CN112379101A - 一种VEGF-CXCL8-Akt的Crosstalk机制的研究方法及其应用 - Google Patents
一种VEGF-CXCL8-Akt的Crosstalk机制的研究方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种VEGF‑CXCL8‑Akt的Crosstalk机制的研究方法及其应用，首先用ELISA检测U87MG、U251及其分泌因子VEGF对HBMEC分泌CXCL8的影响；其次在共培养体系中分别加入CXCL8‑Akt信号通路的特异性激动剂CXCL8和抑制剂LY294002，再用Western blotting检测共培养及激活/抑制对U87MG及U251细胞Akt蛋白及其磷酸化活化状态P‑Akt表达的影响；然后用CCK‑8、Transwell侵袭实验分别检测共培养及激活/抑制对U87MG及U251细胞增殖和侵袭的影响。
Description
技术领域
本发明属于生物医药技术领域，涉及一种VEGF-CXCL8-Akt的Crosstalk机制的研究方法及其应用，具体地说，涉及一种从信号网络的角度对胶质瘤细胞与血管内皮细胞之间VEGF-CXCL8-Akt的Crosstalk机制进行研究的研究方法与应用。
背景技术
胶质瘤是一种血管依赖性极强的实体瘤，当瘤体长到直径超过1～2mm时，就必需周围血管提供额外支持，因此肿瘤和周围血管的互动机制就非常重要。研究证实，血管形成因子是沟通胶质瘤细胞/内皮细胞之间信号crosstalk机制的重要桥梁，而血管内皮生长因子(Vascular endothelial growth factor，VEGF)又是其中最重要的因子之一。胶质瘤细胞诱导VEGF表达升高，可以刺激周围血管内皮细胞内多种细胞因子和生长因子的表达水平，而后者正是激发肿瘤细胞持续恶性生长的信号分子。它们作为配体，与肿瘤细胞表面的特异性受体识别结合后，启动细胞内异常信号转导通路，参与调控肿瘤细胞的克隆性存活、增殖、凋亡、侵袭和血管形成。
目前研究表明，VEGF可以刺激上调表达的细胞因子和生长因子种类非常多，可以激活胶质瘤细胞中多条信号转导通路。CXCL8/Akt通路即其中最重要的细胞内信号转导通道之一。在许多胶质瘤临床标本、原代培养细胞以及细胞系中，可以普遍地检测到持续磷酸化活化的Akt分子。而异常激活的Akt信号通路，可通过诱导下游bcl-xl、bcl-2、c-myc、cyclinD1、survivin、mcl-1和VEGF等多种基因的表达，调节胶质瘤细胞的生长、分化、凋亡及新生血管形成等生物学行为。反之，靶向阻断Akt，可有效下调胶质瘤细胞内多种致癌基因的表达水平，明显抑制肿瘤细胞增殖。这些结果提示，CXCL8/Akt通路在胶质瘤发病中起着非常重要的作用。
胶质瘤是中枢神经系统最为常见的原发性肿瘤，目前治疗效果不佳。新近研究结果表明，恶性胶质瘤患者的中位生存期仅为12～15个月。胶质瘤的发病是一个多因素参与、多基因改变、多步骤演进的复杂过程，也是肿瘤细胞与周围微环境中的各种相关因素相互影响、相互作用的结果。这种相互作用不仅表现为肿瘤细胞与其周围环境的物质交换和能量代谢，更重要的是两者之间的信息传递，即信号Crosstalk机制。研究证实，血管内皮生长因子(Vascular endothelial growth factor，VEGF)是沟通胶质瘤细胞与内皮细胞之间信号crosstalk机制的重要桥梁，可以激活胶质瘤细胞中多条信号转导通路，参与其克隆性存活、增殖、凋亡、侵袭和血管形成的调控，其中就包括CXCL8-Akt信号通路。
