血管生成抑制因子1对胶质母细胞瘤的治疗作用及其机制的实验研究

Therapeutic effect of brain-specific angiogenesis inhibitor 1 on glioblastoma: an animal experiment

目的探讨脑组织特异性血管生成抑制因子1(BAI1)对胶质母细胞瘤的治疗作用及其作用机制。方法采用COS-TPC法进行BAI1-腺病毒载体的构建,病毒重组子的成功构建和对肿瘤细胞的转染通过RT-PCR来验证。采用立体定向的方法将胶质母细胞瘤细胞U87MG接种于裸鼠脑内,待肿瘤形成后向瘤内注射病毒重组子AdeBAI1(AdeBAI1组)或AdeLacZ(Mock)(AdeMock组),两组均为6只裸鼠。观察裸鼠的存活时间。用AdeBAI1或AdeMock转染3个胶质母细胞瘤细胞系SW1783、U87MG和U373MG,48h后收集细胞,用MTT方法进行活细胞计数。用Trizol试剂提取总RNA。用RT-PCR方法研究BAI1及其他血管生成相关因子的mRNA的表达。结果BAI1mRNA的表达仅见于AdeBAI1转染的细胞中。颅内胶质母细胞瘤治疗结果显示,AdeBAI1组平均生成期为(26·0±4·6)d,AdeMock治疗组为(17·3±2·3)d,两组比较P<0·05。AdeBAI1组转染后细胞计数为(2·12±0·18)×105、AdeMock组为(4·23±0·18)×105,两组比较P<0·05。提示胶质母细胞瘤细胞的增殖可被BAI1抑制。AdeBAI1转染后血管生成相关因子Angiostatin和VEGF的表达降低,而VEGF-B和TSP1的表达升高。结论肿瘤内注射AdeBAI1可以抑制胶质母细胞瘤的生长。BAI1的表达升高可以影响其他血管生成相关因子的表达。BAI1的表达升高可以抑制肿瘤细胞的增殖作用。BAI1的抗肿瘤作用可能来自于抑制血管生成以及抑制肿瘤细胞的增殖两个方面。

Objective To study the therapeutic effects of brain-specific angiogenesis inhibitor 1 （BAI1） on human glioblastoma and relevant mechanism. Methods Recombinant adenovirus carrying human BAIl eDNA, AdeBAI1 and recombinant adenovirus carrying LacZ, AdeMock, were constructed with the COS-TPC method. The successful construction of AdeBAIland expression of AdeBAI1 was verified using RT-PCR. Glioblastoma cells of the line U87MG were transplanted into the mice brain using stereotactic technique. AdeBAI1 and AdeMock were injected into the tumors after the tumors were developed. The survival of the mice was observed. Human glioblastoma cells of the lines SW1783, U87MG, and U373MG were cultured and transfected with AdeBAI1 or AdeMock, and then collected 48 hours later and counted using MTT method. The total RNA was extracted using Trizol agent. The mRNA of BAIl and other angiogenesis related genes were detected using RT-PCR. Results The mean survival time of the AdBAI1- treated mice was 26 2 4.6 d, significantly longer than that of the AdMock-treated mice （ 17.3± 2.3 d, P〈0.05）. RT-PCR showed that BAIl mRNA was expressed only in the glioblastoma cells transfected with AdeBAI1. The number of AdeBAI1 treated glioblastoma cells was 2. 12 20. 18 x 105 , significantly less than that of the AdeMock treated cells （4.23 20. 18×10^5, P〈0.05）. The mRNA expression of angiostatin of the AdeBAI1 treated cells was 0.66 ±0.08, significantly less than that of the AdMock-treated cells （0.95±0.12, P〈0.05）. The mRNA expression of vascular endothelial growth factor （VEGF） of the AdeBAI1 treated cells was 0.68±0.07, significantly less than that of the AdMock-treated cells （ 1.02±0. 14, P〈0.05）. The mRNA expression of VEGF-B of the AdeBAI1 treated cells was 1.11±0.10, significantly more than that of the AdMock-treated cells （0.77 ±0.18, P〈0.05）. The mRNA expression of thrombospondin of the AdeBAI1 treated cells was 1.16±0.16, significantly more than that of the AdMock-treated cells （0.60±0.22, P〈0.05）. Conclusion Intratumor injection of AdeBAI1 can inhibit the tumor growth. The anti-tumor effect of BAIl may arise from both anti-angiogenesis and anti-proliferation effects.