【作者】 刘富强；

【导师】 陈建庭； 白岚；

【作者基本信息】 南方医科大学 ， 骨外科学， 2011， 硕士

【摘要】 研究背景胃切除术后的远期并发症之一是骨质疏松症,其发病率文献报道不一,最高可达70%。胃切除术后骨质疏松症的早期症状不明显,许多患者虽有骨质减少,但无临床症状；随着时间的延长,症状会越来越明显,常见的临床症状有：骨痛、甚至骨折,体重减轻,脂肪泻,上腹部不适等。目前对胃切除术后骨质疏松症的药物治疗主要是服用维生素D和钙剂,但其疗效尚不确切。近年来,胃切除术后骨质疏松症逐渐引起国内外学术界的关注与重视。国内、外文献有关胃切除术后骨质疏松症病因学研究的报道逐渐增加,目前认为它与维生素D和(或)钙的吸收不良、蛋白质摄入、术式等因素密切相关,但其具体发病机制仍不清楚。研究发现：胃底是分泌胃酸的主要部位,其提取物具有降低血钙的作用。此提取物经亮氨酸氨基肽酶(extracts of oxyntic mucosa, EOM)消化后,其降低血钙的作用丧失；胃泌素-17和胃底部产酸部分的提取物都具有促进骨钙摄入的作用,胃泌素-17对胃切除术后的小鼠无作用,但是胃底部产酸部分提取物对胃切除术后的小鼠却有作用。这说明胃泌素的降血钙作用是间接的,是通过刺激一种肽类激素的释放而发挥作用的,目前命名为胃钙素(gastrocalcin)。成骨细胞不但分泌大量的骨胶原和骨基质中的其他成分,而且还分泌一些重要的酶类和细胞因子,如基质金属蛋白酶、碱性磷酸酶、骨钙素、骨保护素(OPG)和核因子K B受体活化因子配体(RANKL)等,从而启动骨的形成过程,同时也能通过这些因子将破骨细胞偶联起来,调控破骨细胞的生成、成熟及活化。我们在前期体外实验研究中观察到成骨细胞在胃粘膜提取物刺激下细胞内钙离子增加,同时此提取物不仅能够显著增加成骨细胞的增殖率,而且能够促进成骨细胞的成骨活性。利用蛋白组学对胃泌素刺激胃粘膜后差异蛋白的表达进行分析,发现1,25D3-MARRS Receptor蛋白(ERp57)经胃泌素干预后,在胃粘膜中高度表达,分析蛋白的相互作用和功能,推断它可能是胃钙素。但是1,25D3-MARRS/ERp57是否像胃粘膜提取物一样能促进成骨,以及在胃切除术后骨质疏松症中是否起着重要作用,目前尚不清楚。因此,研究1,25D3-MARRS/ERp57对成骨细胞的生物学作用,将会为验证1,25D3-MARRS/ERp57是否是胃钙素提供重要参考依据,也为研究胃切除术后骨质疏松症的发病机制提供新的思路。目的1.探讨免疫沉淀1,25D3-MARRS/ERp57后胃粘膜提取物对成骨细胞增殖分化的作用。2.探讨1,25D3-MARRS/ERp57对成骨细胞增殖分化的作用。3.探讨1,25D3-MARRS/ERp57联合1,25(OH)2D3对成骨细胞增殖分化的作用。方法1.SD大鼠乳鼠成骨细胞的分离、培养和鉴定取1-2天SD大鼠乳鼠(10只),将骨片剪成lmmxlmm小片后,采用连续酶消化法分离成骨细胞,37℃、5%CO2培养箱中常规培养。倒置相差显微镜下形态学观察、Giemsa染色,碱性酸酶染色和钙结节染色进行鉴定。2.大鼠胃粘膜组织提取物的制备和免疫沉淀实验提取SD大鼠(250-300g)胃粘膜提取物后,采用BCA法蛋白检测试剂盒检测总蛋白浓度。每500μg胃粘膜提取物中分别加入50μl、5μl、0.5μl ERp57抗体和5μl IgG抗体进行免疫沉淀反应。离心去除1,25D3-MARRS—1,25D3-MARRS抗体—Protein A/G bead slurry复合物后,取上清液,稀释至10-4mg/ml后分装,-80℃保存备用。3.大鼠胃粘膜组织提取物中1,25(OH)2D3和ERp57含量的测定。按ELISA试剂盒说明检测大鼠胃粘膜组织提取物中1,25(OH)2D3和ERp57的含量。4.实验分组(1)免疫沉淀1,25D3-MARRS/ERp57后胃粘膜提取物对成骨细胞增殖分化的作用。实验中按照干预因素不同分为6组：生理盐水组(Control)、高沉淀组(HEOMS,用50μl ERp57抗体和50μl Protein A/G bead slurry进行免疫沉淀反应后的胃粘膜提取物上清液)、中沉淀组(MEOMS,用5μlERp57抗体和50μl Protein A/G bead slurry进行免疫沉淀反应后的胃粘膜提取物上清液)、低沉淀组(LEOMS,用0.5μlERp57抗体和50μl Protein A/G bead slurry进行免疫沉淀反应后的胃粘膜提取物上清液)、无关抗体组(IEOMS,用5μl兔抗鼠IgG和50μl Protein A/G bead slurry进行免疫沉淀反应后的胃粘膜提取物上清液)和胃粘膜提取物未处理组(EOM),后五组终浓度为10-4mg/ml。