The strategy of this study is to perform a large-scale analysis of gene expression in order to highlight possible regulation pathways differentiated by traumatic occlusion in early phase. The experiment was conducted with male SD rats (250 ± 10 g) from Laboratory Animal Centre of Shandong University (Jinan, China). The animals were housed under conditions of controlled temperature (23 ± 2 °C) and humidity (60%) with natural light. The experimental protocol was developed according to the selleck chemicals llc institution’s guideline for the care and use of laboratory animals. Anaesthesia was accomplished using chloral hydrate 40 ml/kg (Qilu
Hospital in Shandong University, Jinan, China). In order to create a hyperocclusive state 1 mm MEAW was bonded on the occlusion surface of the first molar at left upper jaw by means of super-bond composite resin accumulation to form the occlusal trauma model of Nutlin-3a datasheet the first molar at the same side of lower jaw, which was taken as the experiment group, whilst the lower jaw of the opposite side was taken as the contradistinctive group. After the treatment for 24 h, the animal was sacrificed by the intraperitoneal injection of chloral hydrate 40 ml/kg (Qilu Hospital), and then the first molars at the both sides of the lower jaw were extracted. Lower jaw bone tissues in the region of the extracted
teeth were ablated, isolated from the mandibles, placed in liquid nitrogen for immediate freezing, and stored in the −70 °C freezer. All the glassware and mortars
were baked at 200 °C for 4 h to inactivate RNA enzyme. The frozen alveolar bone was ground rapidly in liquid nitrogen. Trizol Reagent kit (Gibco BRL Company, USA) was used to extract total RNA of the tissues. Then used gel electrophoresis to test whether the extracted RNA Thymidylate synthase was degradted, and measured the OD value (A260/A280) with spectrophotometer(Agilent, Shanghai, China) to test the content and purity of RNA. For gel electrophoresis the 28S and 18S ribosomal RNA bands should be fairly sharp, intense bands. The intensity of the upper band should be about twice that of the lower band, and for spectrophotometer, the O.D. A260/A280 ratio should be more than 1.8. The extracted RNA was stored at −70 °C. Microarray analysis was performed by rat genome-wide oligonucleotide microarrays in CapitalBio Corp. (Beijing, China).25 Briefly, a Rattus norvegicus genome oligonucleotide set (version 3.0),which was consisted of 269,625 amino acidmodified 70-mer probes representing 22,012 genes and 27,044 gene transcripts, was purchased (Operon, Huntsville, AL) and printed on silanized glass slides using a SmartArray™ microarrayer (CapitalBio). Five micrograms DNase-treated total RNA was prepared and fluorescent dye (Cy5 and Cy3-dCTP)-labelled cDNA, produced through Eberwine’s linear RNA amplification method26 and subsequent enzymatic reaction, were then hybridized to an array.