论文标题:机械刺激后大鼠牙髓HSP70表达的动态变化
The Kinetics of HSP70 Expression in Rat Dental Pulps after Physical Injury
论文作者 谢红玉
论文导师 周杰,论文学位 硕士,论文专业 口腔临床医学
论文单位 中国医科大学,点击次数 48,论文页数 30页File Size1465k
2002-01-01论文网 http://www.lw23.com/lunwen_625691167/ 热休克反应;热休克蛋白70;获得性耐温性;免疫组化;图象分析
Heat Shock Response;HSP70;acquired theromotolerance;immunohistochemical;image pattern analysis
前言 热休克反应(heat shock response)是细胞遭受特殊环境刺激时进行自我保护的防御机制，以热休克蛋白(heat shock protein)表达增加为特点。热休克蛋白70是热休克蛋白家族中重要的组成成分，其某些成员持续表达而另一些成员只有在细胞受到刺激后才有所表达，持续表达的是构成性HSP70，在细胞的各种生理活动中发挥重要作用；受到刺激后才表达的为诱导性HSP70，由于它与细胞受到的各种刺激密切相关，其表达量与细胞的获得性耐温性(acquired thermotolerance)关系密切，所以HSP70近年来成为人们研究的热点。获得性耐温性是指预先给细胞一种低于致死量的热休克处理，该细胞会对继之给予的致死量的热休克处理具有耐受性。细胞的这种一过性的对刺激的耐受性对保护细胞和组织功能极为重要，而由于HSP70表达水平与细胞耐温性存在着量上的伴行关系，因此研究机械刺激后HSP70表达水平的动态变化就可以间接了解预刺激后细胞获得性耐温性的动态改变，从而为进一步利用获得性耐温性保护细胞和组织提供理论依据。本实验的目的即为研究口腔最常见的机械刺激是否引起牙髓内HSP70表达的变化及其表达量与时间的关系。 材料和方法 领取实验动物(体重280-300克的雄性SD大鼠20只)并进行实验动物分组，将实验对象分为5组：正常对照组，刺激后即刻观察组，刺激后4小时观察组，刺激后8小时观察组，刺激后24小时观察组各4只大鼠。将对照组及实验组大鼠均用0 4％戊巴比妥钠按 1毫升 100 克体重剂量进行腹腔麻醉，对照组大鼠麻醉后即处死，实验组大鼠麻醉后固定，用307－6台钻（上海齿科医疗器械厂，转速3000转／分钟）在其左右上颌第一磨牙洽面正中进行机械刺激（刺激时间控制在 1．5 min左右入分别于刺激后即刻，刺激后4小时在小时、24小时各处死4只大鼠。大鼠处死后立即取左右上颌第一磨牙及附近部分牙槽骨组织，生理盐水冲洗，置4％多聚甲醛固定液内并作好标记，4℃冰箱内固定24小时，将标本取出置于 smol／L甲酸－lmoUL甲酸钠复合脱钙液内，4℃脱钙 20天，每天更换脱钙液并修剪组织块。脱钙完成后将组织块进行酒精梯度脱水，二甲苯透明，浸蜡，石蜡包埋，切片机制备冠根纵向石蜡切片，片厚 5 pm。进行 HE染色及 HSP70免疫组织化学染色（兔抗HSpoo多克隆抗体，SP法人空白对照实验以PBS替代1抗，其余染色步骤相同。利用 coolsnapfr显微照相系统（Rop。，Japan）及MetaMorph分析软件（UIC，US）对免疫组化染色标本进行图象分析。 实验结果 免疫组织化学染色结果表明，HSP70在正常未受到刺激的牙髓内及刺激后不同时间的牙髓内均有表达，HSpoo普遍存在于牙髓中各种细胞内，成牙本质细胞及胞浆突起、牙髓成纤维细胞、血管内皮细胞及管壁平滑肌细胞、神经节细胞内均有表达，其中又以成牙本质细胞及胞浆突起内表达更为明显。牙髓受到机械刺激后即刻观察组HSP70表达量及表达强度与正常牙髓无明显差异。刺激后 4小时取材组，HSpoo表达量明显增加，与正常牙髓相比差异明显瞩＜o．05人刺激后8小时取材的牙髓，HS o 0表达达到高峰，成牙本质细胞出现强阳性的着色，与正常牙髓之间差异明显 ·2·p＜0．05人牙髓受到刺激后 24 ／J’时组 HSpoo表达量较 4 ／J’时组及8小时组明显减少，差异显著叩＜0．05入而与正常牙髓之间则无明显差异汀＞0．05八空白对照无阳性着色。兔疫组化染色标本各组图象的积分光密度值＊OD）方差分析结果表明，牙髓受到机械刺激后0小时HSP70表达与正常组无差异，其后随着时间延长HSpoo表达有所增加，至8小时达高峰，其后表达逐渐减少，刺激后24小时HSP70表达量基本恢复至刺激前水平。 讨 论 兔疫组织化学染色结果表明，在正常未受到刺激的牙髓，HSpoo普遍存在于牙髓各种细胞内，其中成牙本质细胞及胞浆突起内表达更为明显。牙髓内各种细胞都不断进行着生理活动，因此都表现出HSpoo阳性着色。成牙本质细胞是牙髓内唯一能形成牙本质的细胞，牙齿发育完成后仍然不断地分泌蛋白质基质以形成继发性牙本质，其细胞内蛋白合成和转运较其它细胞更为活跃，HSP70的表达较其它代谢水平较低的细胞更为明显。 热休克蛋白作为分子伴侣的另一项功能是清除细胞内积聚的异常蛋白。当牙体硬组织受到损害导致牙本质或牙髓暴露时，成牙本质细胞内蛋白合成受到影响，大量异常折叠的蛋白在细胞内积聚，这些异常蛋白成为引起热休克反应的扳机，使细胞逐渐提高HSpoo表达水平来清除异常蛋白，当机体内的HSpoo足够清除掉积聚的异常蛋白时，机体不再产生更多的HSpoo，随着异常蛋白的清除，HSP70逐渐减少直至恢复至正常蛋白代谢所需要的水平，于是出现了HSP70表达的动态变化。 成纤维细胞具有分化为成牙本质细胞的潜能，当成牙本质细胞受到损伤某些细胞走向变性死亡的时候，成纤维细胞加快代谢速度转化为成牙本质细胞，这时就会出现II
PrefaceHeat Shock Response ( HSR) , also known as Stress Response (SR) , is a kind of protect strategy that cells developed to deal with adverse changes in their environment. This response is characterized by the extremely rapid increased expression of Heat Shock Proteins ( HSPs). One major family of the heat shock family is the HSP70 families that are consisted of two kinds of proteins, the constitutive HSP70 and the stress - inducible HSP70. The former continuously express and function as molecular chaperones for newly synthesized proteins. The later express only when cells are stressed, so their appearance is often diagnostic that the cell has experienced some type of trauma. More importantly, the levels of stress - inducible HSP70 have apparently direct correlation with the acquired thermotolerance of cells. Study the