Okazaki R, Conover C A, Harris S A, Spelsberg T C, Riggs B L
Department of Molecular Biology and Biochemistry, Mayo Clinic, Rochester, Minnesota, USA.
J Bone Miner Res. 1995 May;10(5):788-95. doi: 10.1002/jbmr.5650100516.
Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method.(ABSTRACT TRUNCATED AT 250 WORDS)
胰岛素样生长因子I(IGF-I)和II(IGF-II)对成骨细胞具有合成代谢作用。尽管在啮齿类成骨细胞中已证实有IGF-I和IGF-II mRNA的表达,但对于人类成骨细胞模型中的IGF基因表达却知之甚少。在本研究中,我们对以下几种细胞中的IGF-I和-II mRNA表达进行了特征分析:(1)正常人成骨样(hOB)细胞;(2)一种猿猴病毒40永生化的hOB(HOBIT)细胞系;(3)人骨肉瘤细胞系SaOS-2、TE-85、MG-63和U-2。由于IGF cDNA探针与核糖体RNA的交叉杂交会掩盖人类细胞中多种IGF转录本的检测,我们用更特异的核糖核酸酶保护分析(RPA)取代了Northern分析。我们还使用逆转录聚合酶链反应(RT-PCR)来评估mRNA是否以痕量水平存在。仅在正常hOB细胞中通过RT-PCR和RPA一致观察到IGF-I mRNA表达。在IGF-I转录本变体中,正常hOB细胞中Ea IGF-I mRNA比Eb mRNA更丰富。当进行RT-PCR时,在SaOS-2和U-2骨肉瘤细胞中可不同程度地检测到痕量的IGF-I mRNA,但我们在HOBIT、TE-85或MG-63细胞中未发现IGF-I mRNA。通过两种方法评估,IGF-II mRNA在正常hOB、HOBIT、TE-85和U-2细胞中均有表达。(摘要截短至250字)