Oku Takatomi, Iyama Satoshi, Sato Tsutomu, Sato Yasushi, Tanaka Maki, Sagawa Tamotsu, Kuribayashi Kageaki, Sumiyosi Tetsuya, Murase Kazuyuki, Machida Takuro, Okamoto Tetsuro, Matsunaga Takuya, Takayama Tetsuji, Takahashi Minoru, Kato Junji, Hamada Hirofumi, Niitsu Yoshiro
Department of Internal Medicine (Section 4), Sapporo Medical University, School of Medicine, Sapporo, Japan.
Inflamm Bowel Dis. 2006 Jul;12(7):630-40. doi: 10.1097/01.MIB.0000225335.68614.73.
Although the etiology of inflammatory bowel disease has not been fully clarified, reactive oxygen species is speculated to be involved. Extracellular superoxide dismutase (EC-SOD), an isozyme of SODs, is known to function mainly in body fluids. We investigated the efficacy of an ex vivo EC-SOD gene transfer into dextran sulfate sodium (DSS)-induced colitis mice.
Experimental colitis was induced by providing Balb/c mice with DSS in sterile distilled water provided as desired. The syngenic fibroblasts were obtained from Balb/c mice embryos and retrovirally transduced with the hEC-SOD gene. These engineered cells were confirmed to secrete EC-SOD in culture medium by enzyme-linked immunosorbent assay and were inoculated subcutaneously in the backs of DSS-treated mice. Mucosal injury of the colon was evaluated by the disease activity index (DAI: body weight, rectal bleeding, and stool consistency), grading of histologic disease severity, and levels of cytokine (tumor necrosis factor-alpha, interleukin-1beta) production. 8-Hydroxydeoxyguanosine (8-OHdG) levels in the mucosal tissue were assessed by immunohistochemical staining. Malondialdehyde (MDA) was measured using a colorimetric assay.
A significant improvement was observed in DAI score and histologic severity as well as in mucosal tissue levels of inflammatory cytokines, 8-OHdG, and MDA of mice treated with the EC-SOD gene as compared with those without gene therapy, not only in a mild colitis model but also in a severe colitis model. Survival of treated mice in these models was significantly prolonged.
Ex vivo transfer of the EC-SOD gene was feasible for treatment of DSS-induced colitis.
尽管炎症性肠病的病因尚未完全阐明，但推测活性氧参与其中。细胞外超氧化物歧化酶（EC-SOD）是超氧化物歧化酶的一种同工酶，主要在体液中发挥作用。我们研究了将EC-SOD基因体外转移到葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠体内的疗效。
通过按需要向Balb/c小鼠提供无菌蒸馏水中的DSS来诱导实验性结肠炎。从Balb/c小鼠胚胎中获得同基因成纤维细胞，并用hEC-SOD基因进行逆转录病毒转导。通过酶联免疫吸附测定法确认这些工程细胞在培养基中分泌EC-SOD，并将其皮下接种到DSS处理的小鼠背部。通过疾病活动指数（DAI：体重、直肠出血和粪便稠度）、组织学疾病严重程度分级以及细胞因子（肿瘤坏死因子-α、白细胞介素-1β）产生水平来评估结肠的黏膜损伤。通过免疫组织化学染色评估黏膜组织中的8-羟基脱氧鸟苷（8-OHdG）水平。使用比色法测量丙二醛（MDA）。
与未进行基因治疗的小鼠相比，用EC-SOD基因治疗的小鼠在DAI评分、组织学严重程度以及炎症细胞因子、8-OHdG和MDA的黏膜组织水平方面均有显著改善，不仅在轻度结肠炎模型中如此，在重度结肠炎模型中也是如此。这些模型中治疗小鼠的生存期显著延长。
EC-SOD基因的体外转移对于治疗DSS诱导的结肠炎是可行的。
