摘要
目的:神经胶质瘤是颅内最常见的恶性肿瘤,发病率高,呈侵袭性生长并且术后容易复发,传统的各种治疗方法效果均不理想。目前神经胶质瘤的具体发病机制还不十分清楚。随着对神经胶质瘤发病机制的逐渐深入研究以及分子生物学技术的快速发展,分子靶向治疗逐渐成为一种具有广阔前景的治疗新手段。
已有研究表明,趋化因子SDF-1及其受体CXCR4在神经胶质瘤发生和发展过程中发挥着重要而复杂的作用,他们能促进胶质瘤细胞增殖、引起胶质瘤细胞发生迁移、加速胶质瘤细胞的扩散和恶变以及促进血管生成。因此,从趋化因子及其受体的角度来研究神经胶质瘤的侵袭和促进血管生成的机制,对认识胶质瘤发生、发展的机制、胶质瘤的抗血管生成和分子靶向治疗,具有十分重要的意义。
本课题旨在通过研究趋化因子受体CXCR4在神经胶质瘤各病理级别中的表达情况,来分析其表达与神经胶质瘤的侵袭性的内在关系及其与微血管密度(MVD)的关系。
方法:收集河北医科大学第二医院神经外科2009年10月～2010年3月经手术治疗的资料完整的38例神经胶质瘤患者(其中男性22例,女性16例)的手术标本,所有病人均为首发病例,术前未行放化疗等治疗手段,另取5例内减压术后的正常脑组织标本作为对照。每一例标本均取两份,其中一份以4%的多聚甲醛溶液固定,常规石蜡包埋;另一份在标本切除后马上放到冻存管中并于液氮瓶中速冻保存。所有组织均行病理组织学检查确诊,并按照WHO(2000)神经系统肿瘤的分类、分级标准进行临床病理分期。
1运用免疫组织化学方法检测标本中趋化因子受体CXCR4及CD34的表达情况,并计算微血管密度值。
组化结果判定方法:采用阳性细胞百分比结合显色强度法判读结果,阳性细胞的百分比分为以下四级计分:﹤10%计0分;10～25%计1分;25～50%计2分;﹥50%计3分。染色强度按瘤细胞深浅记分:0分,不着色;1分,弱;2分,中等;3分,强。将2个分值相加即得出该例标本的免疫组化阳性分度:0～1分为阴性,记为-;2～4分为弱阳性,记为+;5分以上为阳性,记为++。
测定微血管密度的方法:瘤组织内任何被抗CD34抗体染成棕黄色的单个内皮细胞或内皮细胞簇,有或无管腔,只要与其它结缔组织成分有明显区别,均计为1个阳性血管标记,每例观察切片1张,避开肿瘤坏死或出血区域,在低倍镜(×40)下选择微血管最丰富区,即“热点”(“hot spot”),在高倍视野(×200)下计数,计数5个200倍视野下的血管数,在目镜网格测微尺0.25mm2的范围内计数,其算术平均数计为该例切片的MVD测定值。
2运用实时荧光定量PCR的方法测定标本中趋化因子受体CXCR4在胶质瘤组织中的相对表达量。
采用Trizol法提取总RNA,行琼脂糖凝胶电泳检测,采用分光光度计检测RNA纯度。mRNA反转录成cDNA并进行检测,以β-actin作为内参照,使用严格的复孔法测定。
3统计学方法
根据数据采集的不同方法及统计学处理的不同要求,所有计量资料均用均数±标准差表示,选用x2检验和t检验的统计学方法,使用SPSS17.0统计软件进行统计分析,P<0.05表示差异有显著的统计学意义,P<0.01表示差异有非常显著性的统计学意义。
结果:
1免疫组织化学实验结果显示趋化因子受体CXCR4在正常脑组织中表达阴性,CXCR4在其中的24例胶质瘤组织中表达呈阳性,总阳性表达率为63.16%,在低级别(Ⅱ级)和高级别(Ⅲ、Ⅳ级)胶质瘤组的阳性表达率分别为44.44%、80.00%。CXCR4阳性表达率随着胶质瘤病理级别的增高而呈上升趋势,且高级别组CXCR4的阳性表达率明显高于低级别组,差异具有显著的统计学意义(P<0.05)。
2以CD34单克隆抗体标记的微血管密度(MVD)在正常脑组织和脑胶质瘤组中的表达均值分别为3.76±0.48和35.25±9.75,MVD的表达在脑胶质瘤组明显高于正常脑组织组,差异具有非常显著的统计学意义(P<0.01);在低级别胶质瘤组和高级别胶质瘤组中的表达均值分别为27.38±5.04和42.34±7.12,MVD在高级别胶质瘤组的表达明显高于低级别胶质瘤组,差异具有非常显著的统计学意义(P<0.01)。
3 MVD在14例CXCR4表达阴性的胶质瘤组织中的表达均值为26.37±5.11,在24例CXCR4表达阳性的胶质瘤组织中的表达均值为40.43±7.88,通过统计检验可知MVD在CXCR4表达阴性和阳性的胶质瘤组织中的表达均值具有非常显著的差异(P<0.01)。
4实时荧光定量PCR实验结果显示趋化因子受体CXCR4mRNA在低级别(Ⅱ级)胶质瘤组中的相对正常脑组织组的表达量为2.22士0.80,在高级别(Ⅲ、Ⅳ级)胶质瘤组的相对正常脑组织组的表达量为4.03士1.98,趋势是随胶质瘤病理级别的增高而升高,差别具有显著的统计学意义(P<0.05)。
结论:
1趋化因子受体CXCR4在正常脑组织中表达呈阴性,而在胶质瘤组织中表达阳性,表明CXCR4可能与胶质瘤的侵袭性有关。
2在神经胶质瘤组织中,趋化因子受体CXCR4在蛋白水平和分子水平均有高表达,且随病理级别的升高而表达增加,说明CXCR4可能与胶质瘤的恶性程度相关。
3在神经胶质瘤组织中,能反映新生血管增加的MVD值随着病理级别的增加而增加,提示脑胶质瘤的血管生成与瘤内的MVD关系密不可分;MVD愈高,肿瘤的血管生成愈多,肿瘤的恶性程度愈高。验证了MVD是判定肿瘤恶性程度和预后的重要指标的论断。
4本实验研究结果显示,MVD值在趋化因子受体CXCR4表达阴性和阳性的胶质瘤组织中的表达具有显著的差异,并且CXCR4在胶质瘤的瘤血管内皮细胞上同样存在表达,提示CXCR4可能参与胶质瘤的新生血管生成。
