| Objective: Postmenopausal osteoporosis(PMOP) is due to the ovarian function descends, the oestrogen level drops and also the shortage of calcitonin secretion after menopause, leading to the bone resorption of osteoclasts is greater than the bone formation of osteoblasts, with a low bone mass and bone tissue of degenerative changes in characteristics of microstructure, belonging to primary osteoporosis. The characteristic of the metabolic disease is the osteopsathyrosis and fracture susceptibility increased, and had a high morbidity and mortality. With the increase of aging population, the incidence of the disease is gradually rising, which not only does it reduce the quality of patients’ life, but also brings serious economic burden to the society. For a long time, the prevention and treatment of PMOP predominantly adopts hormone replacement therapy, this therapy is currently recognized as the most effective method of treatment,but it must be strictly controlled the adaptation and contraindication of treatment, following earlier use, the minimum effective dose, individualized treatment and regular follow-up principle, so it only be promoted and used in the appropriate crowd. So we still need to further study the pathogenesis of postmenopausal osteoporosis, to seek a new way for the treatment of PMOP.Connective tissue growth factor(CTGF) is a kind of intercellular matrix protein, participates in a variety of pathophysiological process, its mechanism is very complicated, and also has many arguments. In bone metabolic disease, CTGF can through different signaling pathways, including BMPs. BMPs of similar structure is a set of highly conservative multifunctional protein secretion, through the circulation of the blood to the body, in the form of similar hormone to work in bone inside and outside. And BMP-9 is known as one of the strongest members in family of BMPs osteogenetic activity.This experiment established PMOP model through removing ovaries of rats, using immunohistochemical method to detect CTGF in PMOP rats bone tissue and ELISA method to detect the expression of BMP-9 in serum, to explore the role and correlation between them in PMOP.Methods:1 The grouping and preparation of PMOP animal model30 Clean level female Sprague/Dawley rats at the age of 3 month were randomly divided into 3 groups(10 in each group): ①the control group(Sham); ② the ovariotomy group(OVX); ③ the ovaries treated with Estradiol valerate(OVX+E). The rats were injected with 10% chloralhydrate(0.35ml/100 g weight) through abdominal cavity before surgery. Under sterile condition, through the small of the back in the middle of longitudinal incisio entered into the abdominal cavity, then removed bilateral ovaries of rats in OVX and OVX+E group and an equivalent weight of adipose tissue below bilateral ovaries of rats in Sham group. Spraying cefazolin to prevent infection after operation. 7 days after operation, different groups of rats were administrated intragastricly respectively with different drugs for 12 weeks. OVX+E group was given estradiol valerate at 0.833 mg.kg-1.d-1, Sham group and OVX group with 10 ml/kg body weight were given an corresponding volume of 0.9% sodium chloride solution, once a day. The dosages of drugs were proofread once a week in accordance with their body weights.2 Dual-energy X-ray absorption method measure bone mineral density(BMD)After 12 weeks, under anesthesia,the rats were taken placed on the Osteocore3 dual energy X-ray absorptiometry, the bone mineral density of the femur and lumbar were measured by computer systems center animals software, we measured three times on each and took the average,BMD values expressed in g/cm2.3 Hematoxylin-eosin stain and immunohistochemistry stain, ELISATo get the blood 2ml from rat′heart after death, immediately take the left femoral under aseptic condition for HE staining and CTGF in bone tissue was determined by immunohistochemistry technique, the level of BMP-9 in blood serum was determined by ELISA.4 Result and image analysisWe applied One-Way ANOVA using Statistical Product and Service Solutons 16.0(SPSS16.0) for data analysis. All values were demonstrated by average±standard deviation(-X±S).And two-two comparison among the means were done by Student-Neuman-Keuls. P<0.05 was considered statistically significant.Results:1 The bone mineral densityCompared with sham-operated group, the BMD of the femur and lumbar reduced in OVX group, and the differences were statistically significant(P<0.05),there were no significant differences in OVX+E group(P>0.05). Compared with OVX group, the BMD of the femur and lumbar increased in OVX+E and sham-operated group, and the differences were statistically significant(P<0.05,P<0.05).2 The observation of bone tissue morphology(1)Sham-operation group: The cortical bone was dense, bone trabecula arranged vertically, interconnected net-like structure, arranged densely and regularly, no fracture. The number of bone trabecula was plentiful, and trabecular separation was narrow.(2)Ovariectomized group: Marrow cavity increased, the cortical bone became thinner, bone trabecula was sparse or fractured, no reticulated structure was seen and a large number of trabecular bone blind side was seen, the number of bone trabecula was reduced, and trabecular separation was widened.(3) OVX+E group shared similar changes with sham-operated group in the trabecular number, trabecular thickness, trabecular separation, trabecular length, trabecular distribution.3 The observation result of the immunohistochemistry stainThe expression characteristic of CTGF in bone tissue: The CTGF positive signal is mainly in the cartilage cells, osteoblasts surrounded the bone trabecula, bone marrow, in the majority with fat extracellular matrix in marrow cavity. The expression of CTGF protein was only a small amount in Sham group and OVX +E group, the expression of CTGF protein in OVX group was plentiful. Compared with Sham group, there was no significant difference in OVX+E group(P>0.05), the expression of CTGF protein increased in OVX group(P<0.05); Compared with OVX group, the expression of CTGF protein reduced in OVX+E and Sham group(P<0.05,P<0.05).4 The level of BMP-9 in serumCompared with sham-operated group, the content of BMP-9 in serum decreased in OVX group(P<0.05), there was no significant difference in OVX+E group(P>0.05). Compared with OVX group, the content of BMP-9 in serum enhanced in OVX+E and Sham group(P<0.05,P<0.05).5 The correlation analysis resultThe activity of BMP-9 in serum of each group rats were positively correlated with the BMD of the left femur(r=0.721,P<0.05,r=0.879,P<0.05, r=0.885,P<0.05).The expression levels of CTGF were negatively correlated with the BMD of the left femur in OVX group(r=-0.636,P<0.05). There was no correlation between the expression of CTGF with the BMD of the left femur in OVX+E and Sham group(r=-0.572,P>0.05,r=-0.423,P>0.05).The expression levels of CTGF were negatively correlated with the BMP-9 in OVX group(r=-0.647,P<0.05). There was no correlation between the expression of CTGF with the BM-9 in OVX+E and Sham group(r=-0.353,P>0.05,r=-0.455,P>0.05).Conclusions:1 CTGF is in an excessive expression state in PMOP; Estrogen ca-n prevent the expression of CTGF.2 BMP-9 can promote bone formation.3 The excessive expression of CTGF may inhibit the expression of BMP-9,and then cause the decrease of bone formation, in turn, lead to PMOP, the breakthrough point also prompts CTGF may provide new therapeutic target for the prevention and treatment of PMOP and BMP- 9 also promises as a promote cell osteogenesis cytokines candidate and applied to clinical. |
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