【作者】 陆俭；

【导师】 杨惠林；

【作者基本信息】 苏州大学 ， 外科学， 2011， 博士

【摘要】 目的:探讨Th1、Th2、Treg、Th17在骶骨脊索瘤发生发展中所起的作用,以及与骶骨脊索瘤生物学行为的关系,为骶骨脊索瘤的诊断、预后评测、骶骨脊索瘤免疫学靶向治疗等方面提供理论参考。方法:收集2002年1月～2008年12月间在我院手术的27例骶骨肿瘤患者的新鲜肿瘤组织,包括骶骨脊索瘤11例,骶骨骨巨细胞瘤9例,骶骨神经鞘瘤7例,进行Real-time PCR研究,检测FoxP3 mRNA、ROR-γt mRNA、T-bet mRNA、GATA-3 mRNA的表达,以各基因的表达量以均数±标准误表示,目的基因表达量的比较采用Wilcoxon秩和检验。以P<0.05判定为有统计学意义。结果:①FoxP3基因在骶骨脊索瘤、原发骨巨细胞瘤、神经鞘瘤中相对表达量分别为8.5±7.6%、1.6±0.9%、0.7±0.5%,FoxP3基因在骶骨脊索瘤中表达高于骨巨细胞瘤组及神经鞘瘤组。差异有统计学差异(P<0.05)。②T-bet基因在骶骨脊索瘤、原发骨巨细胞瘤、神经鞘瘤中相对表达量分别为0.7±0.3%、1.7±1.2%、1.6±1.3%,T-bet基因在骶骨脊索瘤中表达低于骨巨细胞瘤组及神经鞘瘤组。差异有统计学意义(P<0.05)。③GATA-3基因在骶骨脊索瘤、原发骨巨细胞瘤、神经鞘瘤中相对表达量分别为2.9±2.3%、0.6±0.5%、0.3±0.1%,GATA-3基因在骶骨脊索瘤中表达高于骨巨细胞瘤组及神经鞘瘤组。差异有统计学意义(P<0.05)。④ROR-γt基因在骶骨脊索瘤、原发骨巨细胞瘤、神经鞘瘤中相对表达量分别为4.1±2.9%、1.6±0.8%、0.2±0.1%,RORγt基因在骶骨脊索瘤中表达明显高于骨巨细胞瘤组及神经鞘瘤组。差异有统计学意义(P<0.05)。结论:原发性骶骨脊索瘤患者肿瘤组织转录因子FoxP3 mRNA、GATA3 mRNA表达增高,T-bet mRNA减低,表明其肿瘤局部组织呈现Th2偏移现象,可能是其发生免疫逃逸的机制之一。ROR-γt mRNA表达增加,提示骶骨脊索瘤患者肿瘤组织存在Th17优势化现象,并可能通过上调肿瘤血管生成因子促进脊索瘤的复发。目的:探讨Th17细胞因子与患者手术后复发率以及无瘤生存时间、总生存时间的相关性。为今后预防或延缓骶骨脊索瘤术后复发,延长术后无瘤生存期提供参考依据。方法:对1998年1月～2008年12月间在我院骨外科完成初次手术的28例骶骨脊索瘤患者进行随访,对病理蜡块行免疫组化。另对我院保存在液氮里27例新鲜骶骨肿瘤组织进行免疫研究,采用Kaplan-Meier法作单因素变量分析并绘制CDFS曲线,并用Log-Rank法检验复发率差异。设P<0.05为差异有统计学意义。结果:①Th17在骶骨脊索瘤组织中阳性表达率为60.7%,而在骨巨细胞瘤组Th17为弱阳性表达,在神经鞘瘤组则不表达。②不同年龄段之间、不同性别之间骶骨脊索瘤Th17表达无统计学差异(P>0.05)。③肿瘤直径≥10cm组Th17阳性表达率为83.33%,直径<10cm组Th17阳性表达率为40.75%,差异有统计学意义(P<0.05)。④肿瘤切缘阳性组Th17阳性表达率为69.23%,高于肿瘤切缘阴性组53.33%,但差异无统计学意义(P>0.05)。⑤术后复发组Th17阳性表达率为78.57%,未复发组为42.86%,两组差异有统计学意义(P<0.05)。其中Th17阳性组5年无复发生存率为(32.4±12.8)%,5年总生存率为(86.9±8.7) %;Th17阴性组5年无复发生存率为(81.8±11.6) %,5年总生存率为100 %,差异有统计学意义(P<0.05)。结论:Th17在骶骨肿瘤局部复发等生物学行为中发挥重要作用,Th17高表达的患者复发率较高,术后CDFS期较短。Th17高表达是病情进展和复发的一个重要信号,可作为判断骶骨脊索瘤复发的指标之一。第三部分CD40、PCNA等在骶骨脊索瘤中表达及其与复发的相关性研究目的:研究骶骨脊索瘤中CD40、PCNA的表达水平及其与脊索瘤复发的相关性,为骶骨脊索瘤的免疫治疗提供一种新的靶点。方法:取1998年1月～2008年12月我院骨科28例临床及病理资料均完整的骶骨脊索瘤患者手术切除的肿瘤组织腊块,采用免疫组化技术检测其中的CD40和PCNA的表达,另9例正常胚胎脊索组织为对照组,并计数CD31标记的肿瘤组织微血管密度(MVD)。所得数据用均数±标准差表示,两组计量数据之间比较采用t检验或方差分析,两组计数资料率的比较采用Fisher确切概率法。设P<0.05为差异有统计学意义。结果:①CD40、PCNA在骶骨脊索瘤中的强阳性表达率分别为66.1%、64.3%,明显高于正常胚胎脊索组织。两者之间的差异有统计学意义(P<0.05);骶骨脊索瘤中CD31标记的微血管密度(24.26±18.68)/mm2,正常胚胎脊索组织微血管密度值为(4.63±2.19)/mm2;差异有统计学意义(P<0.05);②28例骶骨脊索瘤,其中复发组14例,非复发组14例,两组间CD40阳性表达率分别为14.29%、85.71%;PCNA阳性表达率分别为92.86%、50.00%;CD31标记的MVD值分别为(30.66±23.75)/mm2、(17.88±8.52)/mm2;均有统计学差异(P<0.05)。③CD40、PCNA表达及CD31标记的MVD值与患者的年龄、性别、肿瘤大小无统计学相关性。④PCNA阳性表达的骶骨脊索瘤组织中MVD值为(28.92±20.27) /mm2,明显高于PCNA阴性组(12.64±3.70) /mm2,差异有统计学意义(P<0.05)。CD40阴性表达的骶骨脊索瘤组织中MVD值为(31.68±22.81) /mm2,明显高于CD40阳性表达组(16.86±9.28) /mm2,差异有统计学意义(P<0.05)。结论:CD40、PCNA在肿瘤浸润及复发转移等生物学行为中均发挥重要作用,PCNA强表达,CD40弱表达,是骶骨脊索瘤病情进展和复发的重要信号,骶骨脊索瘤中CD40和PCNA表达水平可作为判断病情进展和肿瘤复发的指标之一。更多还原

【Abstract】 Purpose: To discuss the effect of Th1、Th2、Treg、Th17 in the occurrence and development of sacral chordoma, and the relationship with biological behaviour of sacral chordoma, and provide some theoretical basis for the diagnosis, prognostic evaluation and immunological targeted therapy of sacral chordoma.Methods: To collect the fresh tumor tissue of 27 patients with sacral tumors who received surgery in our hospital from January 2002 to December 2008. Among them, there were 11 patients with sacral chordoma, 9 with giant cell tumor of the sacrum, 7 with sacral nerve sheath tumor. Using Real-time PCR to detect the expression of FoxP3 mRNA, ROR-γt mRNA、T-bet