发明内容
本发明的目的在于提供一种VEGF-CXCL8-Akt的Crosstalk机制的研究方法及其应用，该方法从信号网络的角度对胶质瘤细胞与血管内皮细胞之间VEGF-CXCL8-Akt的Crosstalk机制进行研究。建立人脑胶质瘤细胞U87MG、U25 1与人脑微血管内皮细胞HBMEC的Transwell共培养体系，首先用ELISA检测U87MG、U251及其分泌因子VEGF对HBMEC分泌CXCL8的影响；其次在共培养体系中分别加入CXCL8-Akt信号通路的特异性激动剂CXCL8和抑制剂LY294002，再用Western blotting检测共培养及激活/抑制对U87MG及U251细胞Akt蛋白及其磷酸化活化状态(P-Akt)表达的影响；然后用CCK-8、Transwell侵袭实验分别检测共培养及激活/抑制对U87MG及U251细胞增殖和侵袭的影响。
其技术方案如下：
一种VEGF-CXCL8-Akt的Crosstalk机制的研究方法，包括以下步骤：
步骤1、共培养体系建立和药物的干预
1.1检测胶质瘤细胞对血管内皮细胞影响时的分组及干预共分为四组；
1.2检测血管内皮细胞对胶质瘤细胞影响的分组及干预U87MG/U251细胞各分为四组；
步骤2、ELISA检测胶质瘤细胞对血管内皮细胞CXCL8分泌的影响
将步骤1.1中四组细胞各培养24、48、72h时收集HBMEC培养上清液，按ELISA检测试剂盒说明书操作，检测CXCL8含量。取不同浓度标准品和稀释好的样品加入ELISA板孔中，25℃孵育2h，洗板5次，加入兔抗人CXCL8生物素化抗体工作液(100μl/孔)，室温孵育1h，洗板5次，加入HRP标记的山羊抗兔IgG工作液(100μl/孔)孵育20min，加入终止液50μl/孔，酶标仪检测450nm波长处的光密度(D)值。绘制浓度值标准曲线，根据标准曲线计算对照组及实验组相应的浓度值。
步骤3、Western blotting检测血管内皮细胞对胶质瘤细胞Akt/P-Akt表达的影响
将步骤1.2中各组细胞培养至70％～80％的融合度后收集下室肿瘤细胞，加入预冷的细胞裂解液在冰浴中充分裂解细胞，提取全蛋白，BCA法分析定量。取10μg总蛋白样品，用4％～12％TrisGlycine胶进行SDS-PAGE电泳，并转移至PVDF膜上，用TBST配制的牛奶封闭1h，加入一抗(体积稀释比为1∶2000)4℃孵育过夜。其后用TBST洗膜5min×5次，用荧光二抗(体积稀释比为1∶5000)避光室温孵育1h，TBST洗膜5min×5次后用奥德赛(ODYSSEY CLx)曝光。扫描灰度并计算蛋白相应的表达量。
步骤4、CCK-8检测血管内皮细胞对胶质瘤细胞增殖的影响
将步骤1.2中各组细胞按照5×104个接种于24孔板内在37℃、5％CO2条件下培养24h、48h、72h后，每孔加入100μl CCK-8液，继续在培养箱中孵育1h，酶标仪测定450nm波长处吸光度(D)值，代表细胞的增殖水平。
步骤5、Transwell实验检测血管内皮细胞对胶质瘤细胞侵袭的影响