(2) 1,25D3-MARRS/ERp57对成骨细胞增殖分化的作用。实验中按照干预因素不同分为6组：生理盐水组(Control)和2×10-7 ng/ml(ML)、2×10-6 ng/ml(L)、2×10-5 ng/ml(M)、2×10-4 ng/ml (H)、2×10-3 ng/ml(MH) 5个浓度梯度组的1,25D3-MARRS/ERp57。(3)1,25D3-MARRS/ERp57联合1,25(OH)2D3对成骨细胞增殖分化的作用。实验中按照干预因素不同分为4组：生理盐水组(Control)、2×10-4 ng/ml ERp57组(E)、2×10-4ng/mlERp57和100pmol/L1,25(OH)2D3组(E+V)、100pmol/L 1,25(OH)2D3组(V)。5.对成骨细胞增殖分化的作用(1)对成骨细胞增殖的作用SD大鼠乳鼠成骨细胞分别与10μl各组干预因素共孵育0、1、2、3d后,用MTT法检测吸光度(OD)值。(2)SD大鼠乳鼠成骨细胞I型胶原和骨钙素mRNA的表达SD大鼠乳鼠成骨细胞分别与200μl各组干预因素共孵育12h后,收集细胞,提取细胞总RNA,反转录多聚酶链式反应(RT-PCR)检测成骨细胞的Ⅰ型胶原和骨钙素mRNA的表达,以GAPDH为内对照,分析Ⅰ型胶原和骨钙素相对表达量的变化。(3)SD大鼠乳鼠成骨细胞Ⅰ型胶原和骨钙素蛋白的表达SD大鼠乳鼠成骨细胞分别与200μl各组干预因素共孵育24h后,收集细胞,提取细胞总蛋白,免疫印迹法(Western blot)检测成骨细胞的Ⅰ型胶原和骨钙素蛋白的表达,以GAPDH为内对照,分析Ⅰ型胶原和骨钙素蛋白相对表达量的变化。结果1.SD大鼠乳鼠成骨细胞的培养和鉴定倒置相差显微镜下观察到成骨细胞贴壁生长,细胞形态不规则,呈长条形、三角形和多角形等。培养3-5d,细胞体积增大,可见较多细胞分裂相,胞浆丰富、清晰,向外伸展出较多突起,与周围细胞突起相互连接。汇合后,细胞呈铺路石状,并可重叠生长。Giemsa染色表现为胞浆浅蓝色,细胞核明显,呈暗红色,可见核仁。碱性磷酸酶染色可见所有细胞胞质内出现灰黑色颗粒或块状沉淀的阳性反应。钙结节染色后可以看到典型红色钙化结节形成,呈圆形或椭圆形,同心圆状结构。形态学观察,碱性磷酸酶染色和细胞结节染色表明：培养的细胞符合成骨细胞的形态特征,具有分泌碱性磷酸酶和形成矿化结节的生物学行为。2.胃粘膜组织提取物中1,25(OH)2D3和ERp57含量的测定经测定,正常胃粘膜提取物中1,25(OH)2D3和ERp57的含量分别为24.345±0.095ng/l和2.647±0.096ng/ml。另外,随着加入抗ERp57抗体含量的增多,胃粘膜提取物中剩余ERp57的含量减少。3.成骨细胞增殖分析(1)免疫沉淀组胃粘膜提取物和未处理组胃粘膜提取物促进成骨细胞增殖的作用都高于生理盐水组(P<0.05),但各免疫沉淀组胃粘膜提取物与胃粘膜提取物未处理组之间并无显著差异(P>0.05)。(2)与生理盐水对照组相比,不同浓度组1,25D3-MARRS/ERp57对成骨细胞增殖的影响无显著差异(P>0.05)。(3)1,25D3-MARRS/ERp57联合1,25(OH)2D3组显著促进成骨细胞的增殖,与其他3组比较有显著性差异(P<0.05)。4.Ⅰ型胶原和骨钙素mRNA和蛋白的表达(1)胃粘膜提取物中的1,25D3-MARRS/ERp57经免疫沉淀后,成骨细胞Ⅰ型胶原和骨钙素mRNA和蛋白的表达均呈不同程度降低。(2)不同浓度组1,25D3-MARRS/ERp57对Ⅰ型胶原和骨钙素mRNA和蛋白表达的影响无显著差异(P>0.05)。(3)1,25D3-MARRS/ERp57联合1,25(OH)2D3组显著促进Ⅰ型胶原和骨钙素mRNA和蛋白的表达,与其他3组比较有显著性差异(P<0.05)。结论1.免疫沉淀1,25D3-MARRS后胃粘膜提取物相对于正常胃粘膜提取物对成骨细胞的增殖能力无显著影响,但能降低其分化能力。2.单独的1,25D3-MARRS/ERp57对成骨细胞不具有生物学活性。3.1,25D3-MARRS/ERp57联合1,25(OH)2D3能明显促进成骨细胞的增殖和分化。更多还原

【Abstract】 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.更多还原