钙对体外培养大鼠成骨细胞增殖分化及细胞周期的影响

在体外培养大鼠成骨细胞的过程中,添加不同剂量的钙(0、1、2、4 mmol/L),通过检测细胞周期、细胞增殖能力、碱性磷酸酶(ALP)活性及骨桥蛋白表达,探讨了钙对成骨细胞增殖分化及细胞周期的影响.结果表明,2、4 mmol/L钙组在1～8 d均显著或极显著地促进成骨细胞增殖(P<0.05或P<0.01),1 mmol/L钙组则在5、6、8 d有显著或极显著的差异(P<0.05或P<0.01);添加不同浓度的钙除1 mmol/L组第2天外的不同时间均极显著地抑制细胞内ALP活性(P<0.01);1 mmol/L钙能极显著的使成骨细胞滞留在S期(P<0.01);添加不同浓度的钙均能使G2期显著或极显著的增加(P<0.05或P<0.01),G1期极显著的下降(P<0.01),并能极显著地诱导细胞内骨桥蛋白的表达(P<0.01).表明添加不同浓度的钙均能抑制其早期分化,促进成骨细胞的增殖,使细胞滞留在S期或G2期,诱导细胞在基质成熟期分化,有利于细胞的钙化.     

镉对体外培养鸡骨髓基质细胞成骨分化的毒性作用

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚。本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色鉴定成骨分化,RT-PCR检测成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN、RANKL)表达变化。结果显示,1~10μmol/L的镉显著促进BMSCs增殖,20μmol/L的镉显著抑制其增殖(P<0.05);5μmol/L以上的镉可显著抑制ALP活性,并以浓度依赖方式极显著抑制成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN)mRNA表达,上调RANKL mRNA表达(P<0.01)。表明,一定浓度镉可抑制鸡BMSCs体外增殖及其向成骨细胞(OB)的分化。     

1α,25-二羟维生素D_3对大鼠成骨细胞增殖分化及周期的影响

在SD大鼠成骨细胞(Osteoblast,OB)体外培养体系中添加不同浓度1α,25-二羟维生素D_3(0、10~(-9)、10~(-8)、10~(-7) mol/L),作用24、48、72 h,测定OB增殖率、碱性磷酸酶(ALP)活性,作用48 h流式细胞仪测定OB周期.结果显示,10~(-9)mol/L 1α,25-二羟维生素D_3作用24、48、72 h均促进OB增殖(P<0.05或P<0.01),抑制ALP活性(P<0.01);10~(-8)、10~(-7)mol/L作用24、48 h,OB增殖率与对照组差异不显著(P>0.05),但24 h时ALP活性均明显升高(P<0.05或P<0.01),48 h则抑制了ALP活性并使OB滞留在G_2/M期(P<0.05或P<0.01);72 h时10~(-7) mol/L组OB增殖率极显著低于其余各组(P<0.01),并使ALP活性升高(P<0.01).表明低浓度1α,25-二羟维生素D_3能促进OB增殖,抑制其分化;高浓度1α,25-二羟维生素D_3能抑制OB增殖,促进其分化,并使细胞滞留在G_2/M期.     

铅对SD大鼠成骨细胞磷脂酰胆碱特异性磷脂酶C的影响

在体外培养新生sD乳鼠的成骨细胞中暴露不同浓度的醋酸铅（O、20、40、80μmol／L）,作用24h后检测细胞磷脂酰胆碱特异性磷脂酶C（PC-PLC）活性与PC—PLC蛋白的表达量。结果表明：不同浓度的醋酸铅均可使成骨细胞PC—PLC活性和PC-PLC蛋白表达量极显著降低（P〈0．01）,呈明显的剂量-效应关系,表明成骨细胞铅暴露可通过抑制PC—PLC而发挥毒性作用。     