kinetics and the levels of HSP70 expression can give us some messages about the acquired theromotolerance of the stressed cell. The purpose of this study was to research the kinetics and the level of HSP70 in the dental pulps that have exposed to dental physical stress and then offer us the basis to put the theory of acquired thermotolerance into practice.Materials and Methods20 adult Sprague - Dawley male rats (280 -300g) were anesthetized , four were killed immediately after the anesthesia, the rest rats were fixed to the operation table and their maxillary first molars were injured by the dental drill for 1.5 minutes each. Four rats were killed immediately after the injury, four rats were killed 4 hours after, and four rats were killed 8 hours after the injury, the last four rats were killed 24 hours after the injury. The rats"first maxillary molars were extracted and put to the paraformaldehyde. 24 hours later, the molars were taken out and put to decalcification fluid. The specimens were taken out 20 days later, paraffin imbedded, made paraffin section, and then made HSP70 immunohistochemical ( IHC ) staining ( SP method) and HE staining. All the stained sections were studied under the light microscopy and under image pattern analysis; all the data (IOD average) were statistically analyzed.ResultsThe immunohistochemical staining of HSP70 suggested that HSP70 expressed in the non - injured and injured dental pulps. It is apparent that this immunoreactivity for HSP70 is present both within the odontoblasts and the odontoblast processes that occupy the dentin tables. The pulp cells (fibroblasts) appeared HSP70 immunoreactive too. The endothelial cells of the vessels, the smooth muscle cells all express HSP70 although their stain is less intense than that of the odontoblasts. The dental pulps obtained immediately after the physicalinjury expressed HSP70 as much as the pulps that were not injured. The pulps obtained four hours after the injury expressed more HSP70 than the non - injured pulps. The expression of HSP70 in pulps that recovered from injury for eight hours reached the maximum. HSP70 expressed in pulps that obtained twenty - four hours after injury went back to the normal standard.DiscussionThe constitutive HSP70 are continuously expressed and function as molecular chaperones for newly synthesized proteins, assisting in the folding and assembling of protein complexes and in translocation of proteins into organelles. Their degree of expression appears dependent on the metabolic activities of the cell. All cells in the dental pulp have physiological activities, so almost all cells in the pulp express HSP70. The odontoblasts have the ability to form dentin, this means that the odontoblasts exhibit higher secretary activities than the other pulp cells. So the odontoblast expressed more HSP70 than other cells.The other function of heat shock proteins is their ability to clear up the abnormally folded proteins accumulated in the cells. When the hard tissue of tooth was grinded and the dental pulp and odontoblasts were insulted, the synthesis of proteins in the odontoblasts was impacted , they cant experience their normal folding pathway and become abnormall
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