Relativity Research Between Human Cytomegalovirus Infection Balb/c Mice's Intervertebral Disc Tissues And Its Degeneration

Objective:Based on a model in which Balb/c mice in vivo are infected by HCMV to study histology,gene expression and immunology,detect the infection condition and observe the pathological change in Balb/c mice's intervertebral disc(IVD) tissues; preliminarily explore HCMV infecting Balb/c mice's IVD tissues and the degenerative relevance to provide experimental evidence for clinical LBP's etiology,diagnosis and prevention and cure study.Methods:HCMV AD₁₆₉ in five viral infection doses(2×10⁶PFU/ml,2×10⁵ PFU/ml,2×10⁴ PFU/ml,2×10³ PFU/mlå’Œ2×10² PFU/ml),which had been purified in Microbiology Teaching and Research Department,Anhui Medical University,were injected in dose of 1.0ml per each in abdominal cavity 6 or 8-week-old,SPR level mice, including 6 male and female mice respectively.6 male and 6 female mice were injected as inactive control groups with viruses in concentration of 2×10⁶PFU/ml which had been inactivated at 56℃for 30 minutes;another 6 male and 6 female mice were injected as HF cell control groups with HF cell suspension of homologous strain and the same generation.All groups of mice were raised in shielding system.During the 3 months of virus inoculation,tissues were respectively selected to do experiment every one month.Different means were used to detect the following indexes:(1) Observing cytopathic effect(CPE) and the characteristics of pathological changes day by day in mice's IVD tissues attributed to HCMV in the way of inverting microscope; (2)Detecting important genes UL 83 DNA and UL54 mRNA which were connected with the duplication and multiplication of HCMV in IVD tissue cells by means of PCR and RT-PCR;(3) Mice's immune state detection:The antibody level of HCMV IgG and IgM in mice's blood serum were detected by means of indirect ELISA;(4)Relevant pathological detection in degeneration of IVD tissues:mice's IVD tissues construction and pathological change were detected by means of HE dyeing.Results:(1) After continual 4 to 6-week observation,in 2×10⁶PFU/ml and 2×10⁵ PFU/ml infection groups,female mice's IVD tissue bomogenate inoculated with HF cells was detected to reveal viral CPE.IVD cells swelled and became round;particles in cells increased,ablated,had strengthening refraction efficiency,etc.;(2)Scraped pathologically changed cells were detected by means of PCR to display HCMV UL83 genes' specific amplification straps in 766bp,while other groups after blind passage of 3 generations were not detected to reflect CPE and specific amplification straps as well. Detecting HCMV UL54 genetic transcript in IVD tissues by means of RT-PCR:Specific straps,which were detected in 142bp,female mice,2×10⁶PFU/ml and 2×10⁵ PFU/ml infection groups,could verify that HCMV was actively infected in IVD tissues;(3) Mice's immune state detection:The antibody level of IgG,IgM in mice's blood serum were detected by means of indirect ELISA.Mice's antibodies IgG in infection groups and viral inactive groups were masculine,while in HE cell control groups negative;OD values were respectively 0.7±0.06,0.5±0.04,0.12±0.02,and the differences among the three groups were of statistical significance(P＜0.05).Mice's antibody IgG in active infection groups was masculine,while in viral inactive groups and HE cell control groups negative;OD values were respectively 0.53±0.15,0.13±0.02,0.08±0.01,and the differences between active infection groups and viral inactive groups,HE cell control groups were statistically significant(P＜0.05);(4) Pathological observation result of HE dyeing in mice's IVD tissues:Mice's IVD tissues in virus-active infection groups displayed evident pathological changes.Annular fibrosus' layer constitution was disordered;some nucleus gelatinosus tissues shrinked;arrangement in chorda dorsalis cells and cartilage cells of nucleus gelatinosus was uneven;cell population decreasd; transition zone between annular fibrosus and nucleus gelatinosus narrowed;arthrodial calcification cartilaginea layers thickened;plenty of slime and foam cells appeared; blood vessel under cartilage obviously decreasd;lymphocyte infiltration in partial disc and other chronic inflammatory reaction could also be detected.However,such pathological changes could not be tested in mice's IVD tissues in both inactive groups and control groups.Conclusion:HCMV AD₁₆₉ can infect Balb/c mice's intervertebral disc,and the viral infection dose counts to some extent.HCMV active infection is related to degeneration of IVD,and HCMV injection seems to induce the degeneration gradually and pathological changes,which is likely to be one of important risk factors attributed to degeneration of IVD.This model enjoys evident superiority in the molecular level study of degeneration of IVD and it provides experimental proof for clinical LBP's etiology, diagnosis and prevention and cure study.