Objective: Glioma is the most common malignant tumor in brain,which has high incidence, strongly invasive power and recurrence fast after surgery. The effects of a variety of traditional treatment methods are not ideal.At the present time, the specific pathogenesis of glioma is not clear.With the gradually deep study for the pathogenesis of glioma and the rapid development of molecular biology techniques, molecular target treatment of cancer is gradually becoming a promising new means of treatment.
Studies have shown that chemokines SDF-1 and their receptors CXCR4 play important and complex roles in carcinogenesis and development of glioma, they can promote glioma cells prolifertion and migration,accelerate pervasion and canceration of glioma cells or angiogenesis in tumor tissue. Therefore,it is of great significance to study the roles of chemokines and their receptors in the process of carcinogenesis,invasion and angiogenesis of glioma for understanding the mechanism of carcinogenesis and development, anti-angiogenic and molecular target treatment of glioma.
The objective of this study is to analyze the internal relations between the expression of chemokines receptors CXCR4 and invasion of glioma,and the relations with microvessel density (MVD) by studying the expression of CXCR4 in all grade gliomas.
Methods: 38 glioma tissue specimens were collected from the glioma patients who were operated in the Neurosurgery of the Second Hospital of Hebei Medical University from 2009.10 to 2010.3(22 of them were male,16 of them were female),all of the patients are initial cases and not accepted the treatment of radiotherapy and chemotherapy etc. And 5 normal brain tissue specimens of intracranial decompression operation were gathered as control group.Two specimens were taken for each,one of which was fixed by 4% formaldehyde and paraffin-embedded;another was frozen saved in the liquid nitrogen immediately after operation. All of the specimens were confirned by histopathologic examination and clinical pathological staging was taken according to the WHO(2000) classifying and grading criteria of nervous system tumors.
1 Expression of CXCR4 protein and anti-CD34 monoclonal antibody in these specimens were examined by immunohistochemistry.And MVD was counted.
