The Experimental Investigation On The Biological Effects And Its Mechanism Of Platelet-Derived Growth Factor On The OVX Rats

Backgrounds: In recent years, lots of researches indicate that the endocrine estrogen decreases sharply after menopause, which caused the expression of the local growth factors to reduce in the bone and cartilage. The balance of the bone formation and bone resorption was broken, and the chondrocyte lost some prototypes due to the changes of the estrogen level. With the time going on, osteoporosis and cartilage dengeneration occurred. It had been verified that platelet-derived growth factor could stimulate the osteoblasts to divide and proliferate both in vivo and in vitro, and accelerate bone fracture cure. The chondrocytes maintained their prototype and formed new cartilage on scarf in the presence of PDGF in vitro. We also found that osteoblasts regulated the effects of PDGF on the osteoclasts, and PDGF stimulated the osteoblast-osteoclast co-culture system to form new bone. Therefore, it is hypothesized that PDGF would reverse the osteoporosis and cartilage degeneration due to the esteogen loss.Objectives: To investigate the mechanism of the effects of PDGF on theosteoblast-osteoclast co-culture system, and to research the biologicalfunctions of PDGF on the OVX rats. It would provide experimentalevidences for the clinical use of Platelet-derived growth factor (PDGF) toprevent and cure the osteoporosis and degenerative joints.Methods: 1. The biochemistry and image analyzing were used to investigatethe effects of the PDGF-AA at high concentration on the osteoclasticbone resorption in the osteoblast-osteoclast co-culture system. Thechanges of the tartrate-resistant acid phosphatase (TRAP) in theosteoclasts were measured with the immunofluorescence underConfocal microscope. The expression of the Osteoprotegerin Ligand(OPGL) was examined with immunohistochemistry and ReverseTranscriptase-PCR (RT-PCR).2. The NO products of the osteoblasts were measured with chemical colorimetry, and the expression of the eNOS, iNOS andOsteoprotegerin (OPG) mRNA were analyzed with RT-PCR after osteoblasts were stimulated by PDGF-BB. The influence of the NO on the Vitronectin receptor was monitored by immunofluorescence.3. The histological changes of the bone tissues were revealed by the H-E stain and immunohistochemistry. In situ hybridization was used to analyze the effects of PDGF-AA on the expression of collagen type I. In addition, the serum bone marker-bone specific alkaline phosphatase (ALP), osteocalcin (OCN) and TRAP were assayed. The expression of the osteocalcin and PDGF-A mRNA were checked with RT-PCR. The bone mineral density (BMD) was measured at the 3rd, 6th and 9th week.4. The histological changes of the bone tissues were revealed by the H-E stain and immunohistochemistry. In situ hybridization was used to analyze the effects of PDGF-BB on the expression of collagen type I. In addition, the serum bone marker-ALP, osteocalcin and TRAP were assayed. The expression of the osteocalcin mRNA were checked with RT-PCR. The BMD was measured at the 3rd, 6th and 9th week. And the changes of the eNOS and iNOS were analyzed with immunohistochemistry. It was observed that the changes of the bone tissues after the NOS inhibitor (N-Monomethl-L-Arginine , L-NAME).5. The effects of the PDGF (AA&BB) on the histological changes of degenerative cartilage were observed with alcian blue stain and image analyzing. The proliferation of the chondrocytes was assayed with BrdU incorporating into DNA of mitotic cells. The sulfate incorporation assay was conducted for proteoglycan synthesis. The expression of TRAP, Matrix Metalloproteinase-9 (MMP-9) and PDGF in cartilage was analyzed through immunohistochemistry.Results: 1. PDGF-AA could not raise the TRAP activity and osteoclastic bone resorption directly although PDGF-AA was at high-6-concentration. In the co-culture system, both TRAP activity and bone resorption pit area increased with the dose of PDGF-AA (at high concentration). The OPGL mRNA expression was up-regulated by PDGF-AA a...