将BD基质胶置于冰水浴中过夜溶解，在超净工作台中将其用无血清培养基稀释到浓度为10mg/ml，每孔吸取100μl轻轻铺到孔径为8μm的聚碳酸酯滤膜Transwell小室内，摇匀置于37℃孵育箱聚合5h。用胰蛋白酶消化处于对数生长期的U87MG/U251细胞，离心弃上清后用无血清DMEM重悬，调整细胞终浓度为5×105个/ml，在Transwell小室上层加入200μl，并根据分组情况分别加入50ng/m的rhCXCL8或1μmol/L的LY294002干预。小室下层按照1.0×106个/ml接种HBMEC细胞600μl，然后将整个Transwell小室置于37℃、5％CO2的培养箱中孵育72h。取出小室，棉签擦去上层细胞；PBS清洗；4％多聚甲醛固定15min；0.1％结晶紫染色30min；PBS清洗；自然干燥；显微镜下随机取五个视野拍照观察计数穿Transwell小室细胞(200×)。每组实验均重复5次。
步骤6、统计学处理
应用SPSS 18.0软件进行统计学分析，计量资料以均数±标准差(
)表示。各组间比较采用单因素方差分析，以P＜0.05为差异有统计学意义。
进一步，步骤1.1中的四组具体为：(1)对照组：将对数生长期的HBMEC接种于ECM中常规培养，(2)VEGF组：将对数生长期的HBMEC接种于含50ng/ml VEGF165的ECM中培养，(3)U87MG共培养组：将对数生长期的HBMEC接种于Transwell下室ECM中，U87MG接种于上室，置于37℃、5％CO2中培养，(4)U251共培养组：将对数生长期的HBMEC接种于Transwell下室ECM中，U251接种于上室，置于37℃、5％CO2培养箱中培养。
进一步，步骤1.2中的四组具体为：(1)对照组：将对数生长期的U87MG/U251接种于DMEM中常规培养，(2)U87MG/U251+HBMEC组：将对数生长期的U87MG/U251接种于Transwell下室常规DMEM中，HBMEC接种于上室，置于37℃、5％CO2培养箱中培养。(3)U87MG/U251+HBMEC+rhCXCL8组：将对数生长期的U87MG/U251接种于Transwell下室含50ng/ml rhCXCL8的DMEM中，HBMEC接种于上室，置于37℃、5％CO2培养箱中培养，(4)U87MG/U251+HBMEC+rhCXCL8组：将对数生长期的U87MG/U251接种于Transwell下室含1μmol/L LY294002的DMEM中，HBMEC接种于上室，置于37℃、5％CO2条件下培养。
进一步，步骤3中，将步骤1.2中各组细胞培养至75％的融合度后收集下室肿瘤细胞。
进一步，步骤3中，用8％TrisGlycine胶进行SDS-PAGE电泳。
本发明所述方法在治疗人脑胶质瘤的药物制备过程中的应用。
本发明的有益效果：
本发明的研究结果为VEGF干预、分别和U87MG、U251细胞共培养后，HBMEC培养体系的CXCL8分泌量显著高于对照组(P＜0.01)。与HBMEC共培养后，U87MG、U251细胞Akt/P-Akt蛋白的表达明显增高(P＜0.05)；给予CXCL8激活使蛋白表达进一步升高(P＜0.05)，而给予LY294002抑制则使蛋白的表达显著降低(P＜0.05)。与HBMEC共培养后，U87MG、U251细胞的增殖及侵袭能力明显增高(P＜0.05)；给予CXCL8激活使其水平更高(P＜0.05)，而给予LY294002抑制则使两种肿瘤细胞的增殖及侵袭能力显著降低(P＜0.05)。研究结论为：胶质瘤细胞与血管内皮细胞之间存在VEGF-CXCL8-Akt信号网络的交互作用。胶质瘤细胞与血管内皮细胞之间存在VEGF-CXCL8-Akt的Crosstalk机制，形成了肿瘤促进血管、血管进一步供养肿瘤，最终使肿瘤无限增殖侵袭的恶性循环。在目前大家越来越多地倾向于从网络角度认识胶质瘤发病机制的情况下，该Crosstalk机制的揭示为其临床综合治疗提供新的靶点和策略提供依据。
附图说明
