【摘要】 目的:通过观察脑胶质瘤大鼠动物模型中明胶酶的活性变化,为以明胶酶为靶点的脑胶质瘤治疗奠定实验基础。方法:实验于2004-10/2005-12在解放军军事医学科学院野战输血研究所干细胞生物学研究室完成。9L大鼠胶质瘤细胞购自广州南方医科大学。①选取清洁级健康成年雄性Fisher344大鼠27只,24只建立大鼠脑胶质瘤模型,造模后随机数字表法分为建模后7,14,21,28d组,6只/组,每组3只用于灌注固定做免疫组化分析,另3只用于反转录多聚酶链反应和明胶酶谱分析。剩余3只大鼠作为正常对照。②采用反转录多聚酶链反应技术、免疫组织化学和明胶酶谱等方法检测胶质瘤中明胶酶2(matrixmetalloproteinase2,MMP-2)和明胶酶9(matrixmetalloproteinase2,MMP-9)的活性变化。结果:实验选取清洁级健康成年雄性Fisher344大鼠27只,全部进入结果分析。①MMP-2和MMP-9mRNA的表达:9L胶质瘤细胞中均可见MMP-2和MMP-9mRNA的表达,MMP-2的表达量高于MMP-9;大鼠脑胶质瘤组织中可见两者mRNA的表达,而正常脑组织MMP-2mRNA低表达,检测不到MMP-9mRNA的表达;随着肿瘤的生长,MMP-9mRNA的表达量增高。②MMP-2和MMP-9免疫组织化学观察结果:9L胶质瘤细胞的免疫组化显示,MMP-2和MMP-9均呈现阳性染色,主要为胞浆和胞膜染色;肿瘤组织MMP-2和MMP-9染色阳性,为胞浆染色,在血管内皮和肿瘤侵袭边缘细胞胞阳性染色明显。随脑胶质瘤的生长,MMP-2和MMP-9染色程度加深。③MMP-2和MMP-9酶活性检测结果:9L细胞无血清培养上清中可见Pro-MMP2(Mr72000)与Pro-MMP9(Mr92000)及其活化形式activeMMP-2(Mr66000)与activeMMP-9(Mr83000)的表达,MMP-2的表达量较高;MMP-2酶原与活化的MMP-2在脑胶质瘤组织中明显表达,而在正常脑组织低表达。MMP-9酶原与活化的MMP-9在脑肿瘤组织中表达,正常脑组织中未检测到其表达。④MMP-2和MMP-9在肿瘤组织中的定位情况:脑胶质瘤组织冰冻切片原位明胶酶谱结果显示,肿瘤组织因表达活性的明胶酶降解载片上的明胶,致使不被染色,肿瘤部位很透亮。对侧脑组织因不表达明胶酶,不能降解明胶,被染黑。结论:脑胶质瘤模型中表达高活性的MMP-2和MMP-9,且与胶质瘤生长速度密切相关。更多还原

【Abstract】 AIM: To study the change of activity of gelatinase in glioma rat models and provide experimental basis for glioma taking gelatinase as target. METHODS: The experiment was conducted at the Research Center of Stem Cell and Biology, Institute of Transfusion, Academy of Military Medical Science of Chinese PLA from October 2004 to December 2005. 9L glioma cells were purchased from Southern Medical University. ①A total of 27 clean healthy adult male Fisher344 rats were selected. Of them, 24 were used to establish glioma rat models, and then randomly assigned into 7, 14, 21 and 28 days groups with 6 rats in each group. Three rats from each group were fixed for immunohistochemical analysis. Another 3 rats received reverse transcription-polymerase chain reaction (RT-PCR) and gelatine zymography. The left 3 rats were as the normal control. ②Activities of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) were measured with RT-PCR, immunohistochemistry and gelatine zymography. RESULTS: Totally 27 clean healthy adult male Fisher344 rats were involved in the result analysis. ①Expressions of MMP-2 and MMP-9 mRNA: Expressions of MMP-2 and MMP-9 mRNA appeared in 9L glioma cells. MMP-2 mRNA was strongly expressed in cultured 9L glioma cells as compared with MMP-9. They all expressed in brain glioma tissue of rats, but only a trace of expression of MMP-2 mRNA was found in normal tissue, no MMP-9 expression. MMP-9 mRNA increased with the growth of glioma. ②Result of immunohistochemistry in MMP-2 and MMP-9: Immunohistochemistry of 9L glioma cells showed that marked positive staining was seen for MMP-2 and MMP-9, mainly in cytoplasmic and plasma membrane. MMP-2 and MMP-9 in tumor tissues were positive staining in cytoplasm. Vascular endothelium and tumor incursion border cells were significantly positive staining. With the growth of the gliomas the MMP-2 and MMP-9 staining became deep. ③Detection of MMP-2 and MMP-9: In the culture medium without serum of 9L glioma cells, there were Pro-MMP2 (Mr 72 000), Pro-MMP9 (Mr 92 000) and the activation pattern active MMP-2(Mr 66 000) and active MMP-9(Mr 83 000); the expression of MMP-2 was high; MMP-2 zymogen and activated MMP-2 significantly expressed in brain glioma tissues, but low expression of MMP-2 in normal rat brain. MMP-9 zymogen and activated MMP-9 were detected only in the rat brain with 9L glioma cells, but not in normal rat brain. ④Localization of MMP-2 and MMP-9 in tumor tissues: The in situ zymography in brain glioma tissue section showed that actively expressed gelatin in gelatinase degradation carried sheet in tumor tissue resulted in non-staining, and it was bright in tumor region. Because brain tissues in opposite side did not express gelatinase, could not degrade gelatin, were dye dark. CONCLUSION: The high-active expressions of MMP-2 and MMP-9 are in the brain glioma models, which are closely associated with the growth of gliomas.更多还原