| BackgroudDiabetes mellitus (DM) causes endocrine and metabolic disorders due to high blood glucose, including the injury of bone, nerve, kidney and other organs, absolute or relative lack of insulin, the lack of insulin like growth factor, and disorders of mineral tissue metabolism. The patients with diabetes mellitus are prone to occuring fracture because of decreased muscle strength and osteoporosis. Bone mineral reduction leads to high incidence of delayed union or nonunion of fractures [1>2l The diabetic patients with delayed fracture healing can cause paralysis, gangrene, bedsore, urinary tract infection, muscle atrophy and other consequences, and even lead to local or systemic infection How to treat diabetes fracture healing has been paid more and more attention. Recent studies have shown that cytokines affecting bone metabolism play a key role in fracture healing. Fracture may be one of the most suitable diseases for gene therapy, for it only needs one or several related growth factors during local transient fracture and can be effective in promoting fracture healing.Bone morphogenetic protein-2, short for BMP-2, the member of BMP family is as one of the key factors, particularly significant in fracture healing. BMP-2can induce BMSCs (Bone marrow derived mesenchymal stem cells) to differentiate into osteoblast, chondrocyte and promote osteoblast to maturate. BMP-2can transform mouse myoblast cells into bone cells; the BMP-2content of bone cell is regarded as the factor to reflect the osteoblastic bone formation ability. IGF-1(insulin-like growth factor-1) is another important factor with biological activity of bone healing. IGF-1expression in late fracture phage can enhance chondrocyte proliferation, chondrocyte differentiation and accelerate terminal direction of chondrocyte and maintain the phenotype of chondrocytes, suggesting that IGF-1plays important role in the regulation of fracture healing. But at present the application of growth factors on the treatment of fracture exists many problems, such as immunological rejection, extraction difficult, increasing the economic burden on patients. A breakthrough needs to treat bone healing with bioactive factors. The bioactive factors can effectively improve the therapeutic effect, safety and provide clinical selective scheme. BMP-2and IGF-1received a positive comment in promoting fracture healing process. However, what is their role and what kind of the variation in fracture healing process of diabetes mellitus? We intended to observe the relationship between local content of BMP-2, IGF-1and the healing process of fracture by animal experiment of diabetic rats in the fracture healing. BMP-2, IGF-1are the strongest growth factos in the proliferation and differentiation of BMSCs, so we used genetic engineering to transfect BMP-2and IGF-1into BMSCs and observe gene expression, proliferation and differentiation of transfected BMSCs under high glucose environment, to further explore whether the method could be used for gene therapy of fracture healing in diabetes, to detect BMP-2, IGF-1gene interaction when co-transfected in BMSCs, and to provide the theoretical basis and reference strategies for diabetes delayed fracture healing for new treatment and prevention.Research methodsThree sequential steps in this study were as follows:1Animal experiment:establishment of two models of the fracture in diabetic rats and non-diabetic rats; to observe the fracture healing in rats of two groups at different time points and determination of BMP-2,IGF-1in serum, to analysis of possible factors influencing diabetes fracture healing.A:Healthy male Wistar rats were randomly divided into two groups, experimental group and control group. The experimental group was intraperitoneal injected with streptozotocin to prepare diabetic rats. Select diabetic rats according to the standard indexes. All rats with chloral hydrate anaesthetized were opened to exposure of left tibia, wire sawing tibia, closing the wound, then splint external fixation.B:Portal venous blood was obtained at1,2,3,4,6weeks after operation, respectively. Serum were gotten after centrifugation and placed in-20℃refrigerator. The rats were killed for detection of bone density and maximum cross sectional area of fracture callus, and formalin fixed, decalcified, dehydration of alcohol, xylene, embedded in paraffin, sliced, HE stained for analysis of fracture healing.C:Detection of serum BMP-2, IGF-1expression by Elisa.D:Analysis the obtained data by statistical software and drawing the corresponding conclusion.2. Cell culture:BMP-2and IGF-1genes were transfected into BMSCs using the technique of genetic engineering, and observe the proliferation and differentiation of BMSCs in high glucose environment.A:Construction of plasmid vector