Screening Of Serum Protein Markers For Osteoporosis Detection

Background: Osteoporosis(OP) is a metabolic disease of bone with characteristic systemic bone loss and bone microstructure degradation. The main clinical manifestation is increased fracture risk. Osteoporosis can be divided into three categories: primary, secondary and idiopathic. Primary osteoporosis refers to those not accompanied by other disorders when osteoporosis occurs. Postmenopausal and senile osteoporosis are the main types of primary osteoporosis. Secondary osteoporosis is caused by diseases/drugs or organ transplantation which can affect bone metabolism. Idiopathic osteoporosis mainly occurred in teenagers. In addition, osteoporosis can also be divided into localized or generalized osteoporosis according to the affected site. The incidence of osteoporosis is at the head of orthopedic diseases. O ur country has the largest number of the osteoporosis patients, and the incidence will continue to rise due to prolonged average life expectancy. Osteoporosis is a world prominent public health problem, as both of its diagnosis and treatment are costly, bringing economic burden to society and families. Therefore, to deepen the understanding of the pathogenesis of osteoporosis, looking for key factors of its outbreak and to explore the possible targets for early intervention, will become the focus in osteoporosis related researches.Dual energy X-ray bone absorptiometry(DXA) is the major method for bone mineral density(BMD) determination and its result is regarded as the gold standard for clinical osteoporosis diagnosis. BMD T Score <-2.5 is a widely accepted clinical diagnostic criteria(T Score based on peak bone density for the standardization of reference values, according to the corresponding normal race, gender, young people as a reference standard). However, lack of accessibility to the instrument and relatively high cost confined DXA to a limited application. Therefore, looking for a more convenient and economical method to detect osteoporosis has become a hot spot in current research. In recent years, with the development of proteomics technology, large-scale, high-throughput detection technologies have gradually matured. With these up-to-date technologies, cyclophosphamide(CTX) and collagen type I amino-terminal propeptide(PINP) have been identified as specific protein markers for bone formation and resorption. However, there still exists lacks in clinical applicable index for osteoporosis diagnosis. Therefore, the purpose of this study is to use proteomics screening technologies to identify novel serum protein makers that changes significantly in the early onset of osteoporosis and further offer new possibilities for more convenient and economical early diagnosis methods.Experime ntⅠ: Screening osteoporosis-related serum protein marke rs in ratObjective:Screen and identify novel osteoporosis-related serum protein markers in rat. The markers should be more sensitive and specific to early osteoporosis diagnosis so that they can be candidates for further clinical validation.Methods:1)Rat osteoporosis model establishment. 20 healthy adult female SD rats were randomly divided into Sham group and OVX group, with 10 rats each group. O VX group: according to the weight, rats are anesthetized by using of sodium pentobarbital 40 mg/kg through intramuscular injection; then the back skin around surgery region is shaved and disinfected; make a 1 cm longitudinal skin incision on both sides laterally 1.5 cm from spine and longitudinally 1 cm below ribs; separate the underlying soft tissue and muscle; expose ovaries on both sides; ligate and remove both of the ovaries; suture up the peritoneum, muscle and skin respectively. Put the rats back into squirrel cage for recovery. To avoid infections, rats are injected with 40 WU penicillin after surgery for 3 continuous days. Sham group: expose ovaries as described above and remove the adipose tissue around ovaries instead of ovaries. Other manipulation procedures are the same with those of OVX group.2)Serum samples preparation: Collect 1.5 ml of blood through the angular vein from both groups at the time of 2 w, 4 w, 6 w and 8 w after the operation. Put the blood in EP tubes for 4 hours and draw the supernatant serum, save the samples in-80 ℃ refrigerator for further analysis.3)Osteoporosis model verification: Imaging the rats at 2 w, 4 w, 6 w and 8 w after operation using a micro CT scanner. Trabecular bone structures are reconstructed and analyzed for structural parameters.4)Protein chip detection: prepare the samples according to manufacturer’s instructions and detect the fluorescent signals for analyzing.Results:1)Comparing to Sham group rats, the BMD of OVX group rats began to decrease at 4 w after surgery, and significant bone loss was observed at 8 w. There was no obvious BMD change in Sham group rats.2)In OVX group rats, parameters reflecting the cancellous bone morphology change such as BV/TV, Tb.Th and Tb.N were declining from 4 w post surgery and changed dramatically at 8 w post surgery. Tb.Sp showed an opposite trend and started increasing from 4 w. No obvious changes for the above mentioned index were observed in Sham group rats.3)Serum protein levels of B7-2, b-NGF, Fractalkine, IFN g, MCP-1 and TIMP-1 in OVX rats began to increase from 4 w after ovariectomy, and changed dramatically at 8 w. Whereas, no significant changes were detected in Sham rats.Conclusion:The rat osteoporosis model was successfully established. Serum proteins B7-2, Fractalkine, TIMP-1, MCP-1, b-NGF and IFN g were screened out to be potential osteoporosis-related serum markers. Experime ntⅡ: Verification of osteoporosis-related serum markers in patients’ blood samplesObjective:Detect and compare the serum levels of Fractalkine, TIMP-1 and MCP-1in osteoporosis patients, BMD reduced and BMD normal populations. Elucidate the proteins change with the development of osteoporosis and provide potential targets for early osteoporosis clinical diagnosis.Methods:1)Enroll patients for osteoporosis, BMD reduced and BMD normal groups. Collect 2~3 ml of blood from the patients using negative pressure equipment. Blood is centrifuged at 3000 rpm/min for 10 min. Supernatant serum is collected and stored in-80 ℃ refrigerator.2)Serum proteins Fractalkine, TIMP-1 and MCP-1 from patients are quantified by Elisa method following the manufacture’s instructions.Results:Quantification results suggested that all of the three above mentioned proteins, Fractalkine, TIMP-1 and MCP-1, exhibited an increasing tendency from the normal BMD group, the decreased BMD group to osteoporosis group. The changes were statistically significant among the three groups.Conclusion:Serum proteins Fractalkine, TIMP-1 and MCP-1 exhibited different profile in the progression of osteoporosis and may serve as early diagnostic markers.Summary:Both animal experiments and clinical testing results showed that the serum proteins Fractalkine, TIMP-1 and MCP-1 quantities varied with the progression of osteoporosis. These results strongly indicated these serum proteins be associated with osteoporosis development and thus could serve as potential clinical applicable diagnostic markers for early osteoporosis.