神经母细胞瘤及胚胎肾上腺组织端粒酶基因表达的研究

摘要

目的观察神经母细胞瘤和胚胎肾上腺组织中端粒酶基因(hTR/hTRT)的表达，以及神经母细胞瘤细胞在药物诱导分化时端粒酶基因表达的变化，探讨端粒酶在神经母细胞瘤形成和发展中的作用。方法体外转录法制备生物素标记的hTR、hTRT cRNA探针。神经母细胞瘤存档石蜡标本20例，2～5个月胎儿肾上腺组织石蜡标本12例。应用原位杂交组织化学(ISHH)方法检测hTR及hTRT表达。用维甲酸类似物R9158诱导人神经母细胞瘤细胞系SH-SY-5Y及SK-N-SH，细胞涂片经ISHH检测hTR表达。结果20例神经母细胞瘤中hTR 19例(95.0％)阳性；hTRT18例(90.0％)阳性。各月龄胎儿肾上腺组织均有hTR及hTRT表达，神经嵴细胞和继发皮质细胞内信号强于原发皮质细胞。神经嵴细胞形态极似神经母细胞瘤细胞。维甲酸敏感细胞SH-SY-5Y在R9158作用5 d后出现明显形态学变化，表达hTR的细胞明显减少。维甲酸耐受细胞SK-N-SH无明显变化。结论端粒酶基因在神经母细胞瘤和胚胎肾上腺组织中高表达。神经母细胞瘤细胞在药物诱导下分化时端粒酶基因hTR表达明显降低。端粒酶在神经母细胞瘤形成和发展中有重要作用。%Objective To observe telomerase gene (hTR and hTRT) expression in neuroblastoma (NB) and fetal adrenal tissues, and its alterations during NB cell differentiation induced by retinoic acid anologues (R9158), so as to discuss the role of relomerase in the formation and development of neuroblastoma. Methods The tumor samples came from paraffin-embedded archival materials of 20 cases of NB. 3specimens of fetal adrenal tissue were obtained from fetus of 2,3,4,5 month gestation each. The plasmids,pGRN83-hTR and pBS-hTRT, containing full-length hTR and hTRT complementary DNA respectively,were used as template to generate biotin labeled probes through transcription in vitro. The 5μm paraffinembedded tissue sections underwent in situ hybridization (ISHH) to show the expression of hTR and hTRT. Human NB cell lines, SH-SY-5Y and SK-N-SH, induced by R9158 were smeared on slides for hTR expression detection with ISHH. Results Of 20 cases NB,19(95.0%) showed positive hTR expressions, while 18(90.0%) showed positive hTRT expressions. Fetal adrenal tissues of each gestational age had positive hTR and hTRT expressions. Furthermore, the neuroblastic cell foci and the primary cortex cells had higher expression than the secondary cortex cells. The RA-sensitive SH-SY-5Y showed morphological changes by the 5th day of R9158 treatment, and the positive hTR expression cells decreased significantly. The RA-resistant SK-N-SH showed neither morphological change nor hTR expression decreasing. Conclusions The telomerase gene expression was high in neuroblastoma and fetal adrenal tissue,whereas it was depressed significantly in neuroblastoma cells during the differentiation induced by retinoic acid. The telomerase would have to play an important role in the formation and development of neuroblastoma.