| OBJECTIVES:To compare mRNA expression profiles of bone tissues surrounding implant body between normal rats and rats with diabetes using microarray technology and to analysis the involved biological processes. The aim is that to explore the molecular biological mechanisms of osseointegration influenced by diabetes mellitus and to provide the theoretical basis for treatment.METHODS:1. Establish diabetic rat experimental models Using twenty healthy male Wistar rats randomly selected and divided into norm model group and diabetic group, each group has eight animals. The diabetic group was intraperitoneally injected with streptozotocin(STZ)55mg/kg to set up the diabetic rats model, and the norm model group was intraperitoneally injected with the same amount of normal saline. Seventy-two hours laters, the caudal vein’s blood was taken to test its blood-sugar content, if the GLU was higher than16.7mmol/L,we said that the model was successful.2. Placement of implants and collect specimens The two groups of rats were intraperitoneally injected with4%hydration chlorine aldehyde, Titanium implant bodies(1.4mm×4mm) were implanted into the epiphyseal end of the rats’ tibia.After the surgery, the rats were raised routinely and the diabetes rats need to test its blood-sugar content,rejecting the diabetes rats which the GLU is lower than16.7mmol/L.Three months later,bone tissues surrounding implant body were 3. extracted and sampled.The bones were rinsed repeatedly by PBS liquid, and then were put into the cryobiolgical inventory system.4. Microarray technology Using Trizol to extract total RNA from the growing body of the surrounding bone tissue of the diabetes group and normal group respectively, then inspect the quality through spectrophotometer and denaturation agarose gel electrophoresis. Tagging Cy3fluorescent to the cDNA of the experimental group and control group through retrovirus method, making it into a probe, and hybridize with expression profile chips as well as scanning chips fluorescent signals image, using GenePix4000B scanistor to scan the chip and read the original signal through GenePix pro V6.0. Screening differentially expressed genes using two times differentially expressed value(ratio), and analyze the biological process related with the differential expressed gene.5. RT-PCR test to confirm results on core samples Selecting two factors that expressed significantly different from the chip result, and observe the change of the expression using RT-PCR technology in order to verify the results of the chip.RESULTS:1. Wound healing is great in normal rats, no inflammation, no infection. There is occasional lack of energy and the symptoms of more food, polydipsia, polyuria in the diabetic rats and some died from infection.X ray indicated:the bone tissues of the normal rats were contacted with the implants closely. Meanwhile, the bone tissues of the diabetic rats were contacted with the implants discontinuously.2. The results indicated that there were1084differential gene expression. In the diabetic model, there were352plus enhanced expression gene,732suppressed expression. GO analysis involved1154different functional type. Finding out that differential expressed gene involve several biological progress, and bone metabolism and lipid metabolism are closely related with the bone combining of the planting body. CONCLUSIONS:1. Intraperitoneal injection with streptozotocin is a stable and effective method to set up the diabetic rats model.The level of osseointegration is lower in the diabetic rats then in the normal ones.2. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression were increased.3. We found that cDNA microarray technology can be successfully applied to identify differentially expressed genes for characterizing the physiological and pathological processes of bones around the implants of normal rats and diabetic ones. |
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