论文标题:股骨头缺血性骨坏死骨细胞凋亡与Bcl-2、Bax表达的相关性研究
Study of Apoptosis and Expression of Bcl-2、Bax after Ischemic Osteonecrosis of Femoral Head
论文作者
论文导师 吕刚,论文学位 博士,论文专业 外科学
论文单位 中国医科大学,点击次数 332,论文页数 62页File Size4111K
2004-03-01论文网 http://www.lw23.com/lunwen_1167387/
ischemia;; osteonecrosis;; apoptosis;; gene expression
目的 缺血性骨坏死目前已成为一种常见病,尤其是股骨头坏死致残率最高。目前对该病的认识有了很大的提高,但其发病机制尚未完全明了,除已知创伤等病因外,其余病因和发病机理复杂而不明确。对其发生机制国内外学者进行了基础和临床研究,提出了各种学说,从不同的侧面阐述了各自的观点。尽管非创伤性股骨头坏死的病理机制仍不清楚,但与创伤性股骨头坏死的病理改变十分相似,认为均由于局部缺血后,骨及骨髓成分出现坏死。以细胞学及分子生物学为基础来阐述其病理改变有助于对非创伤性股骨头坏死病理机制的了解。缺血是凋亡的常见诱导因素之一,目前脑缺血诱导神经细胞凋亡方面的文献较多,在缺血性损伤研究中,心、肝、肾等脏器的细胞死亡亦与细胞凋亡有关,但缺血后骨组织是否存在凋亡国内外未见报导,了解局部缺血后骨组织内细胞凋亡情况,探明缺血性骨坏死的发病机制具有重要意义。本实验通过建立缺血性骨坏死的动物模型及观察由于股骨颈骨折行人工关节置换时取出的股骨头,检测骨组织中是否存在细胞凋亡及缺血时间与凋亡程度的关系,并在此基础上进一步观察Bcl-2、Bax免疫反应阳性细胞与凋亡细胞的相关性,旨在探讨Bcl-2,Bax对缺血性骨组织细胞凋亡的调控作用。从细胞学和分子生物学方面探讨缺血性骨坏死的发病机制。 材料和方法 1.实验对象及处理因素 SD大鼠35只,雄性,体重280～300g(由中国医科大学实验动物部提供)。2%戊巴比妥钠25mg/kg腹腔注射麻醉,手术在无菌条件下进行,左侧髋部为手术侧,右侧为对照侧,切开左侧关节囊,切断股骨头圆韧节,使髋关节脱位,于大粗隆下20mm处截断股骨,剥离软组织,而后放置股骨近端于原位,缝合皮肤,不做内外固定。分别于术后3h、6h、12h、24h、72h各处死7只,取出两侧股骨近端,4%多聚甲醛固定,20%EDTA(PH7.4),4℃脱钙2-3周,以针刺法检查至脱钙完全,而后石腊包埋。 临床收集21例股骨颈骨折需行人工关节置换术患者的股骨头。病例均来自中国医大一院骨科,男6例,女15例,年龄:62-83岁,取材时距骨折的时间3天-3周。将股骨头沿冠状面剖开,切成厚约5mm骨片,4%多聚甲醛固定24h,20%EDTA(PH7.4),4℃脱钙3-4周,每3天更换脱钙液,以针刺法检查至脱钙完全,而后石腊包埋。 2.试剂来源: 凋亡检测试剂盒及兔抗大鼠Bcl-2单克隆抗体、Bax单克隆抗体及生物素化抗地高辛抗体IgG均购自武汉博士德生物工程有限公司。 3.观察指标: ①HE染色;②透射电镜观察;③DNA凝胶电泳(琼脂糖凝胶电泳分析DNA断裂);④末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记[Terminal deoxynucleotidyl transferase(TdT)dUTP nick end labeling,TUNEL]法检测凋亡细胞;⑤免疫组化检测Bcl-2、Bax表达;⑥计算机显微图像分析系统(型号:MetaMorph/DP10/BX41,生产厂:UIC/OLYMPUS/US/JP)处理,x~2检验和t检验,统计学分析。 结果 1.动物实验部分 HE染色形态学改变:缺血3h,6h骨小梁内骨细胞及成骨细胞形态未见明显改变,缺血12h:骨髓内细胞可见变性坏死,部分骨细胞坏死,缺血24h:成骨细胞消失,缺血72h:可见软骨细胞坏死。(2)透射电镜:缺血12h及24h,细胞固缩,染色质浓聚裂解,胞浆内小泡及凋亡小体形成,细胞核可见边集现象。(3)琼脂糖凝胶电泳:本实验缺血12h、24h标本提取DNA后,经琼脂糖凝胶电泳检测可见到相同的“梯状”条带,梯状条带是凋亡细胞DNA片段化(fragmentation)的结果,内源性核酸内切酶(endonuclease)将核小体间的连接DNA降解,形成长度为180～200bp整数倍的寡核苷酸片段,组蛋白和其它核内蛋白质不降解,核基质也不改变,由于大部分细胞凋亡出现DNA梯状条带,而细胞坏死时DNA随意断裂为长度不一的片段,琼脂凝糖胶电泳呈“弥散状”(Smear)。本实验中缺血72h即可见“弥散状”条带。(4)TUNEL阳性细胞(凋亡细胞)测定:缺血6h、12h、24h组均可检测到TUNEL阳性骨细胞,并与缺血3h、72h组对照,χ~2检验,有显著性差异(p＜0.01)。缺血3h可见Bcl-2及Bax阳性表达,缺血6h时Bcl-2/Bax增高,缺血12h,24h时Bcl-2/Bax下降,缺血72h时,Bcl-2及Bax均呈弱阳性表达。 2.临床研究部分 DNA凝胶电泳可见特征性“梯状”(ladder)条带,骨小梁骨细胞及骨髓细胞均有TUNEL检测阳性细胞,统计学分析凋亡细胞与Bax表达,t检验有相关性(p＜0.01)。 结论 1.大鼠股骨头缺血后骨小梁内可检测出骨细胞凋亡,细胞凋亡是缺血性骨坏死早期骨细胞死亡的途径之一。 2.Bcl-2、Bax可能参与了缺血性骨坏死早期病理过程中细胞凋亡的调控。 3.Bcl-2/Bax比值与骨细胞凋亡程度相关。 4.股骨颈骨折后股骨头因缺血可出现骨小梁内骨细胞凋亡,且与Bax基因表达有相关性。
Objection Ischemic 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 methods Set 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. Reagent All 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. Result Animal 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 rats Clinical experiments There 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). Conclusion 1.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.
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