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VEGF Induced by Hypoxia Inducible Factor-1α in Osteoblasts Cultured under Hypoxia

Author: YangFeng
Tutor: TangXin
School: Dalian Medical University
Course: Surgery
Keywords: HIF-lα VEGF Osteoblast RT-PCR
CLC: R68
Type: Master's thesis
Year: 2009
Downloads: 81
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Abstract

Objective : To investigate the expression of hypoxia inducible factor-lα(HIF-lα) in primary fetal mouse osteoblasts cultured under hypoxia for different time periods,and to explore HIF-1аeffect to the expression of VEGF.Methods:1. Apply modified tissue mess method to obtain osteoblasts:The calvaria harvested from 19 day fetal mouse was stripped off all soft tissues including the periosteum, rinsed and cut to trivial bone block under sterile condition. Then, the bone block was subcultured in culture flask after digested with 0.25% trypsin for 10min. The acquired cells were purified by“repeated adhensive method”.Subsequently the third generation was counted for eight days continuously and the forth generation was identified with alkaline phosphorase stain of calcified nodules by ca-co dye.2.The third generation osteoblasts were divided into two groups to be cultured under normoxia and hypoxia containing cobalt chloride (125umol/L) for different time periods (1,3,5,7days) to observe cell morphology and to count cell number.3.Reverse trancription-polymerase chain reaction was employed to measure the message RNA expression of HIF-1α、VEGF at the time points grouped as mentioned above.4.Statistical treatment:The above experiment was repeated for three times,and the experimental data was analyzed by One-Way ANOVA analisis. Result:1. Through the observation of microscope, the cells obtained from fetal mouse calvaria were fusiform、triangle or polygon with several processes which connect with other cells process . The nucelus with one or more nucleoli appear round or oval-shap.After subculturing、the cells proliferated slowly at the adaption stage, then they accelerated to the climax at the sixth day ,afterward cell population began to decrease .The fourth generation cells were identified as osteoblasts by alkaline phosphorase stain of calcified nodule,and positive cells which appeared fusiform and squamelliform were 95% of 200 counted cells.2. The obscure membrane was shrinkage and cytoplasm was dense morpholocally within cells which were treated with cobalt chloride for three days, and after seven days the apoptotic body was identified. The cell number of hypoxia group was to the climax at the fifth day, but obviously less than that of normxia group at the same time point3. The message RNA expression of HIF-1αand VEGF of osteoblasts increase gradually in hypoxia group, obviously higher than that in normxia groups at the same time point(P<0.01), and there was no obviously difference in normxia groups(P>0.05).Conlusion:1. Cobalt chloride as a chemical inducer of HIF-1 can induce the expression of HIF-1 osteoblasts2. Cobalt chloride as a chemical inducer of HIF-1 can improve the expression of VEGF in osteoblasts. Enhance blood vessel formation , promot differention of osteoblasts accelerate bine formation , and bone reparation under the case of bone necrosis, fracture, poor blood supplication of bone,etc. the paroxysm clinical VEGF gene treatment lay theories foundation.3. The modified tissue mess method is an ideal one to obtain and culture osteoblasts having typical characteristics.

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