图1是HBMEC以及CXCL8、LY294002对U87MG(A)和U251细胞(B)Akt、P-Akt表达的影响；
图2是HBMEC以及CXCL8、LY294002对U87MG(A)和U251细胞(B)增殖的影响；
图3A是HBMEC以及CXCL8、LY294002对U87MG和U251细胞侵袭的影响；
图3B是HBMEC以及CXCL8、LY294002对U87MG细胞侵袭影响的分析；
图3C是HBMEC以及CXCL8、LY294002对U251细胞侵袭影响的分析。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案作进一步详细地说明。
1材料和方法
1.1主要试剂材料
人脑胶质瘤细胞系U87MG、U251购自中科院上海细胞资源中心，均为胶质母细胞瘤单克隆成系，WHO分级标准为IV级。置于含10％胎牛血清的DMEM培养基中培养(DMEM培养液、胰蛋白酶购自美国Gibco公司、胎牛血清购自美国HyClone公司)。人脑微血管内皮细胞(human brain microvascular endothelial cell，HBMEC)购于美国Sciencell公司，置于含10％胎牛血清的ECM培养基中培养(ECM培养液、胎牛血清购自美国Sciencell公司)。VEGF165、rhCXCL8购自美国Pepro Tech公司，LY294002购自美国Selleckchem公司。兔抗人Akt及Phospho Akt单克隆抗体购自英国Abcam公司，兔抗人GAPDH多克隆抗体(AB-P-R001)购自杭州贤至生物科技有限公司，荧光二抗Goat anti-Rabbit 926-32211购自美国li-cor公司。Transwell小室购自美国Coming公司，BD基质胶购自美国Sigma公司，ELISA试剂盒购自上海巧伊生物科技有限公司，CCK-8试剂盒购自北京全式金生物技术有限公司，Westernblotting相关试剂(盒)购自南京凯基生物科技发展有限公司。
1.2共培养体系建立和药物的干预
1.2.1检测胶质瘤细胞对血管内皮细胞影响时的分组及干预共分为四组：(1)对照组：将对数生长期的HBMEC接种于ECM中常规培养，(2)VEGF组：将对数生长期的HBMEC接种于含50ng/ml VEGF165的ECM中培养，(3)U87MG共培养组：将对数生长期的HBMEC接种于Transwell下室ECM中，U87MG接种于上室，置于37℃、5％CO2中培养，(4)U251共培养组：将对数生长期的HBMEC接种于Transwell下室ECM中，U251接种于上室，置于37℃、5％CO2培养箱中培养。
1.2.2检测血管内皮细胞对胶质瘤细胞影响的分组及干预U87MG/U251细胞各分为四组：(1)对照组：将对数生长期的U87MG/U251接种于DMEM中常规培养，(2)U87MG/U251+HBMEC组：将对数生长期的U87MG/U251接种于Transwell下室常规DMEM中，HBMEC接种于上室，置于37℃、5％CO2培养箱中培养。(3)U87MG/U251+HBMEC+rhCXCL8组：将对数生长期的U87MG/U251接种于Transwell下室含50ng/ml rhCXCL8的DMEM中，HBMEC接种于上室，置于37℃、5％CO2培养箱中培养，(4)U87MG/U251+HBMEC+rhCXCL8组：将对数生长期的U87MG/U251接种于Transwell下室含1μmol/L LY294002的DMEM中，HBMEC接种于上室，置于37℃、5％CO2条件下培养。
