Experiment Research On Influence Of Hypoxia Environment On The Formation And Function Of Osteoclast In Vitro

【Research Background】Osteoporosis (OP) is a progressive systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue leading to decreased bone strength,increased bone fragility and susceptibitity to fracture . With the trend of senility of population in the world, OP has become a research focus in the field of medical science at home and abroad. At present there are already about 200 millions of OP patients in the world, and OP ranks the 7th place among the common frequently-occurring diseases. Starting from the year of 1999 China entered into the society of population senility, in the recent years the incidence of OP is on the rise every year, currently there are 88,000,000 persons suffering from this disease, it is estimated that the number will reach one hundred million at the end of this year. In the mainland of China the total incidence of OP is about 12.4%, the rate among the old people exceeds 50%, the occurrence rate of fracture reached nearly one third. As a kind of serious chronic disease, OP not only affects the life quality of the patients, but also leads to fracture in the spine and limbs of the patients. The older the patients are, the higher risk of bone fracture is too, the disability and mortality caused by this disease is high.The provinces and autonomous regions in the west of China are located on the plateau, which covers almost half of the land area, population residing there amounts to over 400 millions, which makes up nearly one third of the total population of the country. Owing to the high altitude in the plateau, oxygen content in the air is obviously lower than that of the plain area, researches on the influence of plateau environment on bone metabolism indicates: the dry, oxygen-lacking environment on the plateau affects bone metabolism, when the people living on the plain go to the plateau, soon they would suffer low bone mass, when a bone fracture happens on the plateau, its heal is relatively slow, even it does not heal at all. Comparing with the region of low altitude, bone absorption in the region of high altitude increases, bone mass decreases.Plenty of evidences proved that receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) of tumor necrosis factor receptor family expressing on stem cells and osteoblasts play an important role in the differentiation of osteoclast, by adjusting the proportion among OPG, RANKL and receptor activator of NF-κB (RANK) many local factors promote the formation of osteoclast and maintain its function. Starting from simulating an external hypoxia environment, this research investigated the effect of various oxygen contents on the formation of osteoclast and bone absorption function, trying to reveal the effect of hypoxia on differentiation of osteoclast with molecular biology technology, and further discover the possible molecular mechanism by which hypoxia causes low bone mass.【Objective】To determine whether osteoclast differentiation and bone absorption function are affected by hypoxia, we design the research by different oxygen concentrations simulating different altitudes.【Materials and Methods】Primary mice osteoblasic cells wered obtained from two-day-old neonatal mice by means of tissue cultivation.The cells were purified,and then were went down to posterity. Mouse bone marrow cells were obtained from the tibiae of six-to-nine-week-old male mice.The cells were cultured in culture medium that contains 1α,25(OH)2D3(10-8mol/L) and dexamethasone(10-8mol/L) in oxygen contents of the various simulated altitudes. When the bone marrow cells were purified, the 3-5th generation osteoblast were added to bone marrow cells, jointly cultured cells were divided into groups at random, each group has 4 samples, the following experiments were done at different times and each experiment was repeated three times: extracting the total RNA;semiquantitating OPG, RANK and RANKLmRNA levels by the method of reverse transcription polymerase chain reaction(RT-PCR); detecting expression of RANK protein by the method of western blotting. After 48 hours , the cell cycles of mouse osteoclasts were assayed by flow cytometry .TRAP-positive multinuclear cells which can form bone absorption pits in the bone,were regarded as the symbol of mature osteoclast; the activity of tartrate-resistant acid phosphatase(TRAP) was measured in various oxygen concentrations. The bone marrow cells were connected with aseptical bone and cultured for 7 days in different oxygen contents, and the bones were stained by toluidine blue.The bone absorption lacunas were scanned by scanning electron microscope,the number and the area of bone absorption lacunas formed by osteoclast in different oxygen concentrations were analyzed by Leica Quantimet 500 graphic analyzor.