Expression And Clinical Significance Of IGF-1 In Degenerated Nucleus Pulposus Of Lumbar Intevetebral Disc

objective: To investigate the expression of insulin-like growth factor 1 (IGF-1) in different degrees of degenerated nucleus pulposus applying the real-time quantitative reverse transcription-polymerase chain reaction (realtime Q-PCR) method ,and explore the relationship between IGF-1 mRNA and the degree of degenerated nucleus pulposus.Methods : 63 nucleus pulposus from patients(aged between 40-50 ) with operation of lumbar vertebrae were collected in Affiliated Zhongshan Hospital of Dalian University, The Second Affiliated Hospital of Dalian Medical University and Peking University Third Hospital. They were divided into three groups (mild degeneration, moderate degeneration, severe degeneration) on the basis of MRI, Another 9 nucleus pulposus from lumbar vertebrae fractured patients (aged between 16-20 ) were set as control group(CON). All of the nucleus pulposus taken out were rinsed thoroughly by saline immediately after biopsy, removed of annulus fibrosus, cut into the size of 1*1*lmm, placed in liquid nitrogen quickly and then deposited to the -80℃refrigerator spare. The total RNA was extracted by Trizol reagent from 50mg nucleus pulposus of all samples weighted respectively. The concentration and purity of RNA were calculated according to the values of OD₂₆₀ and OD₂₈₀ acquired by ultraviolet Spectrophotometer. Total RNA was electrophoresed in 1% formaldehyde denaturalized agarosegel in order to validate integrity of RNA. The mRNA was transcribed into cDNA which integrity is validated by electrophoresed in 2% formaldehyde denaturalized agarosegel using oligo primer. The variable IGF-1 extron 2 gene was chosen to amplify in vitro by real time fluorogcnic quantitative instrument with gene specific primer by PQ-RT-PCR. The threshold of cycle exponential accretionary phage (Ct) standardized by 18S gene of each sample were recorded and used to calculate the incept cDNA template. The statistical software SPSS10.0 for Windows was used to compare and analyze the data of four groups to assay the IGF-1 mRNA differences between each group. Results: (1) OD260/OD280 ratios were detected about 1.856±0.073 of the total RNA in control group , 1.870±0.080 in mild degeneration group, 1.886±0.085 in moderate degeneration group, and 1.859±0.080 in severe degeneration group. There were no significant differences in OD260/OD280 ratio among four groups respectively(P> 0.05). The methods of extraction had no obvious influence on the quality and quantity in total RNA (2) Total RNA electrophoresed by 1 % formaldehyde denaturalized agarosegel showed that the 5S, 18S and 28S RNA strips were all explicit and intact, and had no obvious degradation. (3) The cDNA of the IGF-1 exon 2 and the 18S gene were reservedly transcribed by RT-PCR, and compared with the standard DNA Marker they were 187 bp and 151 bp long respectively, which were in consonance with anticipation. (4) The mRNA expression of IGF-1 in different degrees of degenerated nucleus pulposus were detected by real time fluorogenic quantitative reserve transcription polymerize reaction and stated by SPSS10.0 for Windows in one-way ANOVA and LSD-t test. Statistical analysis showed that each among the groups has significant difference (P<0.01) except no significant difference (P>0.05) between severe degeneration group and control group. Conclusion: The expression of IGF-1 in mild degeneration group and moderate degeneration group is significantly higher but less in severe degeneration group than that in control group abnormally. The expression of IGF-1 is related to the degree of degenerated nucleus pulposus, and the expression of IGF-1 is weaker with the degeneration of nucleus pulposus.