The Influence Of Exogenous NGF On CGRP Expression In Osteoporosis Rat Under CGRP Gene Transgenic MSCs Transplantation Therapy

BackgroundOsteoporosis is a multifactorial common disease which has been threating the health of most the elder,showing obviously sexual orientation that female incidence rate is 5-7 times higher than male.Its main pathological change is manifestations of bone mass reduction,leading to bone tissue microstructure degeneration,and bone structure strength will degenerate lastly.Unconspicuous clinical symptoms is a feature of osteoporosis early stage.When it comes to the progress and later,chronic pain in part or the whole body will occur.The pain can be aggravated as developing and produce secondary in lesions related,such as spinal compression fractures,fracture risk increasing in femoral neck,trochanter,wrist,proximal humerus,even leaving the body limited mobility bedridden,etc.Long-term inevitably may cause some common complications including pneumonia,deep vein thrombosis,peripheral nerve atrophy and motor system has been a serious threat to health and make serious effect on living standard of the elderly population.The occultting of osteoporosis and slow progress make people ignore it easily,when clinical symptoms occur,it has already progressed to the advanced.So advanced diagnosis of osteoporosis and prevention is very important.Currently treatment for the disease is mainlysymptomatic treatment.With the development of medical technology,people come to realize that the direct or indirect and predisposing factors including hereditary factors,traumatic factors,endocrine disorders,nervous system disorders,immune system disorders,physical braking,but the aging physical function degradation caused by physiological status and hormone function decline are an insurmountable problems in osteoporosis treatment.Currently,the treatment options we choose are mainly controlled drug,dietary modification program and according to the individual patient differences need to make specific programs plan.The final purpose of the treatment is to increase bone mass,while clinical practice is often difficult to achieve this for those symptomatic treatment,calcium or mineral salt intaking,may be a little treated efficacy or caused by a partial remission of body pain or other clinical discomfort not effectively prevent further progression of osteoporosis.Asking the patient most of the time to maintain the appropriate activities to avoid simultaneous fracture complications instead.And then fewer activities accelerated the process of natural progression of osteoporosis in turn.Current drug treatment programs,often has a long period,spends a bigger fare,and gets unsure result of therapy and there are different degrees of toxicity which increases risks in other medical conditions.Therefore,many scholars and health workers are committed to finding new solutions to be able to slow down osteoporosis process,alleviate clinical symptoms,reduce the complications and improve the quality of life of the elderly population.Recent years,with great development of bone tissue engineering research on bone MSCs in vitro and in vivo animal experiments,bone marrow mesenchymal stem cell transplantation research has achieved good results.Bone marrow mesenchymal stem cell transplantation therapy has been used in clinical treatment of some diseases,and achieved good effect,such as traumatic bone defects,nervous system repair,soft tissue defects,cardiovascular disease,ect.While basical research of bone cell activity and metabolism continue to achieve new breakthroughs,it has been found that a variety of related factors on bone metabolism regulating activities,such as insulin-like growth factor B(IGF-B),nerve growth factor(NGF),vascular endothelial growth factor(VEGF),transforming growth factor(TGF-B),calcitonin gene-related peptide(CGRP)and so on.Recent studies have demonstrated that the metabolic activities of bone can be regulated by the nervous system which is closely related to osteoporosis occurrence.Meanwhile immunocytochemical study also confirmed the periosteum,cortical bone,trabecular bone,the presence of sensory and autonomic nerve fibers in the bone marrow,passing a variety of neurological biological factors(such as neuropeptides,catecholamines and neurotrophic factors)play important roles in bone metabolism.Calcitonin gene-related peptide on osteoblast,osteoclast activity are affected can promote bone formation and inhibit bone resorption,the CGRP gene transplanted into mice,can be found in osteoblasts CGRP overexpressed small bone beam density increases.Gene therapy may significantly alleviate osteoporosis has gradually been recognized by the majority of scholars.Currently,the osteoporosis treatment study focused on the development of new drug,cell gene therapy study levels,and the osteoporosis model establishment of various types of diseases causes,which also layed the technical foundation for osteoporosis research.CGRP gene therapy promote osteoblasts CGRP overexpressed,which occurred in the prophylactic treatment of ovariectomized rats with osteoporosis by promoting bone formation and inhibiting bone resorption