1.3 ELISA检测胶质瘤细胞对血管内皮细胞CXCL8分泌的影响
将1.2.1中四组细胞各培养24、48、72h时收集HBMEC培养上清液，按ELISA检测试剂盒说明书操作，检测CXCL8含量。取不同浓度标准品和稀释好的样品加入ELISA板孔中，25℃孵育2h，洗板5次，加入兔抗人CXCL8生物素化抗体工作液(100μl/孔)，室温孵育1h，洗板5次，加入HRP标记的山羊抗兔IgG工作液(100μl/孔)孵育20min，加入终止液50μl/孔，酶标仪检测450nm波长处的光密度(D)值。绘制浓度值标准曲线，根据标准曲线计算对照组及实验组相应的浓度值。
1.4 Western blotting检测血管内皮细胞对胶质瘤细胞Akt/P-Akt表达的影响
将1.2.2中各组细胞培养至70％～80％的融合度(本数值范围内优选数值点为：75％，)后收集下室肿瘤细胞，加入预冷的细胞裂解液在冰浴中充分裂解细胞，提取全蛋白，BCA法分析定量。取10μg总蛋白样品，用4％～12％TrisGlycine胶(本数值范围内优选数值点为：8％)进行SDS-PAGE电泳，并转移至PVDF膜上，用TBST配制的牛奶封闭1h，加入一抗(体积稀释比为1∶2000)4℃孵育过夜。其后用TBST洗膜5min×5次，用荧光二抗(体积稀释比为1∶5000)避光室温孵育1h，TBST洗膜5min×5次后用奥德赛(ODYSSEY CLx)曝光。扫描灰度并计算蛋白相应的表达量。
1.5 CCK-8检测血管内皮细胞对胶质瘤细胞增殖的影响
将1.2.2中各组细胞按照5×104个接种于24孔板内在37℃、5％CO2条件下培养24h、48h、72h后，每孔加入100μl CCK-8液，继续在培养箱中孵育1h，酶标仪测定450nm波长处吸光度(D)值，代表细胞的增殖水平。
1.6 Transwell实验检测血管内皮细胞对胶质瘤细胞侵袭的影响
将BD基质胶置于冰水浴中过夜溶解，在超净工作台中将其用无血清培养基稀释到浓度为10mg/ml，每孔吸取100μl轻轻铺到孔径为8μm的聚碳酸酯滤膜Transwell小室内，摇匀置于37℃孵育箱聚合5h。用胰蛋白酶消化处于对数生长期的U87MG/U251细胞，离心弃上清后用无血清DMEM重悬，调整细胞终浓度为5×105个/ml，在Transwell小室上层加入200μl，并根据分组情况分别加入50ng/m的rhCXCL8或1μmol/L的LY294002干预。小室下层按照1.0×106个/ml接种HBMEC细胞600μl，然后将整个Transwell小室置于37℃、5％CO2的培养箱中孵育72h。取出小室，棉签擦去上层细胞；PBS清洗；4％多聚甲醛固定15min；0.1％结晶紫染色30min；PBS清洗；自然干燥；显微镜下随机取五个视野拍照观察计数穿Transwell小室细胞(200×)。每组实验均重复5次。
1.7统计学处理
应用SPSS 18.0软件进行统计学分析，计量资料以均数±标准差(
)表示。各组间比较采用单因素方差分析，以P＜0.05为差异有统计学意义。
2结果
2.1 VEGF及U87MG、U251细胞共培养促进HBMEC分泌CXCL8
如表1所示，VEGF干预或者分别与U87MG、U251细胞共培养24h后，ELISA检测结果即显示HBMEC培养液中CXCL8的分泌量显著增高(P＜0.01)。其后时间分别延长到48h、72h，CXCL8分泌的检测值进一步随着时间逐渐增高(P＜0.01)。
表1 VEGF及U87MG、U251细胞共培养促进HBMEC分泌CXCL8[ρB/(ng·L-1)]
