The Expression Of Nuclear-κappaBP65 And The Effect Of NF-κappaBP65 SiRNA On Its Expression During Fracture Healing Process Of Experimental Osteoporosis Model

ObiectiveWith the population aging, the morbility of osteoporosis gradually increase. Its main pathogenesis is that with the absence of estrogen, various bone absorption stimulus factors such as TNF - α, RANKL, NF - kB etc act on osteoblast and osteoclast, which promote the activation of osteoclast and inhabit the apopto-sis of osteoclast, consequently result in an excess of the rate of bone absorption over that of bone formation that cause osteoporosis. For osteoporosis patient , the risk of fracture increases and the healing of fracture prolonged.IL - 1, IL - 6, TNF - a secreted by osteoblast are strong and major bone absorption stimulus factors. They can directly promote the differentiation and formation of osteoclast, inhabit its apoptosis and activate nuclear factor - Kappa B. Conversely, activated NF - kB can also promote the expression of IL - 1, IL - 6 and TNF - a. So NF - kB plays an important role in the pathogenesis of osteoporosis.Is there relationship between the regulation of NF — kB and the prolonged healing process of fracture? Whether can we improve the healing of fracture by the regulation of NF - kB? We duplicate model of osteoporosis (ovariectomy) and design fracture model of distal end of radius fracture to observe the expression of NF - KBp65 in broken end of bone tissue during fracture healing process of experimental osteoporosis mice and investigate the relationship between NF -KBp65 expression and fracture healing of osteoporosis.RNA interference (RNAi) is first discovered in Arabidopsis, C. elegans in 1998 and demonstrated that it is belong to post - transcriptional level gene silen-cing mechanism. The discovery of RNAi change the traditional understand of cellular gene regulation and provide new method for blocking gene expression. Recent research indicates that the siRNA can not only inhabit and degrade homologous single - stranded RNA, but also avoid non - specific gene degradation of long - stranded dsRNA and cell death. On the base of the result that in intro NF - KBp65 siRNA can specifically inhabit the expression of NF - KBp65 on the osteoblast, this experiment is designed to further investigate the effect of NF -KBp65 siRNA on osteoporotic fracture healing.Methods1. The grouping and establishment of osteoporosis model and osteoporotic fracture model.80 four month female Balb/c mice, weight 20 -23g.1) 40 four - month female Balb/c mice were randomly divided into ovariec-tomized group and sham - operated group. Distal ends of radius were made broken for fracture model in all mice after three months.2 ) 40 four — month female Balb/c mice after establishment of osteoporotic fracture model were randomly divided into siRNA interference group and mismatching siRNA interference group.2. Molybdenum target ray film, histology observation, immunohistochemis-try stain and in - situ hybridization observation.All mice were taken molybdenum target ray film to observe the establishment of fracture model. After sacrifice of the animal at 1 ^2^4^6 week after fracture , we cut the distal end of right radius by sterile operation and ophthalmic operating microscope. Observation of the expression of NF - KBp65 was done by in - situ hybridization detection and immunohistochemistry stain.3. Result assessment and image analysisObservation of NF - KBp65 and NF - KBp65 mRNA expression was done by light microscope. Photodensity values of positive cell in local tissue of fracture were measured by computer image analysis. The values were demonstrated by average ± standard deviation and analyzed by t test, p < 0. 05 was consideredthat there was significant difference.Results1. Histology observationAt 1 - 2 week after fracture, HE stain showed that in SHAM - operated group, mesenchymal cell, early cartilaginous callus and nascent capillary were seen in broken end of fracture. Large amount of regular osteoblasts were seen on the surface of primitive trabecula. In ovariectomized group, the formation of cartilaginous callus and the number of osteoblast were less than that of SHAM - operated group. At 4 - 6 week after fracture, disarrangement of trabecula and broaden interval among trabecula were seen in ovariectomized group.2. The immunohistochemistry stain and observationThe positive signals of NF - i0. 05) .ConclusionThis research confirms that the expression of NF - KBp65 significantly increased in broken end of bone tissue during early stage of osteoporotic fracture healing. NF - KBp65 which promotes the expression of various bone absorption stimulus factors may produce a marked effect on the process of osteoporotic fracture healing. NF - KBp65 siRNA can specifically block the expression of NF -KBp65 mRNA and decrease the formation of osteoclast stimulus factors such as TNF - a. It may improve the process of the osteoporotic fracture healing.