【Results】1. Oxygen contents and oxygen-lacking period have marked effects on the expression of RANKmRNA and RANK protein(P<0.01).In the first oxygen-lacking day, there is no significant difference between hypoxia groups and normoxia group; in the third day, expression of RANKmRNA and RANK protein in hypoxia groups increased compared with normoxia group(P<0.05),but there was no significant difference among groups of hypoxia; in the fifth day,the expression of RANKmRNA and RANK protein in the hypoxia groups increased markedly compared with normoxia group,especially in 3%oxygen concentration ,the increase of expression was very obvious; in the 7th day,the expression of RANKmRNA and RANK protein in hypoxia groups increased markedly compared with normoxia group(P<0.01),but compared with the result of the fifth day, the expression of RANKmRNA and RANK protein relatively decreased.2. Oxygen contents and oxygen-lacking period have significant effect on the RANKLmRNA on osteoblast (P<0.01). In the first oxygen-lacking day,the expression of RANKLmRNA in hypoxia groups obviously increased compared with normoxia group(P<0.01),there is no significant difference between groups of hypoxia; in the third day, expression of RANKmRNA in hypoxia groups increased markedly compared with normoxia group,its expresstion is most obvious in 3% oxygen concentration; in the 5th and 7th day,the expression of RANKmRNA in the hypoxia groups increased obviously compared with normoxia group (P<0.01),but compared with the expression in the three days before, it relatively decreased.3. Oxygen contents and oxygen-lacking period have obvious effect on the expression of OPGmRNA(P<0.01).In the first oxygen-lacking day, the expression of OPGmRNA in hypoxia groups markedly decreased compared with normoxia group(P<0.01),there is no significant difference between hypoxia groups; in the third day, expression of OPGmRNA in hypoxia groups decreased markedly(P<0.01); decrease in 3%,1% is more obvious than in 6%, but there was no significant difference between them. In the 5th and 7th day the expression of OPGmRNA in hypoxia groups decreased markedly, and the difference among the groups of hypoxia was marked compared with normoxia group,decrease in the content of 1% was most obvious.4. With decrease of the content of oxygen,bone absorption function of osteoclast becomes stronger. The number of bone absorption lacuna increased from 7.58±1.08/piece to 9.67±0.98,12.50±1.45,11.00±0.85/piece;bone absorption lacuna area increased from 341.86±195.46μm2 /piece to 470.62±207.89, 688.08±429.02, 583.27±224.54μm2 /piece;activity of StrACP increased from 3.87±1.58IU/L to 4.99±0.25,7.40±0.53,6.11±0.37IU/L.5. With decrease of the content of oxygen,the cell content proportion of osteoclast in phase G1 decreased from 81.13±1.31% to 66.77±0.70%,73.60±0.76% and increased to 84.87±1.60%(P<0.01); in phase G2 the cell content proportion increased from 8.57±0.48 % to 13.13±3.18 % ,14.20±0.44 % and 11.10±3.08 %(P<0.01 );the cell content proportion in phase S increased from 10.63±1.17% to 20.17±3.36 % ,12.20±0.33 % ,on the condition of 1% oxygen content the cell proportion decreased to 6.56±0.39%(P<0.01).6. With decrease of the content of oxygen,the number of TRAP-positive multinuclear cells which was counted by 200-time light microscope increased from 3.30±0.45 /glass piece to 6.10±0.55,7.90±0.42,5.00±0.61/ glass piece(P<0.01).【Conclusion】 1. Oxygen content and oxygen-lacking period have significant effect on the formation of osteoclast. With decrease of oxygen contents, differentiation from osteoclast precursors to osteoclast increased markedly;when compared with normoxia condition,the number of cell nucleus in osteoclast increased and the bulk also increased, when the oxygen content is 3%, the increase was most obvious;with extension of oxygen-lacking period, the formation of osteoclast gradually increased, it is most obvious in the 5th day, but after that it gradually decreased with extension of oxygen-lacking period.2. Oxygen content has significant effect on bone absorption function of mouse osteoclasts,with decrease of oxygen content, bone absorption function of osteoclast became gradually stronger, it was most obvious when the oxygen content was 3%,bone absorption function of osteoclast reached a peak phase, after that the function gradually became weak with decrease of oxygen content.