| BackgroundGastrectomy bone disease is one of the long-term complications that can be expressed as osteoporosis or osteomalacia, or both of them. The reported incidence is 70%. The early symptoms of gastrectomy osteoporosis and osteomalacia are not obvious, usually first manifested as waist and leg pain. There will be a series of symptoms as the disease progresses. Currently the treatment of bone disease after gastrectomy is mainly vitamin D and calcium therapy, there is no effective method. In recent years, gastrectomy bone disease draw growing concern and emphasis. Domestic and foreign literature about bone disease after gastrectomy is increasing. Althoμgh that vitamin D and (or) calcium absorption, protein intake, the impact of surgical factors are thoμght due to bone disease s after gastrectomy,however, the specific pathogenesis remains unclear.The extracts of oxyntic mucosa (EOM) can reduce the level of calcium.when the EOM is digested with leucine aminopeptidase, blood calcium decreased activity was destroyed.Both gastrin-17 and the extract of the stomach play a role in calcium intake. Gastrin-17 had no effect on mice after gastrectomy,however,the extract of oxyntic mucosa had obvious effect on mice after gastrectomy.lt indicates that the calcium-lowering effect of gastrin is indirect, it may throμgh stimμlating the release of a peptide hormone, which is currently named as gastrocalcin.Osteoblasts are the cells that can promote bone formation, they not only secrete large amounts of bone collagen and other bone matrix, but also can secrete a number of important cytokines and enzymes, such as matrix metalloproteinases, alkaline phosphatase, osteocalcin, osteoprotegerin, RANKL, etc., in order to initiate the process of bone formation.Moreover osteoblasts can even with the osteoclasts throμgh these factors and control the formation, maturation and activation of osteoclasts.In vitro, an increase in intracellμlar calcium response was observed in osteoblasts stimulated by the extract of oxyntic mucosa, and the extract not only significantly increased the proliferation rate of osteoblasts, but also promoted bone formation of osteoblast activity. Proteomics of gastric mucosa after gastrin stimμlation to analyze differences in protein expression was performed, and protein disμlfide isomerase A3 (Protein disulfide-isomerase A3, PDIA3, also called ERp57, the 1,25D3-MARRS Receptor protein) was highly expressed in the gastric mucosa after the intervention with gastrin. PDIA3 was concluded as the gastrocalcin from protein interaction and functional analysis. It is not clear whether 1,25 D3-MARRS/ERp57 can promote osteogenic activity as EOM,which play an important role in bone disease after gastrectomy. Therefore, the study of the effect of 1,25D3-MARRS/ERp57 on the biological role of osteoblasts will verify whether 1,25 D3-MARRS/ERp57 is the gastrocalcin.It will provide new ideas for the study of the mechanism of gastrectomy bone disease.Objectives1.To study the effect of the extract of oxyntic mucosa on the proliferation and differentiation of osteoblast after immunoprecipitation of 1,25D3-MARRS/ERp57. 2.To study the effect of 1,25D3-MARRS/ERp57 on the proliferation and differentiation of osteoblast.3.To study the effect of 1,25D3-MARRS/ERp57 combined with 1,25(OH)2D3 on the proliferation and differentiation of osteoblast.Methods1.Osteoblast isolation, cμlture, and identificationOsteoblasts were isolated from calvariae of 1-day-old Sprague-Dawley rats (10) with the way of sequential enzymatic digestion, and than were identified by morphology, ALP staining and Alizarin red staining.2. Preparation of EOM in rats and immunoprecipitation experimentsEOM in SD rats (250-300g) was prepared according to the instruction of literature and the solution was quantified according to the instruction of BCA protein assay kit.50μl,5μl,0.5μlERp57 antibodies and 5μl normal IgG antibody were added to EOM after pre-cleaning and immunoprecipitation was performed according to the instructions. The supernatant was transferred to another four EP tubes after centrifugal removal of the complex of 1,25 D3-MARRS-1,25D3-MARRS antibody-Protein A/G bead slurry and was diluted to a concentration of 10"4 mg/ml, and then kept at-80℃.The content of ERp57 was quantified according to the instruction of a ELISA assay kit.3. The content of 1,25(OH)2D3 and ERp57 in EOMThe content of 1,25(OH)2D3 and ERp57 in EOM was quantified according to the instruction of a ELISA assay kit.4. Grouping of experiment(1)The effect of the extract of oxyntic mucosa on the proliferation and differentiation of osteoblast after immunoprecipitation of 1,25D3-MARRS/ERp57. 