Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
To detect the cell density, apoptotic incidence and the expressions of Bax and Caspase-3in human lumbar intervertebral discs, so as to further understand the mechanism of human lumbar intervertebral discdegeneration and provide a new idea for biologic treatment of it in future. Methods From May to December in 2006,30 human lumbar intervertebral discs in experimental group(L2 to S1)were surgically collected from 27 patients undergoing posterior lumbar intervertebral discoidectomy and fusion. All the cases were affirmed by MRI and they never experienced discography, collagenolysis of nucleus pulposus and percutaneous laser disc decompression. The control group consisted of 20 human lumbar intervertebral discs(L2 to S1)harvested from 5 young men without spine-related condition immediately after their accidental death. Apoptotic disc cells were detected by TUNEL and histomorphology, and immunohistochemical staining with SP method was performed to examine the expressions of Bax and Caspase-3 in all specimens. Results HE staining disclosed that the average cell density in control group (17.16 ± 1.22)/HP was higher than that in experimental group (12.41 ± 0.95)/HP (P lt; 0.01). However, TUNEL staining observed that the average TUNEL positive incidence in control group (6.97% ± 0.92%) was lower than that in experimental group (12.59% ± 0.95%), (P lt; 0.01). Immunohistochemical staining with SP method showed that the Bax and Caspase-3 positive incidence of nucleus pulposus in control group (11.02% ± 1.18%, 9.01% ± 1.00%) were lower than those in experimental group (19.29% ± 1.18%, 15.07% ± 0.97%), (P lt; 0.01). The results of the average gray scale value of nucleus pulposus in control group were 187.33 ± 7.88 and 185.68 ± 3.26, respectively, with 124.98 ±6.69 and 160.13 ± 4.37 in experimental group. There was significant difference between the two groups (P lt; 0.01). When thetotal 50 specimens in the two groups were analyzed, TUNEL positive incidence showed significant inverse correlations with their respectively corresponding cell densities (r = － 0.88, r = － 0.93, P lt; 0.01). The Bax and Caspase-3 positive incidence of nucleus pulposus showed significant positive correlation with the TUNEL positive incidence of nucleus pulposus (r = 0.83, r = 0.91, P lt; 0.01). Conclusion The decrease of cell density is involved in the development of human lumbar intervertebral disc degeneration. Bax and Caspase-3 might play a role in disc cell apoptosis in nucleus pulposus of human lumbar intervertebral disc.