Experimental Study On Gene Therapy Of Postmenopausal Osteoporosis In Rat With HOPG Gene

With social development and population aging, incidence of primary osteoporosis has been climbing, resulting in high incidence rates and certain mortality rates of osteoporotic fracture and huge economic burdens for the society as well as individuals. It has become an imperative issue to prevent and treat osteoporosis in the graying world. Of the many causes of osteoporosis, increased numbers and function of osteoclasts are fundamental, which leads to the rate of bone resorption exceeds that of bone formation. Hence, most strategies for treating osteoporosis aim at inhibition of osteoclasts. hOPG, found in 1997, is a member of the TNFR superfamily and a secretory glucoprotein, which is generated by marrow stroma cells or osteoblasts and is capable of suppressing differentiation of and inducing apoptosis of osteoclasts. Administration of recombinant hOPG effectively prevents loss of bone mass in animal models of osteoporosis or postmenopausal women. hOPG can be recombined in vitro and administered as proteinaceous preparations. However, osteoporosis is a chronic disease, requiring a long therapeutic course and repetitive administration of hOPG. It is difficult to obtain bioactive hOPG. In addition, hOPG has many drawbacks, such as high molecular weight, unstable properties, short half-life time in vivo, immunological rejection. In light of these problems in the treatment of osteoporosis with hOPG protein, we attempted at a gene therapy of osteoporosis by introducing hOPG gene into animal body using gene recombination techniques, with adenovirus as the vector. The study is composed of three sections: 1. Construction, preparation, titration, and in vitro expression of Ad-hOPG; 2. Charaterization of the in vivo expression of Ad-hOPG in rats; 3. Observation on the therapeutic effects of Ad-hOPG on osteoporosis in OVX rats. Methods: 1. The target gene was cut from plasmid PUC19-hOPG that contains hOPG cDNA. Plasmid Adtrack-CMV-hOPG was recombined using the target gene along with shuttle plasmid AdTrack-CMV. Recombinant adenoviral DNA containing hOPG gene was constructed through homologous recombination of hOPG gene and adenoviral genome in BJ5183-Adeasy-1 bacteria. 2. Ad-hOPG was packaged and amplified in the 293 cells, viral titer determined, recombinant adenoviral DNA identified using PCR, in vitro expressed protein identified using Western blotting analysis. 3. Ad-hOPG was injected into rats via vena caudalis. Visceral specimens, including the liver, spleen, kidney, and heart were obtained at different time points, and the specimens were subjected to frozen section. Expression of Ad-hOPG reporter gene, i.e., GFP was detected under a fluorescent microscope. The characteristics of Ad-hOPG expression were observed at various titers, and the prime Ad-hOPG expression and corresponding titer were selected to use in the following experiments. 4. Non-pregnant female SD rats (age 10 months) were randomly assigned to the Sham, NS, and Ad-hOPG groups. Rats in the former group underwent sham operations and those in the latter two groups underwent OVXs. 8 weeks later, the Sham group received no interventions; whereas, the NS and the Ad-hOPG groups received normal saline and 3.16×109pfu/ml Ad-hOPG injections, respectively. From 48h after the interventions onward, serum hOPG concentrations were periodically monitored. On day 35, radiography, determination of bone density and bone biomechanics parameters, bone morphological studies, and osteoclast counting were performed. Results: 1. Ad-hOPG was successfully constructed, as confirmed by identification of enzymatic cutting and PCR. 2. Ad-hOPG was successfully packaged. TCID50 assay revealed the highest titer of 3.16×109pfu/ml of recombinant adenovirus. Sufficient Ad-hOPG was amplified in the 293 cells. PCR results confirmed that recombinant adenoviral DNA contained hOPG gene, and Western blotting analysis indicated that OPG could be stably expressed in vitro. 3. Injection of Ad-hOPG at the titers of 3.16×107 and 3.16×108pfu/ml produced its weak expression in vivo, and the expression lasted a short time (10d and 16d, respectively). Injection of Ad-hOPG at the titer of 3.16×109pfu/ml produced its strong expression in the liver and spleen 2d later and its mild expression in the heart and kidney. The expressionreached a peak on day 6. Even on day 26, GFP expression could be detected. 4, ①Determination of serum hOPG concentration: Serum hOPG was not detected in the Sham and the NS groups because it was lower than the detectable low threshold of the ELISA kit (0.2 ng/ml). 2h after intervention, 2.511μg/ml of Ad-hOPG was detected in the Ad-hOPG group. On day 6 the expression reached a peak of 62.342μg/ml. On day 30 the expression was 0.0354μg/ml. But on day 34, the expression was not detected. ②Radiography: In the Sham group, rat bone density changed in significantly; in the NS group, it was slightly reduced, more obvious in the vertebrae than in the limbs. Bone density was higher in the Ad-hOPG group than in the NS or the Sham group. Changes mainly developed in the vertebral body, distal end of the femur, upper tibial metaphysis, and proximal end of the ilium. ③Bone density as determined DEXA: There were significant differences in femoral and lumbar BMD between the NS and sham groups(P<0.05); The Ad-hOPG group showed compared with the NS group, there were significant differences in femoral and lumbar BMD(P<0.01), But no significant differences were found between the Sham and the Ad-hOPG groups (P>0.05). ④Femoral biomechanics properties: There were significant differences in femoral structure mechanics (maximal bias, maximal load, maximal energy absorption), material mechanics (maximal stress, elastic modulus) (P<0.01) between the NS and the Ad-hOPG groups.But no significant differences were found between the Sham and the Ad-hOPG groups (P>0.05), with these parameters for the latter slightly lower than those of the former. Vertebral biomechanics properties: There were significant differences in vertebral structure mechanics (maximal bias, maximal load, maximal energy absorption) and material mechanics(maximal stress, elastic modulus) between the Ad-hOPG and the NS groups(P<0.01). But no significant differences were found between the Sham and the Ad-hOPG groups (P>0.05), with these parameters of the latter slightly lower than those of the former. ⑤Bone morphology: Bone trabeculae were microscopically observed to densely align and form reticular structures in the Sham group. In the NS group, bone trabeculae obviously reduced and sparsely aligned and did not form reticular structures. Widened bone trabeculae interspace was also obvious. The number of bone trabeculae, reticular network, and bone trabeculae links were significantly more in the Ad-hOPG group than in the NS group. ⑥Osteoclast counting: There were many red positive TRAP-stained cells between the bone trabeculae and around the epiphyseal plate in theSham group; there were also many positive cells between the sparse bone trabeculae in the NS group; but positive cells were nearly absent in the tibial metaphysis of the Ad-hOPG group. Conclusions: 1.Ad-hOPG DNA was successfully constructed using shuttle plasmid Adtrack-CMV-hOPG and Adeasy-1 bacteria, and a high titer of Ad-hOPG(3.16×109pfu/ml) was prepared. 2. Various titers of Ad-hOPG can be expressed in rats, which positively correlate to the strength and time frame of Ad-hOPG expression. Higher titer of Ad-hOPG(3.16×109pfu/ml) can induce 30 days of Ad-hOPG expression in rat. 3. Treatment of rat osteoporosis with Ad-hOPG at the titer of 3.16×109pfu/ml markedly ameliorates BMD, biomechanics properties, bone microscopic morphology, and significantly reduces osteoclasts, indicating that Ad-hOPG exerts sound therapeutic effects on postmenopausal osteoporosis.