6 groups were divided in this part. Control(saline),HEOMS(The EOM supernatant collected after the polyclonal antibody to ERp57 (10μg) were incubated with 500μl (500μg) sample of EOM),MEOMS(The EOM supernatant collected after the polyclonal antibody to ERp57 (1μg) were incubated with 500μl (500μg) sample of EOM),LEOMS(The EOM supernatant collected after the polyclonal antibody to ERp57 (0.1μg) were incubated with 500μl (500μg) sample of EOM), IEOMS (The EOM supernatant collected after normal rabbit IgG (1μg) were incubated with 500μl (500μg) sample of EOM) and EOM.(2)The effect of 1,25 D3-MARRS/ERp57 on the proliferation and differentiation of osteoblast.6 groups were divided in this part. Control(saline),ML(ERp57 at 2×10-7 ng/ml), L(2×10-6 ng/ml), M(2×10-5 ng/ml), H(2×10-4 ng/ml) and MH(2×10-3 ng/ml).(3)The effect of 1,25D3-MARRS/ERp57 combined with 1,25(OH)2D3 on the proliferation and differentiation of osteoblast.4 groups were divided in this part. Control(saline),E(2×10-4 ng/ml ERp57), E+V (2×10-4ng/mlERp57 and 100pmol/L1,25(OH)2D3), V (100pmol/L 1,25(OH)2D3).5.Effect on the proliferation and differentiation of osteoblasts.(1)Effect on the proliferation of osteoblasts.Osteoblasts were incubated with 10μl different stimμlus respectively for 0,1,2,3 days, then MTT assay was used to detect the absorbance values.(2)Reverse transcriptase polymerase chain reaction (RT-PCR) testThe cells were inocμlated with 200μl different stimμlus.12 hours later, the cells were collected and total cellμlar RNA of them was extracted. Then reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of collagen type I, osteocalcin and GAPDH of osteoblast. GAPDH was used as an internal control to the analysis of relative expression changes of collagen type I and osteocalcin.(3)Western-blot testThe cells were inoculated with 200μl different stimulus.24 hours later, the cells were collected and total cellμlar protein of them was extracted. Then western-blot test was used to detect the protein expression of collagen type I, osteocalcin and GAPDH of osteoblast. GAPDH was used as an internal control to the analysis of relative expression changes of collagen type I and osteocalcin.Resμlts1.Osteoblast identificationObserved by inverse phase contrast microscope, osteoblast cells adhered to the bottom of culture bottle.They showed irregular shapes such as rectangle, triangle and polygon. After three to five days, most cells became bigger and cell division coμld be easily observed. Osteoblasts with Giemsa staining exhibitted light blue kytoplasm,dark red nucleus.Black particle and massive precipitation appeared in all osteoblast cells after ALP staining. The calcified nodules, which were stained by AZR solution, became red round bone nodules. According to the morphology observation and the staining of ALP, AZR, the isolated and cultured cells from rat cranial bone presented the characters of osteoblasts.2. The content of 1,25(OH)2D3 and ERp57 in EOMThe content of 1,25(OH)2D3 and ERp57 in EOM is 24.345±0.095ng/land 2.647±0.096ng/ml respectively.With the addition of anti ERp57 antibody levels gradually increased, the content of ERp57 in EOM decreased.3. The proliferation of osteoblasts(1)Immunoprecipitation EOM and untreated EOM promoted the proliferation of osteoblasts than the saline(P<0.05), but there is no difference between immunoprecipitation EOM and the untreated EOM (P>0.05). (2)1,25D3-MARRS/ERp57 did not promote the proliferation of osteoblasts compared with saline.(3)1,25D3-MARRS/ERp57 combined with with 1,25(OH) 2D3 promoted the proliferation of osteoblasts compared with 3 other groups.4. mRNA and protein expression of collagen type I and osteocalcin(1) EOM treated with immunoprecipitation of 1,25D3-MARRS/ERp57 reduced mRNA and protein expression of collagen type I and osteocalcin in osteoblast.(2)Different concentrations of 1,25D3-MARRS/ERp57 had no difference in mRNA and protein expression of collagen type I and osteocalcin.(3) 1,25D3-MARRS/ERp57 combined with with 1,25(OH)2D3 promoted mRNA and protein expression of collagen type I and osteocalcin compared to 3 other groups P<0.05).Conclusions1. EOM treated with immunoprecipitation of 1,25D3-MARRS/ERp57 had no effect on the proliferation of osteoblasts, but inhibited the differentiation of osteoblasts.2. 1,25D3-MARRS/ERp57 had not biological effect on osteoblasts.3. 1,25D3-MARRS/ERp57 combined with with 1,25(OH)2D3 promoted the proliferation and differentiation of osteoblasts. |
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