Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200040, China.
Chin Med J (Engl). 2013;126(9):1720-5.
Angiogenesis is a prerequisite for tumor growth and plays an important role in rapidly growing tumors, such as malignant gliomas. A variety of factors controlling the angiogenic balance have been described, and among these, the endogenous inhibitor of angiogenesis, tumstatin, has drawn considerable attention. The current study investigated whether expression of tumstatin by glioma cells could alter this balance and prevent tumor formation.
We engineered stable transfectants from human glioma cell line U251 to constitutively secrete a human tumstatin protein with c-myc and polyhistidine tags. Production and secretion of the tumstatin-c-myc-His fusion protein by tumstatin-transfected cells were confirmed by Western blotting analysis. In the present study, we identify the anti-angiogenic capacity of tumstatin using several in vitro and in vivo assays. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine the statistical significance in this study.
The tumstatin transfectants and control transfectants (stably transfected with a control plasmid) had similar in vitro growth rates compared to their parental cell lines. However, the conditioned medium from the tumstatin transfected tumor cells significantly inhibits proliferation and causes apoptosis of endothelial cells. It also inhibits tube formation of endothelial cells on Matrigel. Examination of armpit tumors arising from cells overexpressing tumstatin repress the growth of tumor, accompanying the decreased density of CD31 positive vessels in tumors ((5.62 ± 1.32)/HP), compared to the control-transfectants group ((23.84 + 1.71)/HP) and wild type U251 glioma cells group ((29.33 + 4.45)/HP).
Anti-angiogenic gene therapy using human tumstatin gene may be an effective strategy for the treatment of glioma.
血管生成是肿瘤生长的前提，在快速生长的肿瘤中发挥着重要作用，如恶性神经胶质瘤。已经描述了多种控制血管生成平衡的因素，其中内源性血管生成抑制剂 tumstatin 引起了广泛关注。本研究探讨了胶质瘤细胞表达 tumstatin 是否可以改变这种平衡并阻止肿瘤形成。
我们通过稳定转染人神经胶质瘤细胞系 U251 来构建 tumstatin 稳定转染细胞，使其持续分泌带有 c-myc 和多组氨酸标签的人 tumstatin 蛋白。通过 Western blot 分析证实了 tumstatin 转染细胞产生和分泌 tumstatin-c-myc-His 融合蛋白。在本研究中，我们使用多种体外和体内实验来鉴定 tumstatin 的抗血管生成能力。使用 Student's t 检验和单向方差分析（ANOVA）检验来确定本研究中的统计学意义。
与亲本细胞系相比，tumstatin 转染细胞和对照转染细胞（稳定转染对照质粒）的体外生长速度相似。然而，tumstatin 转染肿瘤细胞的条件培养基显著抑制内皮细胞的增殖并诱导其凋亡，还抑制内皮细胞在 Matrigel 上的管形成。检查过表达 tumstatin 的细胞形成的腋窝肿瘤发现，与对照转染细胞组（(5.62 ± 1.32)/HP）和野生型 U251 神经胶质瘤细胞组（(29.33 + 4.45)/HP）相比，肿瘤的生长受到抑制，肿瘤中 CD31 阳性血管的密度降低（(5.62 ± 1.32)/HP）。
使用人 tumstatin 基因的抗血管生成基因治疗可能是治疗神经胶质瘤的有效策略。
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