17)脊椎々間板手術症例の検討 : とくに椎間板障害例における手術適応について(第399回千葉医学会整形外科例会,第8回千葉整形外科災害外科臨床懇談会,千葉県労災指定医集談会)

<p>Data are expressed as medians and interquartile ranges; comparisons among time points, as determined by Friedman-ANOVA test, and between L-PRP and HA treatments, as determined by the Mann-Whitney U test, are not significant. w = week, m = month</p

Karyotype.

<p>Data are summarized in the table (above) and refer to 30 analyzed mitoses for each sample. Three abnormal karyotypes are shown (below). N = normal karyotype; n.d. = not determined.</p

Gene expression analysis.

<p>Dendrogram obtained by hierarchical clustering of 6 ASC samples (A–F) at different culture passages (P, from 1 to 14) based on the expression of 84 genes involved in DNA damage signaling pathways. Gene expression levels are shown as a heat map for each gene.</p

Effect of culture expansion on DNA damage signaling pathway gene expression.

<p>Results from RT-PCR array analysis are shown as fold changes between P1 (n = 6) and P5 (n = 4) or P14 (n = 1).</p><p>n.a. = not applicable; n.s. = not significant.</p

Telomere length dynamics in culture.

<p>Geometric mean of fluorescence intensity is reported at different culture passages relative to sample A, as representative example.</p

Characteristics of ASC analyzed at different culture passages.

<p>Cumulative Population Doublings and [days of culture] of all the samples at the different culture passages analyzed are reported.</p

Microsatellite instability (MSI) analysis.

<p>On the left: MSI analysis of sample A is shown as an example. Allele patterns for each of the five analyzed microsatellite sequences (CD4, VWA, Fes, Tpox and P53) were identified by acrylamide gel separation and compared among culture passages (P1, 2, 4, 6, 9 and 14). Each allele pattern appears to be stable throughout the culture period. On the right: Table summarizing MSI analysis of the 6 ASC samples. Allele nomenclature refers to the number of repeats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077895#pone.0077895-Neri1" target="_blank">[47]</a>. For each ASC sample, allele patterns for the five analyzed microsatellite loci (CD4, VWA, Fes, Tpox and P53) were maintained at all the different analyzed culture passages.</p

Growth curves.

<p>The continuous line represents the regression fit of all the samples. The upper dashed line represents the regression fit of samples excluded sample D. The lower dashed line indicates the regression fit of sample D alone.</p

<i>In vitro</i> pharmacological inhibition of GSK3β determines ROS production, oxidative damage, stress dependent growth inhibition and activation of an intra-S checkpoint.

<p><b>2A</b>, Compared to control (NS), 5 mM LiCl and 10 μM SB216763 increases ROS production and mitochondria activation at 4 hours treatment. Merged and separated signals of Hoechst 33258 nuclear counterstaining, DCHF-DA and Mitotracker Orange CMTMRos mitochondrial staining. Right: high magnification detail of a LiCl treated cell. Right Graph: at 16 hours, LiCl (black columns) but not SB216763 (grey columns) induced a significant increased (n = 5) accumulation of 8-oxo-dG compared to controls (white columns) on the basis of a flow cytometry analysis. <b>2B</b>, LiCl (black columns) and SB216763 (grey columns): longitudinal assessment of the effects of GSK3β inhibition on cell growth versus the control (white columns). Upper graphs: counts normalized versus the 8 hours count; lower graph: percentage count reduction due to either LiCl or SB216763 at each time point. <b>2C</b>, longitudinal assessment of the effects of siRNA mediated GSK3β silencing (squared columns) on cell growth versus the control non targeting siRNA (white columns). Right graph: Population doublings reduction following either 5 mM LiCl or 10 μM SB216763 stimulation as percentage of each unstimulated control at 8, 16 and 24 hours of both siCTL and siGSK3β treated cells <b>2D</b>, Left: LiCl determines a significant increased () percentage of cells in the S phase at 24 hours, as evidenced by DNA staining (Sytox green, n = 9 different experiments with LiCl and 4 with SB216763). Right: a representative example with cell cycle analysis of control (left) versus 5mM LiCl (right) treated cells at each time point. *P < 0.05; **P < 0.01;***P < 0.001.</p

Association of oxidative damage, markers of DDR and senescence in mid-deep layers of cartilage of obese patients.

<p>8-oxo-dG, GADD45β, p21 and SA-β-Gal assessed with immunohistochemistry and colorimetric detection and bright field images at 4x original magnification: cases representative of non obese (upper panel) or obese (lower panel) patients. High magnification inset shows the prevalent cytoplasmic localization of GADD45β signal (red staining) with sybr green as a nuclear counterstaining. For each marker, experiments were carried out in the same experimental session to rule out biases in comparing the signal intensity. Below the pictures for each marker a cumulative assessment (with mean and standard error of mean) of the percentage of positive cells in superficial, intermediate and deep layers in non obese (NO, white column) or obese patients (O, black columns) is shown. *P < 0.05; **P < 0.01;***P < 0.001.</p