Raffetto J D, Mendez M V, Phillips T J, Park H Y, Menzoian J O
Department of Surgery, Boston University Medical Center, Massachusetts, USA.
Am J Surg. 1999 Aug;178(2):107-12. doi: 10.1016/s0002-9610(99)00134-8.
Fibroblasts (fb) cultured from venous ulcer patients and patients with venous reflux disease without ulcer demonstrate characteristics of cellular senescence, such as increased fibronectin level and senescence-associated beta-galactosidase (SA beta-gal) positive cells. Cellular senescence is an in vitro event characterized by the progressive loss of proliferative capacity with increased passage number, and has been associated with impaired healing in vivo. This report examines progressive stages of cellular senescence in fb from the distal area (du-fb) and proximal fb (pu-fb) of patients with venous ulcer, as well as in distal fb (dr-fb) and proximal fb (pr-fb) from patients with venous reflux without ulcer, by comparing the population doubling time (T) and percent SA beta-gal expression.
The mean value of T over 6 passages for fb in the ulcer group was 132.5 +/- 29.0 hours for pu-fb and 492.9 +/- 146.2 hours for du-fb (P = 0.0009). For fb in the reflux group the mean value of T over 5 passages was 79.3 +/- 12.8 hours for pr-fb and 94.2 +/- 16.8 hours for dr-fb (P = 0.8). Comparing ulcer and reflux fb, no difference in T was observed between pu-fb and pr-fb (P = 0.6), but a difference was noted between du-fb and dr-fb (P = 0.0004). The mean percent SA beta-gal activity for fb in the ulcer group was 11.2% +/- 3.1% for pu-fb and 63.8% +/- 8.9% for du-fb (P = 0.0001). Individual passages demonstrated significant difference (P <0.05) in SA beta-gal activity between pu-fb and du-fb at early and late passages. No difference was noted in SA beta-gal activity for fb in the reflux group or between pu-fb and pr-fb, but comparison between du-fb and dr-fb was significant (63.8% +/- 8.9% versus 7.8% +/- 2.9%; P = 0.0001).
The in vitro passage of du-fb and pu-fb in chronic venous ulcer patients has an effect on T and cellular senescence as measured by SA beta-gal activity. Our data further suggest that du-fb are at a more progressive stage of cellular senescence when compared with pu-fb, and more importantly with fb cultured from patients with venous reflux without ulcer. These findings are consistent with impaired wound healing of venous stasis ulcer. The accumulation of senescent fb and a more advanced stage of cellular senescence of du-fb may explain why repeated episodes of venous ulceration are resistant to conservative treatment and require more aggressive measures of therapy.
从静脉溃疡患者以及无溃疡的静脉反流疾病患者中培养出的成纤维细胞（fb）表现出细胞衰老的特征，如纤连蛋白水平升高和衰老相关β半乳糖苷酶（SAβ-gal）阳性细胞增多。细胞衰老为一种体外现象，其特征是随着传代次数增加，增殖能力逐渐丧失，且与体内愈合受损有关。本报告通过比较群体倍增时间（T）和SAβ-gal表达百分比，研究静脉溃疡患者远端区域（du-fb）和近端fb（pu-fb）以及无溃疡的静脉反流患者远端fb（dr-fb）和近端fb（pr-fb）中细胞衰老的进展阶段。
溃疡组fb传代6次以上时，pu-fb的T平均值为132.5±29.0小时，du-fb为492.9±146.2小时（P = 0.0009）。反流组fb传代5次以上时，pr-fb的T平均值为79.3±12.8小时，dr-fb为94.2±16.8小时（P = 0.8）。比较溃疡组和反流组的fb，pu-fb和pr-fb之间的T无差异（P = 0.6），但du-fb和dr-fb之间存在差异（P = 0.0004）。溃疡组fb的SAβ-gal活性平均百分比，pu-fb为11.2%±3.1%，du-fb为63.8%±8.9%（P = 0.0001）。在早期和晚期传代时，pu-fb和du-fb之间的SAβ-gal活性在各传代中均显示出显著差异（P <0.05）。反流组fb或pu-fb与pr-fb之间的SAβ-gal活性无差异，但du-fb和dr-fb之间的比较具有显著性（63.8%±8.9%对7.8%±2.9%；P = 0.0001）。
慢性静脉溃疡患者du-fb和pu-fb的体外传代对T以及通过SAβ-gal活性测定的细胞衰老有影响。我们的数据进一步表明，与pu-fb相比，du-fb处于细胞衰老的更进展阶段，更重要的是与无溃疡的静脉反流患者培养的fb相比。这些发现与静脉淤滞性溃疡伤口愈合受损一致。衰老fb的积累以及du-fb更高级的细胞衰老阶段可能解释了为什么静脉溃疡的反复发作对保守治疗有抵抗性，需要更积极的治疗措施。