BMP-2, IGF-1.B:Isolation and purification of BMSCs:extraction of healthy Wistar rats bone marrow from femur and tibia by density centrifugation and adherence screening methods for the separation and purification of BMSCs.C:Gene transfection:BMP-2andIGF-1gene were transfected into BMSCs respectively, and divided into control group (not including the target gene), BMP-2group, IGF-l,groupBMP-2and IGF-1co-infection group.D:All groups were cultured in high glucose DMEM medium and normal sugar culture medium,37degrees,5%CO2, the moisture saturated culture incubator.E:Quantitative detection of RT-PCR were used to detect the mRNA expression of BMP-2, IGF-1in BMSCs cultured for5days,10days.F:Expression of BMP-2, IGF-1protein were detected by Western-blot after transfection experiment.G:Analysis the obtained data by statistical software and drawing the corresponding conclusion.3Animal experiment:to prepare diabetic rat fracture model again, and injected seed cells of BMSCs containing BMP-2and IGF-1gene into the fracture site and detection local BMP-2, IGF-1expression at different time points and the fracture healing.A:Construction of seed cells BMSCs with IGF-1and BMP-2gene..B:Diabetic rat fracture model was prepared as the above method.C:Inject BMSCs with IGF-1and BMP-2gene into the fracture siteD:Portal venous blood was obtained at1,2,3,4,6weeks after operation, respectively. Serum were gotten after centrifugation and placed in-20℃refrigerator. The rats were killed for detection of bone density and maximum cross sectional area of fracture callus, and formalin fixed, decalcified, dehydration of alcohol, xylene, embedded in paraffin, sliced, HE stained for analysis of fracture healing.E:Detection of serum BMP-2, IGF-1expression by Elisa.F:ALP activity was determined quantitatively, to judge the osteoblast changes.G:Analysis the obtained data by statistical software and drawing the corresponding conclusion.Results1The blood glucose of diabetic rats induced by STZ was higher than16.7mmol/L, and urine glucose appear positive. At the same time, the diabetic rats appear polydipsia, polyphagia, reduced activity, unresponsive, loose fur. And the symptoms aggravated over time, but the symptoms on the fracture mental status after1week to ease and activity increased. All of these proved that the model of diabetic rats prepared successfully.2Local bone callus density of diabetic rats group increased somewhat, but the increase was significantly lower compared with the normal control group,, with significant difference (P<0.05), and with the extension of time, significant further increased. 3The maximum cross-section callus in diabetic group was significantly higher than that in the normal control group(P<0.05), and with the extension of time, significant further increase.4The histological results showed each postoperative time point, the fracture callus cartilage cells and the healing process of cartilage cell hypertrophy and mature of osteoblasts less delayed in diabetic group than those in the control group of rats.5Trend of BMP-2and IGF-1were similar after operation week1-3. The concentration gradually increased and was significantly different compared with the control group (P<0.05); then the concentration decreased of the two factors, with no significant difference (P>0.05).6During the high glucose environment, BMP-2and IGF-1was respectively transfected and co-transfected into BMSCs. The expression of BMP-2and IGF-1of co-transfection group increased more obviously than the individual transfection group. The difference was statistically significant (P<0.05).7BMP-2and IGF-1were co-transfected into BMSCs and were given local genetherapy. Bone mineral density and bone callus maximum area significantly improved (P<0.05). Histological results showed that, after treatment, the mature cartilage cell differentiation and proliferation of osteoblasts can effectively improved, significant in promoting fracture healing.8Expression of BMP-2and IGF-1after treatment increased significantly, compared with the diabetic group (P<0.05). The serum ALP level can be changed obviously, indicating that gene therapy of BMP-2and IGF-1with BMSCs are beneficial to the healing of fracture.Conclusions1Diabetes mellitus can lead to a decrease in bone density, and seriously impact fracture callus of fracture site.2In the healing process of fracture, BMP-2and IGF-1in the early serum of diabetic rats decrease, which is one of the most important factors for treatmeng of poor fracture healing. 3In the high glucose environment, BMP-2, IGF-1co-transfected into BMSCs can give a positive feedback to promote BMP-2, IGF-1expression.4BMP-2, IGF-1co-transfection of BMSCs gene therapy can improve the fracture healing in diabetic rats. |
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