c-fos介导下颌功能前伸后髁突骨改建的研究

发布时间：2018-04-28 16:04

  本文选题：下颌功能前伸 + 髁突软骨　； 参考：《河北医科大学》2014年硕士论文

【摘要】：目的：髁突软骨在整个生命过程中始终处于改建状态,改建除了受到机体自身遗传、体液、神经等因素控制之外,其局部所受的各种应力刺激也起着重要作用。髁突软骨细胞能够感受各种应力刺激，在生物机械力的作用下，髁突软骨内会产生生物力学信号的转换，表现出细胞增殖、分化和程序性细胞死亡等一系列的反应。然而,髁突软骨受到机械应力作用后,体内软骨细胞如何应答机械应力刺激,并将刺激信号转导进入细胞内,从而调节髁突骨改建的细胞机制仍未能得到清楚的认识。近年来发现c-fos基因参与髁突骨改建的信号转导和调控过程,并受到学者的关注。c-fos属于细胞癌基因中的核内蛋白类,集中分布于细胞核内,细胞处于静止期时不表达,它的表达一般与细胞生长、增殖同步出现。原癌基因的主要功能是调节细胞生长、增殖和分化,有研究发现该基因与软骨细胞增殖以及骨形成有着密切的关系。 本实验采用免疫组织化学染色的方法检测大鼠下颌功能前伸后髁突软骨中c-fos的表达及其变化规律，探讨细胞内c-fos介导功能矫治前伸下颌后髁突骨改建的机制，为临床上功能矫治提供实验依据。 方法：选用4周龄健康雄性Sprague-Dawley(SD)大鼠70只，体重约80克，随机等量分为实验组和对照组，组内根据实验周期（1、3、7、14、21、28和35天）分为7组（每组5只），共14组。适应性饲养一周后，实验组利用3M玻璃离子将自制下颌前伸斜面导板矫治器粘固于大鼠上切牙，通过链状橡皮圈经鼻上颌复合与两侧口外弓连接，使得斜面导板获得稳定的固位，将大鼠四肢用医用橡皮膏紧紧包裹有效地防止大鼠前爪对装置的破坏及对面部组织的伤害，矫治器24h戴用；对照组不做任何实验操作。所有动物均以软食饲养，自由饮水，同环境下饲养至实验结束。实验后1、3、7、14、21、28、35天分别取材，常规固定，脱钙，脱水，包埋。石蜡切片后进行常规HE染色观察髁突组织学变化；免疫组化方法（二步法）检测FOS蛋白的表达，采用ZKPACS-G型中科恒业细胞图象分析系统进行免疫组化染色评分，以IHS值表示染色强度。所有数据结果均应用SPSS13.0统计软件进行处理，，统计分析时实验组与对照组组间比较采用t检验，组内比较采用单因素方差分析，以P＜0.05为有统计学差异。检验水准α=0.05。 结果： 1大体观察 实验组大鼠下颌处于适度前伸状态，上下颌前牙呈对刃关系，摘除矫治器后，下颌不能后退。而对照组大鼠前牙覆盖约3mm。 2髁突形态学变化 对照组随着观测时间的延长，髁突软骨呈现增龄性变化，表现为软骨厚度变薄，其中软骨中部和后部变化更为显著；实验组自实验第3天起,髁突软骨各层组织变厚,软骨细胞数量增多,髁突中后份有明显变化,而前份变化不明显。之后到21天时，软骨软骨各层逐渐稳定，移行层较对照组仍明显增厚，并可见处于末期的软骨细胞，成骨细胞以及大量原始骨小梁及发育不全的骨髓腔。 3FOS蛋白表达水平及分布 空白对照（PBS代替一抗）组未见阳性着色颗粒。对照组髁突软骨中仅肥大层少量细胞FOS蛋白表达阳性，且一直到第21天都维持在较低的表达水平，第21天以后表达逐渐下降到极低的水平，组间比较差异无统计学意义（P0.05）;实验组第3天时,髁突软骨中FOS蛋白表达明显增强,IHS=2.2±1.10,与对照组IHS=1.0±0.70显著差异（P＞0.05）,第7天时，实验组IHS=6.0±1.22阳性表达持续增强，与对照组IHS=1.0±0.70比较有明显差异（P 0.05）；第14天，实验组IHS=6.0±1.58,与第7天基本维持在同一表达水平，第21天,实验组IHS=3.6±1.67，大部分样本阳性表达细胞开始减少,但与对照组相比仍有显著差异（P0.05）；第28天，实验组IHS=2.4±1.14，对照组IHS=0.8±0.45，差别仍具有统计学意义；第35天，髁突软骨FOS蛋白表达回落至极低水平，实验组IHS=0.6±0.55，与对照组IHS=0.6±0.55相比无统计学差异。 结论： 1c-fos参与了大鼠下颌前伸后髁突软骨改建过程的调节。 2c-fos在髁突软骨细胞增殖和分化中起到重要作用。 3c-fos介导了大鼠髁突软骨骨改建过程中机械外力-生物化学信号的转导，在生物信号由细胞核外转入核内的过程中起到重要的作用。
[Abstract]:Objective: the condylar cartilage has been rebuilt throughout the life process. In addition to the control of the body's own inheritance, body fluid and nerve, the local stress stimulation also plays an important role. The condyle cartilage cells can feel a variety of stress spines. Under the effect of biological mechanical force, the condylar cartilage will be within the condylar cartilage The transformation of biomechanical signals shows a series of responses to cell proliferation, differentiation and programmed cell death. However, after the condylar cartilage is subjected to mechanical stress, how the cartilage cells in the body respond to mechanical stress stimulation and transduce the stimulus signal into the cell, thereby regulating the cell mechanism of the remodeling of the condyle bone still failed. In recent years, the c-fos gene has been found to be