Expression and promoter-alternation of osteoblastic insulin-like growth factors

以人软骨肉瘤软骨细胞HCS-2/8为研究对象，采用RNase protection Assay (RPA)，建立了胰岛素样生长因子基因表达及启动子变化的定量分析方法。RPA实验探针是针对IGF-2的启动子2P-1、2P-3和2P-4以及IGF-1的启动子1P-1和1P-2构建的，但由于HCS-2/8中转录产物很少，IGF-2的启动子2P-2未能构建。现在，我们将继续研究使用他们的探针确定RPA法的检测限制，我们将在积累rp -2转录物的RT-PCR产物后重新构建探针。在目前的发展调查中，我们发现了一些新的事实：1。四种IGF-2启动子在HCS-2/8和人正常软骨细胞中同时表达。HCS-2/8中IGF-2的启动子2P-4转录本在第6外显子的3'端存在4个碱基对缺陷，该位置位于翻译起始点上游9 ~ 6个碱基处。这种缺陷可能是由于软骨肉瘤特有的选择性剪接所致。与HCS-2/8和人正常软骨细胞比较，我们发现IGF-2和H19（肿瘤抑制基因）表达平行相关。在HCS-2/8中，IGF-2基因的限制性片段长度多态性表明在基因组水平上只有父本等位基因存在，而在软骨肉瘤中不存在母本印迹等位基因。这可能与肿瘤的形成有关。

Using a chondrocytic cell line HCS-2/8 derived from human chondrosarcoma, the quantitative analytical method adapting the RNase protection Assay (RPA) was developped in order to examine the expression and promoter-alternation of insulin-like growth factor genes. The probe of the RPA experiments was constructed for the promoters 2P-1,2P-3 and 2P-4 of IGF-2 and the promoters 1P-1 and 1P-2 of IGF-1, but the promoter 2P-2 of IGF-2 failed to be constructed because the transcriptional product was very little in HCS-2/8. Now, we continue to investigate the determination of detectable limitation of the RPA method using their probes, and we will retry to construct the probe asfter the accumulation of RT-PCR product for 2P-2 transcript.During the present developmental investigations, we found some new facts as following :1.The simultaneous expression of four promoters of IGF-2 were induced in HCS-2/8 and human normal chondrocytes.2.A defect of four base pair in the promoter 2P-4 transcript of IGF-2 in HCS-2/8 was observed at the 3'-end of exon 6, at which the position is upstream of 9 to 6 base from the translation-starting point. This defect may be due to the alternative splicing specific to the chondrosarcoma.3.Comparing with HCS-2/8 and human normal chondrocytes, we found a parallel correlation between IGF-2 and H19 (tumor reppressor gene) expressions.4.The restriction fragment length polymorphism of IGF-2 gene in HCS-2/8 suggested that only the paternal allele (s) existed at the genomic level, but the maternal imprinting allele was absent in the chondrosarcoma. This perhaps correlate with the tumor formation.