Judgment of immunohistochemistry result:By the methods of the percentage of positive cells and color intensity,(1) percentage of postive cells is divided into the following four grade:0 score:postive cell<10%;1 score:postive cell 10%～25%;2 scores:postive cell 25%～50%;3 scores:postive cell>50%. (2)dyeing intensity: 0 score: not stained;1 score:poor positive; 2 scores:moderate positive;3 scores:strong postive; The results can be gotten by adding (1)and(2):0～1score as negative,marked“-”;2～4 scores as minute positive,marked“+”;5 scores above as positive,marked“++”. Method of counting MVD:Any endothelial cell or endothelial cell cluster which is stained brown by anti-CD34 monoclonal antibody in the tumor tissue is signed one positive blood vessel marker,with or without lumen,if only it is obvious different from other connective tissue components. We observe one slice of every specimen,avoid the tumor necrosis region or bleeding region, and select the most abundant area of micrangium under low power field(×40),which is“hot spot”. we count the average of blood vessels under five high power fields(×200),and count in eyepiece micrometer grid range of 0.25mm~2,the arithmetic mean is signed as MVD measured value of this slice.
2 The relative expression of chemokines receptors CXCR4 in glioma tissue specimen was determined by Quantitative real-time PCR.
Use Trizol to extract total RNA,then make agarose gel electrophoresis detection, and RNA purity is detected by spectrophotometer.The mRNA is reversed transcription into cDNA ,and cDNA were detected,β-actin as internal reference.It is measured by strict re-hole. Statistical methods:According to the different methods of data acquisition and various requirements of statistical analysis,all of the measurement data is shown with Mean±SD. we choose x2-test,t-test,and t '-test. We use SPSS17.0 statistics software to processing data.When P<0.05,the data has significant statistics significance. When P<0.01,the data has very significant statistics significance.
Results:
1 Immunohistochemistry experiment showed that chemokines receptors CXCR4 was negatively expressed in normal brain tissues and positively expressed in 24 cases glioma tissue,the total positive expression rates in tumor group are 63.16%,and the positive expression rates were separately 44.44% and 80.00% in low-grade (gradeⅡ)glioma group and high-grade (gradeⅢandⅣ) glioma group.The positive expression rates of CXCR4 increased along with the pathological grades of glioma,and the positive expression rates of high-grade glioma group were significantly higher than low-grade glioma group, the data has significant statistics significance(P<0.05).
2 The expression average of MVD labelled with anti-CD34 monoclonal antibody in normal brain tissues group and tumor group respectively were 3.76±0.48 and 35.25±9.75, the expression of MVD is higher in glioma tissues group than in normal brain tissues group, the data had very significant statistics significance(P<0.01).The expression of MVD in low-grade glioma group and high-grade glioma group respectively were 27.38±5.04and 42.34±7.12, the expression of MVD is higher in high-grade glioma group than in low-grade glioma group, the difference had very significant statistics significance(P<0.01).
3 The expression average of MVD respectively were 26.37±5.11and 40.43±7.88 in 14 cases negative expression of CXCR4 and 24 cases positive expression of CXCR4 in glioma tissues, we can know that the data has very significant statistics significance through the statistical test (P<0.01).
4 Quantitative real-time PCR detection showed that the normalized chemokines receptors CXCR4 mRNA amount relative to normal brain tissues group was 2.22±0.80 in low-grade glioma group ,and 4.03±1.98 in high-grade glioma group,the data increased along with the pathological grades of glioma, the difference had significant statistics significance(P<0.05).
Conclusion:
1 Chemokines receptors CXCR4 expresses negatively in group of normal brain tissues,and expresses positively in group of glioma tissues,it suggests that CXCR4 is related to the invasiveness of glioma.
2 In glioma tissue, chemokines receptors CXCR4 has higher expression at protein and molecular level,and the expression increased along with the pathological grades of glioma,,this suggests that CXCR4 is related to the malignancy of glioma.
3 In glioma tissue, MVD which can reflect the increase of neovascularization, increased along with the pathological grades of glioma.It suggests that the relation between the angiogenesis of glioma and MVD is inseparable.The higher MVD,the more tumor angiogenesis,the higher malignancy of the tumors.It was proved that MVD was an important indicator of determining malignancy and prognosis of tumors.
4 The experimental results show that the expression of MVD has significant statistics significance between the negative expression of CXCR4 and positive expression of CXCR4 in glioma tissue,and CXCR4 can also express in vascular endothelial cells of glioma,which suggests that CXCR4 might take part in the angiogenesis of glioma.
引文
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