**P＜0.01 vs control group.
2.2 HBMEC对U87MG和U251细胞Akt及P-Akt表达的影响
Western blotting检测结果(图1)显示，与HBMEC共培养后，U87MG、U251细胞P-Akt蛋白的表达明显增加；如果同时给予CXCL8干预使P-Akt蛋白表达进一步升高；而给予LY294002干预则使P-Akt蛋白的表达受到抑制。
2.3 2.4内皮细胞及其分泌因子对胶质瘤细胞增殖影响
CCK-8检测结果(图2)显示，与HBMEC共培养后，U87MG、U251细胞的增殖能力显著提高(P＜0.01)；给予CXCL8干预能够使其增殖能力更高(P＜0.01)；而给予LY294002抑制则使两种肿瘤细胞的增殖能力降低(P＜0.05)。
2.4内皮细胞及其分泌因子对胶质瘤细胞侵袭影响
Transwell侵袭实验检测结果(图3A-图3C)显示，与HBMEC共培养后，U87MG、U251细胞的增殖能力显著提高(P＜0.01)；给予CXCL8干预能够使其增殖能力更高(P＜0.01)；而给予LY294002抑制则使两种肿瘤细胞的增殖能力降低(P＜0.05)。
本研究首先探讨了VEGF能否促进微血管内皮分泌CXCL8。作为过量表达对照组，采用外源性VEGF165(50ng/ml)刺激后，HBMEC分泌的CXCL8明显增高。而和U87MG、U251细胞共培养后，HBMEC培养体系中CXCL8的分泌量也显示了类似的显著增高，表明胶质瘤细胞诱导产生的内源性细胞/组织因子能够促进血管内皮细胞CXCL8的分泌，而结果分析可知VEGF正是这些因子中主要发挥作用的成分之一。从而验证了胶质瘤细胞通过影响诱导VEGF表达促进周围血管内皮细胞增大CXCL8的分泌量，由此形成了细胞间信号由胶质瘤细胞向周围血管内皮细胞的“传话”。
接着探讨了CXCL8能否使胶质瘤细胞Akt/P-Akt表达增加？结果显示，与HBMEC共培养使U87MG、U251中Akt/P-Akt的表达显著提高，加入CXCL8则使其进一步提升，而加入Akt通路的抑制剂LY294002则可以逆转这个提高。这个结果表明CXCL8(不管是内源性还是外源性)能够激活胶质瘤细胞Akt信号通路。在此基础上对其效应进行进一步探讨。与HBMEC共培养使U87MG、U251的增殖和侵袭均显著增加的结果表明，与血管内皮细胞相互作用能够有效促进胶质瘤细胞增殖和侵袭。而加入CXCL8和LY294002后，增殖和侵袭分别受到了增强和抑制，说明这两种效应是通过CXCL8/Akt通路来实现的。从而验证了血管内皮细胞通过分泌CXCL8，激活胶质瘤细胞的Akt信号通路，促进其增殖和侵袭，由此形成了细胞间信号由瘤周血管内皮细胞向胶质瘤细胞的“传话”。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (6)
1.一种VEGF-CXCL8-Akt的Crosstalk机制的研究方法，其特征在于，包括以下步骤：
步骤1、共培养体系建立和药物的干预
1.1检测胶质瘤细胞对血管内皮细胞影响时的分组及干预共分为四组；
1.2检测血管内皮细胞对胶质瘤细胞影响的分组及干预U87MG/U251细胞各分为四组；
步骤2、ELISA检测胶质瘤细胞对血管内皮细胞CXCL8分泌的影响
将步骤1.1中四组细胞各培养24、48、72h时收集HBMEC培养上清液，按ELISA检测试剂盒说明书操作，检测CXCL8含量；取不同浓度标准品和稀释好的样品加入ELISA板孔中，25℃孵育2h，洗板5次，加入兔抗人CXCL8生物素化抗体工作液，室温孵育1h，洗板5次，加入HRP标记的山羊抗兔IgG工作液(100μl/孔)孵育20min，加入终止液50μl/孔，酶标仪检测450nm波长处的光密度(D)值；绘制浓度值标准曲线，根据标准曲线计算对照组及实验组相应的浓度值；
步骤3、Western blotting检测血管内皮细胞对胶质瘤细胞Akt/P-Akt表达的影响