mRNA and GATA-3 mRNA, using mean±standard error to represent the expression of all genes, and using Wilcoxon rank test to compare the expression of genes. It has statistical significance if P <0.05.Results:①The relative expression of FoxP3 in sacral chordoma, giant cell tumor of the sacrum and nerve sheath tumor were 8.5±7.6%、1.6±0.9%、0.7±0.5%. The expression of FoxP3 in sacral chordoma was higher than that in giant cell tumor of the sacrum and nerve sheath tumor. The difference has statistical significance (P<0.05).②The The relative expression of T-bet in sacral chordoma, giant cell tumor of the sacrum and nerve sheath tumor were 0.7±0.3%、1.7±1.2%、1.6±1.3% . The expression of T-bet in sacral chordoma was lower than that in giant cell tumor of the sacrum and nerve sheath tumor. The difference has statistical significance (P<0.05).③The relative expression of GATA-3 in sacral chordoma, giant cell tumor of the sacrum and nerve sheath tumor were 2.9±2.3%、0.6±0.5%、0.3±0.1%. The expression of GATA-3 in sacral chordoma was higher than that in giant cell tumor of the sacrum and nerve sheath tumor. The difference has statistical significance (P<0.05).④The relative expression ROR-γt of in sacral chordoma, giant cell tumor of the sacrum and nerve sheath tumor were 4.1±2.9%、1.6±0.8%、0.2±0.1%.The expression of ROR-γt in sacral chordoma was obviously higher than that in giant cell tumor of the sacrum and nerve sheath tumor .The difference has statistical significance (P<0.05).Conclusion: The high-expression of FoxP3 mRNA、GATA3 mRNA and low- expression of T-bet mRNA in the primary sacral chordoma patients indicated that the local tissue of the tumors appeared migration phenomenon of Th2, and it may be one of the reasons for the immunologic escape. The increased expression of ROR-γt mRNA indicated that there was advantage phenomenon of Th17 in the primary sacral chordoma tissue, and it may cause the recurrence of chordoma by upexpressing the angiogenic factors of the tumors.Part two The study of Th17 and its relationship with prognosis in primary sacral chordomaPurpose: To discuss the correlation between Th17 and the recurrence after operation, survival time without tumor, overall survival time. And to provide some reference for prevent or delay the recurrence of sacral chordoma after operation, prolong the survival time without tumor after operation in the future.Methods: To follow up the 28 patients with sacral chordoma who received the first operation in our orthopaedic department from January 1998 to December 2008. Using the immunohistochemisty with the pathology wax. Besides, using the immunity study for the 16 fresh tissue of giant cell tumor of the sacrum and nerve sheath tumor which were stored in the liquid nitrogen. Using Kaplan-Meier to get the single factors analysis of variance and draw the curve of CDFS, and using the Log-Rank to check the difference of recurrence. It has statistical significance if P <0.05.Results:①The positive expression of Th17 in sacral chordoma was 60.7%,and weakly positive expressed in group of giant cell tumor of the sacrum, no expression in group of nerve sheath tumor.②There were no statistical significance for the expression of Th17 in sacral chordoma between different age and gender (P>0.05).