilitate the Generation of Tenomodulin-Positive Tenocytes in a Rat Rotator Cuff Healing Model.

Background: Fibroblast growth factor (FGF)–2 has the potential to enhance tendon-to-bone healing after rotator cuff (RC) injury. Hypothesis: FGF-2 stimulates tenogenic differentiation of progenitors to improve the biomechanical strength and histological appearance of repaired RCs in rats. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 156) underwent unilateral surgery to repair the supraspinatus tendon to insertion sites. The FGF-2-treated group (gelatin hydrogel containing 5 μg of FGF-2) and a control group (gelatin hydrogel only) were compared to investigate the effects of FGF-2 at 2, 4, 6, 8, and 12 weeks postoperatively. Biomechanical testing was performed at 6 and 12 weeks. Semiquantitative histological analysis and immunohistochemical analysis for the proliferating cell nuclear antigen (PCNA) were performed, and the expression of tendon-related markers, including Scleraxis (Scx) and Tenomodulin (Tnmd), was monitored by real-time reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. SRY-box containing gene 9 (Sox9) expression was monitored by RT-PCR and immunohistochemical analysis. At 2 and 4 weeks, immunohistochemical analysis for mesenchymal stem cell (MSC) markers was also performed. Results: The FGF-2-treated group demonstrated a significant improvement in mechanical strength at 6 and 12 weeks and significantly higher histological scores than the control group at ≥4 weeks. The average incidence of PCNA-positive cells was significantly higher at 2 and 4 weeks, and more cells expressing MSC markers were detected at the insertion site in the FGF-2-treated group. The expression level of Scx increased significantly in the FGF-2-treated group from 4 to 8 weeks, while the Tnmd level increased significantly from 4 to 12 weeks postoperatively. The localization of Tnmd overlapped with the locations of reparative tissues accompanying collagen fibers with an aligned orientation. Sox9 expression was significantly upregulated at 4 weeks in the FGF-2-treated group. Conclusion: FGF-2 promotes growth of the tenogenic progenitor cells, which participate in tendon-to-bone healing, resulting in biomechanical and histological improvement of the repaired RC. Clinical Relevance: These findings provide clues regarding the clinical development of regenerative repair strategies for RC injury

オウショク ジンタイ ヘンセイ ニ オイテ Matrix metalloproteinase ワ ダンセイ センイ ノ ゲンショウ オ ソクシン スル

熊本大

Pathological changes in LF tissue.

<p><b>A:</b> Hematoxylin-eosin (HE), picrosirius, trichrome, and Elastica-van Gieson (EVG) staining of LF in LSCS and control samples. Arrowheads indicate hyaline degeneration. Type 1 collagen appears red or orange and type 3 collagen appears green in color. Each scale bar, 100 μm. <b>B:</b> Average number of fibroblasts in the randomly selected field (×400). <b>C, D:</b> Average occupation of collagen fibers (C) and elastic fibers (D). <b>E:</b> Ratio of elastic and collagen fibers. <b>F, G:</b> Mean grade of LF fibrosis (F) and elastin degradation (G) by Park’s scoring systems. Lower grade (0 and 1) indicates less degeneration and grade 4 indicates severe degenerative change. <b>H:</b> Resorcin-fuchsin staining of LF samples in LSCS-fibrosis, LSCS-chondrogenic, and the representative control area. Arrowheads indicate elaunin-like fibers and arrows indicate oxytalan-like microfibers. Scale bar, 50 μm. <b>I:</b> Quantitative proteoglycan levels of LF tissues in LSCS and control. Data represent the mean ± SEM (n = 13 per group for histological assay; and n = 20 per group for proteoglycan assay, selected randomly). **P < 0.01 vs. control group.</p

Demographic data of the study population.

<p>Demographic data of the study population.</p

MMP-2 and -9 expressions in LF tissue.

<p><b>A, B:</b> Comparison of MMP-2 (A) and MMP-9 (B) mRNA and protein expression in the LF of the LSCS and control groups. mRNA levels were normalized to <i>18S rRNA</i> levels. The value in the control group was set as 1. <b>C:</b> Immunohistochemical analysis of MMP-2 and -9 expressions in LF tissue from patients with LSCS (left) and control subjects (right). The inset in the left panel for MMP-2 shows a high-magnification view of the area enclosed by the dashed line. Arrowheads indicate MMP-positive cells. Scale bar, 100 μm. <b>D:</b> Correlation between MMP-2 mRNA (left) and protein (right) expression and LF thickness. Data represent the mean ± SEM (LSCS, n = 31 and control, n = 21 for mRNA assay; and n = 14 per group for protein assay). **P < 0.01 vs. control group. N.S., not significant.</p

Sequences of primers used for RT-PCR analysis.

<p>Sequences of primers used for RT-PCR analysis.</p

Elastic fiber assay in LF tissues.

<p><b>A:</b> Correlation between <i>MMP-2</i> mRNA expression and elastic fiber area. The minimum value of MMP-2 expression in analyzed samples was set to 1 (LSCS, n = 13 and control, n = 11). <b>B:</b> <i>Elastin</i> mRNA expression in LF tissue (LSCS, n = 31 and control, n = 21). Data represent the mean ± SEM. N.S., not significant.</p

MMP-2 activation and elastolysis induced by IL-6 in LF fibroblasts.

<p><b>A:</b> Representative gelatin zymography analysis of LF fibroblasts in response to IL-6/sIL-6Rα stimulation for 24 h with or without Stattic. <b>B:</b> Quantitative analysis of data shown in panel A (n = 3). <b>C:</b> Concentration of soluble elastin following incubation for 6 h with the supernatant of IL-6-stimulated cultures with or without Stattic (n = 3). Data represent the mean ± SEM. **P < 0.01.</p