Abstract
Introduction
Hypoxia-inducible factor-1α (HIF-1α) is one of the most important factors that mediate direct cellular response to hypoxia. Intervertebral disc is the largest avascular organ in human body, nucleus pulposus cells originating from mesenchymal tissue are in the hypoxia environment. HIF-1α has been reported to express in normal and degenerated intervertebral disc cells. But there is no intervertebral disc HIF-1α deficiency mice available for degeneration study till now.
Materials and Methods
Shh-Cre+/− and HIF-1αflox/flox mice were used to generate HIF-1α-deficiency mice in intervertebral disc which was confirmed by RT-PCR and in situ hybridization. Five mice of HIF-1α−/− and wild-type (WT) group received the radiographic and MRI study at 2, 4 weeks after birth. Histological observation, Safranin O/Fast green staining of intervertebral disc were performed in each group. QRT-PCR and Western blot were used to assay the changes of collagen II and aggrecan with growth in the discs of mutant and wild type mice.
Results
RT-PCR of tail snip confirmed the genotype of HIF-1α deficiency mice and in situ hybridization of HIF-1α showed the ablation of HIF-1α in the disc. Radiographic study showed narrowing of intervertebral disc was noted on 4 weeks age mice in gene deletion, compared with the WT group, MRI study showed signal in the nucleus pulposus area disappeared and became “dark disc” in the HIF-1α−/− groups, starting from 2 weeks age, while the signal intensity WT group discs did not show appreciable degeneration. Histological observation indicated early degeneration in 2 weeks age of mutant mice (2.53 ± 0.97), and no degeneration of disc in WT mice (1.67 ± 0.52). mRNA expression and protein secretion of maim matrix in disc: collagen II (col2α) and aggrecan were both decreased in HIF-1α deficiency mice compared with WT mice.
Conclusion
Shh can be used as promotor for Cre recombinase to delete HIF-1α in mice nucleus pulposus cells successfully. HIF-1α deficiency mice in the disc were phenotypically normal as the WT, but manifest degeneration of intervertebral disc as early as 2 weeks age.
None declared