regulates bone metabolism and bone tissue phenotype.NGF is a important neurotransinitter and other regulatory factors,osteoblasts secrete spontaneously NGF,inducing osteoblasts autophosphorylation reaction,adjust the balance of bone metabolism,is conducive to bone formation activity in bone metabolism.NGF can induce bone metabolic activity of nutrition to improve bone repair rate and strengthen callus biomechanical in fracture repair,and also can stimulate the formation of sensory nerve regeneration around the bone-like tissue type.Present study confirmed that CGRP and NGF gene therapy in the treatment of osteoporosis showed different potential.In animal models of osteoporosis,CGRP and NGF gene therapy protocols combined applications rarely reported.The influence of Exogenous NGF on CGRP expression in osteoporosis rat under CGRP gene transgenic MSCs transplantation therapy is not clear,whether the effect can obtain cumulative effect or not.There is no report yet.Objective1.To evaluate the efficacy of exogenous(NGF)application in CGRP transgenic bone marrow stem cell transplantation in osteoporosis treatment;2.To explore Exogenous NGF on CGRP expression in osteoporosis rat under CGRP gene transgenic MSCs transplantation therapy.Methods1.Bone marrow mesenchymal stem cells(BMSCs)isolation,purification,and culture in vitro.Select 6 4-week-old female SD rats,harvest lower limbs long bone marrow tissue,make steps in vitro separation and purification,to obtain the right amount of the amount of bone marrow MSCs cells and subculture to the third generation,save spare.2.Construction of eukaryotic gene expression vector of CGRP.Choose homologous experiment SD rat genome as a template,PCR amplification of the coding sequence of CGRP gene were pBaBb-puro-CGRP eukaryotic expression vector was constructed by gel electrophoresis and the results of gene sequencing vector.3.Transfected MSCs and the preparation of stable transgenic CGRP-MSCs stable cell lines.We got pBaBb-puro eukaryotic expression vector with 293FT(human embryonic kidney)cell line proliferation using retrovirus(Retrovirus),then transfected it into the prepared bone marrow mesenchymal stem cells to keep confluent cell proliferation by virus infection,further screening CGRP stable transgenic bone marrow mesenchymal stem cells stable cell line(CGRP-BMSCs).Amplifying culture cryopreservation standby.4.Establishment of a rat model of osteoporosis,and making animal group plant and processing the experimental treatment group.Select 60 four-month female SD rats were gavage retinoic acid to construct animal models of osteoporosis,were randomly divided into four groups averagely:Group A,the control group(normal saline);Group B,simple bone marrow mesenchymal stem cell transplantation in the treatment group(MSCs transplantation treatment);Group C,switch between gene calcitonin gene-related peptide in bone marrow mesenchymal stem cell transplantation in the treatment group(CGRP-MSCs transplantation treatment);Group D,exogenous nerve growth factor transgene down jiont calcium gene related peptide bone marrow mesenchymal stem cell transplantation in the treatment group(CGRP-MSCs + NGF migration process).All animals in the experimental treatment on appeal under the same conditions and raising the level of observation.5.Test specimen collection,data processing and analysis.After the first 1,2,3w appeal experimental treatment after treatment,using Real-Time fluorescence quantitative PCR and Western Blot relative expression levels in three time periods in CGRP femur bone tissue of rats mRNA and protein,to collect data,use spss19.0 software for data analysis and processing.Result1.Comparison the Group A(control group),the expression of Group C(CGRP-MSCs transplantation treatment)and Group D(CGRP-MSCs + NGF transplant treatment)of the CGRP CGRP mRNA and protein at three periods are all significantly increased(P<0.01).2.After one week of treatment experiment,Group C(CGRP-MSCs transplantation treatment)and Group D(CGRP-MSCs + NGF transplant treatment)group raised the level of significant difference(P<0.01),low expression levels in group D of CGRP CGRP mRNA and protein when compared group C;2 weeks after the experimental treatment,remains a significant difference(P<0.05)between Group C and D,D group of CGRP CGRP mRNA and protein expression levels are still lower than in Group C.3.After experiment 3 weeks,the level of CGRP mRNA and protein expression of CGRP between Group C and Group D upregulated significantly reduced the level difference,the difference was not statistically significant(P>0.05).Conclusion1.Whether or not use of exogenous NGF,CGRP gene transgenic bone marrow mesenchymal stem cell for the treatment of osteoporosis in rats,the expression levels of CGRP CGRP mRNA and protein are both improving revealed positive effects.2.The use of exogenous NGF joint CGRP transgenic-MSCs transplant to treat osteoporosis in rats,at least in CGRP mRNA and protein expression levels of CGRP did no promotion and did show no cumulative effect,while the simple CGRP gene transgenic-MSCs transplantation therapy efficacy of osteoporosis in rats superior combination of exogenous NGF treatment.