Pathogenesis Of Hearing Loss Induced By Cytomegalovirus Infection And Preliminary Research On In Utero Gene Therapy

PART ONEPathogenesis of Hearing Loss Induced by Infection of MCMV【Objective】To establish a murine model of hearing loss due to MCMV infection and probeinto the role of HMGB-1 cellular signal pathway in the mechanism of CMV-inducedhearing handicap.【Methods】The Smith strain of MCMV was passaged in NIH/3T3 cells in vitro and TCID₅₀of the virus was quantitated in the light of CPE. MCMV-free BALB/c mice were screenedby ELISA assay of MCMV-specific IgM/IgG antibodies. After overnight mating,pregnantmice were separately bred. The newborn pups were randomly divided into two groups:MCMV-infected group(infected group),which was intracerebrally inoculated with viralsuspension in the first 24 hours after birth,and experimental control group(control group),which was injected with sterile 0.9% sodium chloride of equal volume. We recorded theliving and developing condition of the pups,measured the ABR thresholds of the mice twoweeks later. MCMV and HMGB-1 mRNA in the cochleae were assayed by RT-PCR,in themeantime the expression of RAGE and IκBαwere measured by Western Blot analysis.【Result】TCID₅₀ of the virus was 10^5.22/0.1mL. 44 in the 121 pups of the infected groupsurvived with notable anomaly 2 weeks after birth,while all the pups of the control groupnormally developed. The ABR thresholds of the two groups were 28.42±3.03 dB (infected)and 15.66±0.38 dB (control),respectively(P<0.01). In the cochlear extract,MCMV mRNAwas detected in the infected group and the relative abundance of HMGB-1 mRNA were 3.124±1.836 and 1.077±0.085,respectively(P<0.05). According to the results of WesternBlot,the RAGE level was conspicuously higher while the IκBαapparently decreased in theinfected group(P<0.01).【Conclusion】The murine model of hearing loss induced by MCMV was successfullyestablished. The HMGB-1/RAGE/NF-Κb pathway might play an important role in thepathogenesis of hearing handicap caused by CMV infection. PART TWO Construction and In Vitro Expression of Double-geneVector PshHMGB-1-EGFP【Objective】To construct an eukaryotic vector co-expressing shRNA specific to HMGB-1and EGFP,and transfect NIH/3T3 cells to screen out the most effective interfering plasmidfor IUGT in vivo.【Methods】We designed and synthesized five double-strand oligoneucleiotides of DNA,respectively including inverted repeat sequences of four different gene fragments ofHMGB-1 spaced by a 9bp loop sequence and a negative control sequence NC. The doublestrands were inserted into the plasimid pGenesil-1 carrying EGFP gene to constructrecombinant plasmid vectors: pshHMGB-1-EGFP a,b,c,d,and pNC-EGFP. Therecombinant plasmids were digested by Sal I and identified by electrophoresis on agarosegel,meanwhile the plasmid DNA was sequenced. Then we transfected NIH/3T3 cells withthe plasmid/liposome complex. The transfection efficiency was estimated by fluorescencemicroscope and flow cytometer,and the HMGB-1 level was assessed by RT-PCR andWestern Blot analysis.【Result】The restrictive digestion and identification as well as DNA sequencing allindicated that the recombinant vectors were correctly constructed. The green fluorescenceof EGFP could be observed in transfected NIH/3T3 cells,and the transfecting efficacy is57.25% according to flow cytometry assay. The mRNA and protein level of HMGB-1 in theNIH/3T3 cells transfected by pshHMGB-1-EGFP a,b,d obviously decreased,among whichpshHMGB-1-EGFP d displayed the strongest inhibitory effect(P<0.05).【Conclusion】The double-gene eukaryotic expression vector pshHMGB-1-EGFP wassuccessfully constructed. The recombinant interfering plasmid could efficaciously transfectNIH/3T3 cells and perform RNAi effect,which set up foundation for applying RNAi inIUGT in vivo. PART THREE Preliminary Research on Application of the RecombinantPlasmid Vector PshHMGB-1-EGFP in IUGT【Objective】To probe into the possibility of treating congenital diseases combining RNAitechnology and IUGT by virtue of observing and evaluating the capability of passingthrough the placenta and transfecting the fetuses of pshHMGB-1-EGFP,as well as theRNAi effect in the fetuses.【Methods】The dams at day 12.5 post coitus were randomly divided into four groups:Group 1 inoculated with pshHMGB-1-EGFP+liposome via the tail vein; Group 2 withpNC-EGFP+liposome; Group 3 with pNC-EGFP alone; Group 4 with aseptic normal saline.The dams were sacrificed 72 hours after inoculation,meanwhile the placenta weredissected together with the fetal organs. EGFP expression in the placenta and fetal organswas examined by fluorescence microscope; HMGB-1 mRNA was measured by RT-PCR;the activity of NF-κB was determined by EMSA.【Result】Green fluorescence could be seen in all the offsprings of the dams in Group 1 and2; none of the fetuses in Group 3 and 4 presented visible fluorescence. Fluorescence inGroup 1 and 2 was particularly obvious in the chorionic plate and labyrinth zone of theplacenta,fetal liver and kidney,local cerebral region especially proximal to the ventricles,capillaries among cardiac fibers,the wall of main vessels,the interstitial vessels of thelung,the cortex and sinus of spleen. Fluorescence was rather feeble or even could not beseen in corresponding regions of Group 3 and 4. The HMGB-1 mRNA and the NF-κBactivity were clearly lower in Group 1 compared with the other three groups(P<0.05).【Conclusion】PshHMGB-1-EGFP could efficaciously get through the placenta andtransfect the fetues with the help of liposome. HMGB-1 expression and NF-κB activitywere inhibited by RNAi effect caused by the plasmid,which presented a novel potentialremedy for congenital diseases due to intrauterine infection of CMV.