③The positive expression percentage of Th17 in the group of tumor diameter≥10cm was 83.33%,for the group of tumor diameter<10cm was 40.75%,there was statistical significance (P>0.05).④The positive expression percentage of Th17 in the group of positive incisal margin was 69.23%, which was higher than that of negative incisal margin(53.33%), but no statistical significance(P>0.05).⑤The positive expression percentage of Th17 in the group of recurrence was 78.57% and 42.86% in the group of no-recurrence. There was statistical significance (P<0.05). The survival rate of 5-year no recurrence in the group of positive Th17 was (32.4±12.8)%, overall survival rate of 5-year was (86.9±8.7) %. The survival rate of 5-year no recurrence in the group of negative Th17 was (81.8±11.6)%, overall survival rate of 5-year was 100 %. Conclusions: Th17 played an important role in the local recurrence of sacral tumors.The high expression of Th17 indicated the high recurrence and short CDFS after operation. The high expression of TH17 was an important sign for the progress and recurrence,and could be one of the indexs to determine the recurrence of the sacral chordoma. Purpose: To study the expression level of CD40、PCNA in sacral chordoma and their relationship with recurrence, and to provide a new target for the immunization therapy of sacral chordoma.Methods: To collect tumor tissue of 28 cases with sacral chordoma who had full information of clinical and pathology for the wax. To detect the expression of CD40 and PCNA by using immunohistochemisty, another 9 cases with normal notochordal tissue were set as control group and count the MVD marked by CD31. All the data was showed by mean±standard error and using T-test to compare two groups. It has statistical significance if P <0.05.Results:①The strong positive expression of CD40、PCNA in the sacral chordoma were 66.1% and 64.3%, obviously higher than that in normal notochordal tissue. There was statistical significance (P<0.05). The MVD marked by CD31 in sacral chordoma was (24.26±18.68)/mm2 and (4.63±2.19)/mm2 in normal notochordal tissue. There was statistical significance (P<0.05).②Among the 28 sacral chordoma, 14 had recurrence and 14 had no recurrence, the positive expression of CD40 were 14.29% and 85.71%.For the PCNA, the positive expression were 92.86% and 50.55%. MVD marked by CD31 were (30.66±23.75)/mm2 and (17.88±8.52)/mm2. All had statistical significance (P<0.05).③There were no statistical correlation between the expression of CD40、PCNA and the value of MVD with the age, gender and the size of tumor.④The MVD in the group of positive expression of PCNA was (28.92±20.27) /mm2, significantly higher than the negative group (12.64±3.70) /mm2. And there was statistical significance(P<0.05). The MVD in the group of negative expression of CD40 was (31.68±22.81 /mm2, significantly higher than the positive group (16.86±9.28) /mm2. And there was statistical significance (P<0.05).Conclusions: CD40 and PCNA had vital role in the tumor invasion, recurrence and metastasis. Strong expression of PCNA and weak expression of CD40 was important sign for the progress and recurrence of sacral chordoma. The expression level of CD40 and PCNA in the sacral chordoma was one of the index to determine the progress and recurrence of the tumor.更多还原