involved in the signal transduction and regulation of the reconstruction of the condylar bone, and the attention of the scholars is concerned that.C-fos belongs to the intracellular protein of the cell carcinoma gene, which is concentrated in the nucleus and the cell is not expressed at the stationary phase. Its expression is in synchro with cell growth and proliferation. Its main function is to regulate cell growth, proliferation and differentiation. Some studies have found that this gene is closely related to chondrocyte proliferation and bone formation.
In this experiment, immunohistochemical staining was used to detect the expression of c-fos in the mandibular condylar cartilage and the changes in the mandibular protrusion in rats. The mechanism of c-fos mediated function in the reconstruction of the posterior condyle of the mandibular protrusion was explored to provide the experimental basis for the clinical functional correction.
Methods: 70 healthy male Sprague-Dawley (SD) rats of 4 weeks old, weighing about 80 grams, were divided into the experimental group and the control group randomly. The group was divided into 7 groups according to the experimental period (1,3,7,14,21,28 and 35 days) and 14 groups. After a week of adaptation, the experimental group used the 3M glass ion to stick the self-made mandibular protrusive inclined plane guide appliance. The rat upper incisor was fixed on the upper incisor by the chain like rubber ring through the nasal maxillary compound and the two side pantograph, making the oblique guide plate stable. The rat limbs were tightly wrapped with medical rubber cream to effectively prevent the damage of the rat's front claw and the injury of the facial tissue. The orthodontic 24h was worn and the control group did not do any experimental operation. All animals were fed with soft food, free drinking water, and kept in the same environment to the end of the experiment. After 1,3,7,14,21,28,35 days, the animals were harvested, routinely fixed, decalcified, dehydrated and embedded. After paraffin section, routine HE staining was used to observe the histological changes of the condyle; the immunohistochemical method (two step method) was used to detect the expression of FOS protein, and ZKPACS-G medium was used. The industrial cell image analysis system was used to perform immunohistochemical staining score with IHS value. All data were processed with SPSS13.0 software. The experimental group was compared with the control group by t test, and the single factor variance analysis was used in the group. The statistical difference between the group and the P < 0.05 was statistically significant. The test level was alpha =0.0. Five
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