| ObjectionIschemic osteonecrosis has been recognized as a common disease presently, especially does the osteonecrosis of femoral head.With the development of med-ical research,people have made great progress on the cognition of the disease. Except the pathogeny of trauma,the mechanism of the disease is so complex that the true pathogeny of ischemic osteonecrosis has not been identified.The scien-tists of home and abroad have drawn some conclusion by the basic research and clinic experiments.Each theory has its own viewpoint,but any of them could not explain the mechanism absolutely.The mechanism of nontraumatic osteone-crosis of femoral head is dimness,but the pathologic change is similar to the traumatic one.It has been consider that the osteonecrosis was due to the ische-mic.Demonstrate the change of pathology based on the cytology and molecular biology which is beneficial to know the true mechanism of nontraumatic osteone-crosis of femoral fead.Ischemic is one of the induced factor of apoptosis.There are so many literature about the apoptosis of neural cells which is induced by is-chemic in the brain.In the research of ischemic necrosis,the cells of organ such as heart,liver and kidney,the death of them is associated with apoptosis. But whether there will be apoptosis in the osseous-tissue has not been reported at home and abroad.It will be of great signality to prove up the mechanism of is-chemic osteonecrosis.In this experiment,we established the animal models of ischemic osteonecrosis compare with the femoral heads which were took from the operation after the fracture of femoral neck fracture.Investigate the relationship between the extent of apoptosis and the spatio-temporal change.In advance to discuss the effect of Bcl-2,Bax in apoptosis of the ischemic osteonecrosis,then discuss the function of Bcl-2,Bax in the apoptosis of ischemic osteonecrosis. To sum up,we investigated the mechanism of ischemic osteonecrosis based on cytology and molecular biology.Materials and methodsSet up animal models of ischemic osteonecrosis with 35 SD rats,male, weight 280～300g,(They are all supplied by the animal department of China Medical University).The operation was going on in the fully integral asepsis en-viromnent after general anesthesia.The left hip is experimental side and the right one is control side.Open the capsula articularis of hip joint,severed the ligament of capitis femoris,then the hip joint was dislocation artificially.After cutted the thighbone off at the site of 20mm away from the trochanter major, closed the incision without any fixation.Put seven rats to death separately at 3 h,6 h,12 h,24h,72 h after operations,then take out of both proximal fe-murs,fixation with 4%formaldehydum polymerisatum for 24 h.Decalcification with EDTA(PH 7.4)at 4℃for 2 to 3 weeks,changes the decalcification fluid every 3 days.Test with needle until complete decalification,then imbed with paraffin.Collect 21 clinical cases in which the patients have artificial joint after hav-ing the femoral head resected for femoral neck fracture,All of the clinical cases come from China Medical University first affiliated hospital,in which 6 is male, 15 is female,and from 62 to 83 years old.Draw the materials in 3 days to 3 weeks after fracture.Dissect the femoral head along coronal face,each piece is about 5mm in thickness,fixation with 4%formaldehydum polymerisatum for 24 h.Decalcification with EDTA(PH 7.4)at 4℃for 3 to 4 weeks,changes the decalcification fluid every 3 days.ReagentAll of the reagent such as In Situ Cell Apoptosis Detection Kit I,Bcl-2,Bax monoclonal antibody were bought from Wu han Boster Biological Technology Ltd. Observation target(1)HE stain,(2)Observation by transmission electron microscopy(TEM), (3)DNA gel electrophoresis(analyze DNA fragment by agarose gel electrophore-sis),(4)dUTP nick end labeling(TUNEL)mediated by terminal deoxynucleoti-dyl transferase(TdT).(5)test Bcl-2,Bax by immunohistochemical.(6)man-aged by computer microgram analysis system(CMAS),and analyzed by statistic methods.ResultAnimal experiments[1]changes in morphology under HE stain:there is no obvious morphology changes of the osteocyte and osteoblast in bone trabecula in the 3 h and 6 h is-chemic rats;in the 12 hours ischemic rats:there is degenerative necrosis in bone marrow,and some osteocyte necrosis;in the 24 h ischemic rats:there is no osteoblast;in the 72 h ischemic rats:there is necrosis of chondrocyte.[2]TEM:in the 12 h and 24 h ischemic rats,cell pycnosis,chromatin gather and burst,the apoptotic body and cytoplasmic vesicle form,and there is marginal gather phenomenon of nucleus.[3]Agarose gel electrophoresis:by experiment:there is speific "ladder" zone in DNA electrophoresis of 12 h and 24 h ischemic rats,and in 72h ische-mic rats,there is "smear" zone.And there is not this phenomenon in the con-tract group.[4]Test of the TUNEL positive cell(apoptotic cell):there are TUNEL positive cells in the 6 h,12 h,24 h ischemic rats,and there is obvious differ-ences with the 3 h,72h group.(p＜0.01).[5]there is positive expression of Bcl-2 and Bax in 3 h ischemic rats, and Bcl-2/Bax increases in 6 h ischemic rats,Bcl-2/Bax decreases in 12 h and 24 h ischemic rats,and there is weak positive expression of Bcl-2 and Bax in 72 hours ischemic ratsClinical experimentsThere is specific "ladder" zone in DNA gel electrophoresis,and there are TUNEL positive cells in bone trabeculla cells and bone marrow cells,and have a relationship with the expression of Bax(p＜0.01).Conclusion1.The apoptosis of osteocyte can be tested in the bone trabeculla of the is-chemic osteonecrosis rats.Apoptosis is one of the ways in which cell necrosis in the early times of ischemic osteonecrosis.2.Perhaps Bcl-2 and Bax take part in the control of apoptosis in the early times of ischemic osteonecrosis.3.The value of Bcl-2/Bax has a relationship with the apoptosis degree of the osteocyte.4.There is apoptosis in bone trabeculla of femoral head after femoral neck fracture for ischemia,and it is related with the expression of Bax gene.
|
PDF Full Text Request