将步骤1.2中各组细胞培养至70％～80％的融合度后收集下室肿瘤细胞，加入预冷的细胞裂解液在冰浴中充分裂解细胞，提取全蛋白，BCA法分析定量；取10μg总蛋白样品，用4％～12％TrisGlycine胶进行SDS-PAGE电泳，并转移至PVDF膜上，用TBST配制的牛奶封闭1h，加入体积稀释比为1∶2000的一抗4℃孵育过夜；其后用TBST洗膜5min×5次，用体积稀释比为1∶5000的荧光二抗避光室温孵育1h，TBST洗膜5min×5次后用奥德赛ODYSSEY CLx曝光；扫描灰度并计算蛋白相应的表达量；
步骤4、CCK-8检测血管内皮细胞对胶质瘤细胞增殖的影响
将步骤1.2中各组细胞按照5×104个接种于24孔板内在37℃、5％CO2条件下培养24h、48h、72h后，每孔加入100μl CCK-8液，继续在培养箱中孵育1h，酶标仪测定450nm波长处吸光度(D)值，代表细胞的增殖水平；
步骤5、Transwell实验检测血管内皮细胞对胶质瘤细胞侵袭的影响
将BD基质胶置于冰水浴中过夜溶解，在超净工作台中将其用无血清培养基稀释到浓度为10mg/ml，每孔吸取100μl轻轻铺到孔径为8μm的聚碳酸酯滤膜Transwell小室内，摇匀置于37℃孵育箱聚合5h；用胰蛋白酶消化处于对数生长期的U87MG/U251细胞，离心弃上清后用无血清DMEM重悬，调整细胞终浓度为5×105个/ml，在Transwell小室上层加入200μl，并根据分组情况分别加入50ng/m的rhCXCL8或1μmol/L的LY294002干预；小室下层按照1.0×106个/ml接种HBMEC细胞600μl，然后将整个Transwell小室置于37℃、5％CO2的培养箱中孵育72h；取出小室，棉签擦去上层细胞；PBS清洗；4％多聚甲醛固定15min；0.1％结晶紫染色30min；PBS清洗；自然干燥；显微镜下随机取五个视野拍照观察计数穿Transwell小室细胞200×；每组实验均重复5次；
步骤6、统计学处理
应用SPSS 18.0软件进行统计学分析，计量资料以均数±标准差
表示；各组间比较采用单因素方差分析，以P＜0.05为差异有统计学意义。
2.根据权利要求1所述的VEGF-CXCL8-Akt的Crosstalk机制的研究方法，其特征在于，步骤1.1中的四组具体为：(1)对照组：将对数生长期的HBMEC接种于ECM中常规培养，(2)VEGF组：将对数生长期的HBMEC接种于含50ng/ml VEGF165的ECM中培养，(3)U87MG共培养组：将对数生长期的HBMEC接种于Transwell下室ECM中，U87MG接种于上室，置于37℃、5％CO2中培养，(4)U251共培养组：将对数生长期的HBMEC接种于Transwell下室ECM中，U251接种于上室，置于37℃、5％CO2培养箱中培养。
3.根据权利要求1所述的VEGF-CXCL8-Akt的Crosstalk机制的研究方法，其特征在于，步骤1.2中的四组具体为：(1)对照组：将对数生长期的U87MG/U251接种于DMEM中常规培养，(2)U87MG/U251+HBMEC组：将对数生长期的U87MG/U251接种于Transwell下室常规DMEM中，HBMEC接种于上室，置于37℃、5％CO2培养箱中培养；(3)U87MG/U251+HBMEC+rhCXCL8组：将对数生长期的U87MG/U251接种于Transwell下室含50ng/ml rhCXCL8的DMEM中，HBMEC接种于上室，置于37℃、5％CO2培养箱中培养，(4)U87MG/U251+HBMEC+rhCXCL8组：将对数生长期的U87MG/U251接种于Transwell下室含1μmol/L LY294002的DMEM中，HBMEC接种于上室，置于37℃、5％CO2条件下培养。
4.根据权利要求1所述的VEGF-CXCL8-Akt的Crosstalk机制的研究方法，其特征在于，步骤3中，将步骤1.2中各组细胞培养至75％的融合度后收集下室肿瘤细胞。
5.根据权利要求1所述的VEGF-CXCL8-Akt的Crosstalk机制的研究方法，其特征在于，步骤3中，用8％TrisGlycine胶进行SDS-PAGE电泳。
6.一种权利要求1所述的方法在治疗人脑胶质瘤的药物制备过程中的应用。
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