| Osteoporosis is a common metabolic bone disease characterized by lowbone mass and microarchitectural deterioration of bone tissue with a subsequentincrease in bone fragility and a susceptibility to fracture caused by interaction ofmany factors such as gene, hormone, environment, et al. Osteoporosis is a majorpublic issue with the process of society aging and is called the fourth diseasethreatened human health. It frequently happens among elderly people andsignificantly influences their living quality by causing pain, loss of life abilityand other complications such as fracture, which can increased mortality to15%～30%.Bone is a living tissue. It undergoes a continuous process of remodelingwith bone resorption, following by deposition of new bone. Bone remodeling iscarried out by teams of osteoclasts and osteoblasts and regulated by manyfactors, especially partial cytokines, which plays an important role inosteogenesis and bone development in forms of paracrine and autocrine directlyto bone cells. The couple process between resorption by osteoclast and boneremodeling by osteoblast involves in cellular information communication,during which cytokines act as information molecular. Both bone partial factorsand other cytokines related to bone remodeling can influence sclerotin, theconsequences include negative effect caused by bone resorption, positive effect108by bone formation and bidirectional effects between them.During the research of growth hormone (GH) in 1957, Salmon andDaughaday observed that it was a kind of cytokine not GH itself could stimulatethe growth of cartilage by promoting the synthesis of 35-phosphate. Therefore,this new cytokine was named as sulphation factor while changed to insulin-likegrowth factor-1,2 (IGF-1, IGF-2) after Rinderknecht and Humbel found theirsimilar structure to insulin in 1976 when isolating them from human serum.Based on the development of separation of IGF-1cDNA and expression ofhuman recombinant IGF-1 since 1980s, the research about IGF-1 got more andmore attention around the world. It has been conformed that IGF-I can promoteskeleton growth, differentiation, participate in glycometabolism, proteinmetabolism, skeleton and fat metabolism and other biological processes.Therefore, IGF-1 is one of most important cytokines during bone remodelingand probably plays a key role in the pathogenesis of metabolic bone disease andosteoporosis. The effects of IGF-1 are less clear so far, although it has beenapplied in clinic for the treatment of osteoporosis.Experimental protocolIn vitroGenetic engineering technique was used. IGF-1 gene was cloned,recombinant eukaryotic expression plasmid pcDNA3.1-rhIGF-1 was constructedand detected by gene sequencing. Cultured osteoblast of neonate rat wereobserved by inverted microscope and testified by the assays of alkaliphosphatase staining (Gomori method) and Alizarin Red S(ARS) staining. Ratosteoblast was transfected with pcDNA3.1-rhIGF-1 through liposome,examination of the quantity of hIGF-1 in supernatant liquid and western boltwere performed to confirm the successful transfection. PcDNA3.1-rhIGF-1plasmid (μg) and Lipofectamine (μl) were used to transfect osteoblast and MTTassay was performed to observe cell proliferation, ALP activity and osteocalcinquantity was detected to determine the biologic influence of IGF-1 gene toosteoblasts.The results showed increase in cell proliferations, ALP activity andquantity of osteocalcin compared to control group. Therefore, it demonstratedthat IGF-1 can promote proliferation and differential of normal ostroblasts,improve the activity of ALP and increase the synthesis of osteocalcin leading tostable biological transfection of IGF-1 gene and the expression of ossificatedcharacters of osteoblasts.In vivoThe experimental ovariectomized osteoporosis rat model was establishedand rats were randomly divided into 3 groups: control group, sham group andovariectomized group. After 3 month of surgery, 10 rats of each group wereexecuted to examine whether the model was successful by detecting thedifferences of some biochemical indicators level, wet weight of wombs, bonedensity and bone histology. After that, the therapeutic effect of IGF-1 onovariectomized osteoporosis rats was detected by following steps. Differentdosage of gene recombinant IGF-1 were injected subcutaneously toovariectomized osteoporosis rats, after 3 months, biochemical indicator bonedensity and bone tissue morphometry parameter significant difference compared toovariectomized group. These results indicated that gene recombinant IGF-1 afterovariectomization could improve bone density and cause changes inhistomorphology, promote bone mineralization and turnover the loss of bonemass after ovariectomization.Conclusions:1.Human IGF-1 gene was cloned and recombinant eukaryotic expressionplasmid pcDNA3.1-rhIGF-1 was constructed successfully.2.Rat osteoblast was obtained;recombinant eukaryotic expression plasmidpcDNA3.1-rhIGF-1 could be transfected into osteoblast and express IGF-1efficiently.3.IGF-1 can promote the proliferation and differential of osteoblast innormal rats, enhance the process of actived osteogenesis and the synthesis ofosteocalcin, lead to the expression of ossification characters of transfectedosteoblast.4.Ovariectomized osteoporosis rat model was established.5.Recombinant IGF-1 can improve bone density and cause changes of bonehistomorphology, promote bone mineralization and overturn the loss of bonemass after ovariectomization.In this study, we conclude biologic characters and functions of IGF-1 basedon references and experiments in domestic and abroad. We confirm tentativelythe function and mechanism of IGF-1 in promoting ossification, and provideevidences in the treatment of osteoporosis, but we still need further animal andclinic research to explain its long-term side effect.
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