| Objective and background: How to cure the ANFH (Avascular necrosis of the femoral head) is a tough problem in the clinically of orthopedics. No effective therapy be find can stop or retroconverse the pathology procedure,accordingly further prohibit the collapse of the femoral head. Femoral head collapse base on the absorption of the necrotic bone,The distance and speed of reparation can influence the absorptive Procedure.Rapid vascularize and bone formation can decrease the absorption. High-blood flow is nessensary for the healing procedure of the necrotic area of femoral head.Change the ischemic condition of the necrotic is the basis to enhance the healing procedure.VEGF is a specific cytokine that act on the vascular ehdothelial cell.It can differentiate and prolixferate the antecedent of vascular endothelial cell and osteocyte Improve the blood flow in the necrotic region ,Enhance the bone formation and remoulding.It hint that VEGF possible act significant role in the healing procedure in necrotic femoral head. Therapy of ANFH need to solve three problem:1.Reconstruct the blood circulation of the necrotic region of femoral head.2.Enhance the new bone formation;3.The quality and quantity of the reconstructive bone trabecular can support the bone under the cartilage. To achieve our objective,we study the model that use liquid nitrogen frozen necrotic femoral head in rabbits. First,we use gene recombination technique to construct adenovirus vector with huVEGF121 gene,Then we establish japan albino rabbits necrotic femoral head model which is frozen by liquid nitrogen.The huVEGF121 gene is transfected in necrotic femoral head mediaed by adenovirus.We observed the effect on construction of blood vessel and bone formation by RT-PCR,IHC,histomorphology,blood vessel quantitative analysis,SPECT,X-ray,bone densitometry technique. The experiment content three parts:1. The model of necrotic femoral head;3.the necrotic femoral head was transfected by AD-huVEGF121.;2. Construct adenovirus vector with huVEGF121 gene We observed the expression of huVEGF121.3.The effection of improve blood vessel reconstruct in necrotic region and enhanced necrotic femoral head reparation. Methods and results: PART ONE: Establish animal model We result in the avascular necrosis of femoral head through frozen by liquid nitrogen.The bone sclerosis was deteced by X-ray examination after 8W and 12W after operation Bone density was uneven.Accordingly,we found the new bone formation on the surface of the necrotic bone trabecula that near the neck of femur by histologyical technique. PART TWO Construction of adenovirus vector with huVEGF121 gene 1. The huVEGF121 gene was proved to have correct base pair sequence by sequencing the pUC18-huVEGF121 plasmid. 2. The huVEGF121 gene was cloned into shuttle plasmid PTrack-CMV We contructed recombinant adenovirus plasmid Padeasy-huVEGF121 by gene homologous recombination technique. We gained recombinant adenovirus Ad-huVEGF121 by Padeasy-huVEGF121 transfected the 293 cell 3. The objective gene was identified in recombinant adenovirus Ad-huVEGF121 by PCR. The titer of virus is generally up to 1.56X1010 phaque forming units (pfu) per milliliter 4. Recombinant adenovirus vectors expression were identified by RT-PCR and and ELISA..It can express huVEGF121 for 21 days in NIH 3T3 cells. PART THREE Ad-huVEGF121 gene transfection and its effect 1. The huVEGF121mRNA and protein was expressed in femoral head for 21days after gene transferction,and no expression was detected in control groups.Many kinds of cell express huVEGF121. 2. After 2W,New blood vessel invaded into necrotic region after gene transfection,and invaded into full-thickness of bone under cartilage after 4W.Immunohistochemistry stain by CD105 and Indian ink infuse show that the quantity of the blood vessel in gene transfective group is significant higher than in the control group.The radiocounting in gene transfectivegroup is significant higher than in control group. 3. Osteogenesis effect hematoxylin-eosine stained show:2W after operation, Bone trabecular spaces near the hole and the hole full with mesenchymal in gene transfective group.4W after operation,a little of osteogenesis in hole.The necrotic spongy bone and subchondral was surranded by osteoblast.6W after operation,a great quantity of new bone in the hole.8W after operation,Normal myeloid tissue can be find in hole.12W after operation,normal morphous of trabecular can be find in hole in gene transfect group.The new bone surround the necrotic bone is more mature,The morphology measurement show that the TBS and TBW of subchondral in gene transfect group is significant high than in control group.BMD measurement show that the BMD in gene transfect group is significant high than in control group.at 8 and 12 w after operation The ALP activeity,lag phase radiocounting,collagenâ… and BMP2 expression in gene transfect group is significant high than in control group.at every phase. Conclusion: 1. liquid nitrogen frozen cause femoral head necrosis and its repairitive procedure similar to human femoral head necrosis.The absorptive necrotic bone is naked which is not surround with new bone.In the repairitive procedure of femoral head.the sclerosis region on X-ray graphic is necrotic bone surround with new bone. 2. The recombinete adenovirus we construct content objective gene,complete sequence, correct open-reading frame,high valence,Can express objective gene in enkaryocyte,refer to gene therapy. 4. VEGF can accelerate the revascularizative procedure in necrotic femoral head.and enhance the metabolic activity of bone salts in osteoblast,accelerate the new bone formation on the necrotic bone trabecula,the new bone can barrier the osteolysis of osteoclast.
|
PDF Full Text Request