褪黑素对鸡成骨细胞增殖和分化的影响

采用组织块贴壁法分离鸡成骨细胞并进行体外培养,细胞纯化后经姬姆萨、甲苯胺蓝、碱性磷酸酶(ALP)和茜素红染色鉴定;在成骨细胞培养液中分别添加0,10,50,100μmol/L褪黑素,连续14 d测定成骨细胞增殖情况;在第1,3,7,14天测定诱导分化培养的成骨细胞ALP活性,并在第14天检测成骨细胞BSP基因表达水平。甲苯胺蓝染色结果与小鼠胚胎成骨细胞前体细胞系MC3T3-E1一致;ALP染色呈阳性,可见大量黑色颗粒;茜素红染色后矿化结节呈深红色。成骨细胞ALP活性在第3天有所降低,但随培养时间延长,ALP活性逐渐升高;添加褪黑素对细胞增殖的影响差异不显著(P0.05),但在第1天和第14天显著提高成骨细胞ALP活性(P0.05);添加褪黑素提高了BSP基因的相对表达量(P=0.09)。结果表明,利用组织块贴壁法,分离得到鸡颅骨成骨细胞,确定了褪黑素促进成骨细胞分化,且具有时间和浓度依赖性,但不影响增殖。     

胰岛素样生长因子-1对奶牛成骨细胞增殖及分化的影响

研究了外源性胰岛素样生长因子-1(IGF-1)对体外培养的奶牛成骨细胞增殖、分化及矿化的影响。应用组织块移行法分离奶牛成骨细胞,姬姆萨染色及碱性磷酸酶(ALP)染色进行鉴定。MTT法检测不同质量浓度IGF-1对成骨细胞增殖的影响,比色法检测及放射免疫法(RIA)分别测定细胞基质ALP活性及骨钙素(OC)水平,评价细胞分化功能。结果显示,1～200μg/LIGF-1均能促进奶牛成骨细胞增殖,100μg/L组具有最大刺激效应,且IGF-1干预组细胞光密度值(D值)从第1天到第5天逐渐增加,第7天降低。与对照组相比,10μg/L与100μg/LIGF-1均可显著增强ALP活性及OC水平,增幅达20%～180%(P0.05或者P0.01)。结果表明,IGF-1是奶牛成骨细胞增殖和分化代谢促进剂,因此推测IGF-1可以成为动物骨代谢性疾病潜在治疗手段。     

caspase-3、caspase-9在醋酸铅诱导NRK细胞凋亡中的作用

将体外培养的大鼠肾小管上皮细胞NRK分别用0、0.5、1.0、2.0μmol/L醋酸铅作用12h后,MTT法检测不同剂量的醋酸铅对其增殖的抑制率,Annexin V-FITC/PI双染法检测醋酸铅引起的NRK凋亡,罗丹明123检测线粒体膜电位的变化,Western blot检测caspase-3和caspase-9蛋白的表达,以及caspase广谱抑制剂和caspase-9的抑制剂(Z-VAD-FMK和Ac-LEHD-FMK)对醋酸铅诱导细胞凋亡的抑制作用。结果显示,与对照组相比,细胞存活率明显下降(P<0.05或P<0.01),醋酸铅明显抑制NRK细胞增殖,且呈一定剂量效应,IC50为(2.44±0.32)μmol/L;凋亡检测表明,随着醋酸铅浓度的升高,细胞凋亡现象越明显;明显减低线粒体膜电位;激活体caspase-3和caspase-9的表达水平呈剂量依赖性增加;caspase抑制剂能显著抑制醋酸铅诱导的细胞凋亡。结果表明,醋酸铅抑制NRK细胞增殖,可能通过激活caspase依赖性的线粒体信号通路而诱导NRK细胞凋亡。     

1α，25-二羟维生素D3对大鼠成骨细胞增殖分化及周期的影响

在SD大鼠成骨细胞（Osteoblast,OB）体外培养体系中添加不同浓度1α,25-二羟维生素D3（0、10^-9、10^-8、10^-7mol／L）,作用24、48、72h,测定OB增殖率、碱性磷酸酶（ALP）活性,作用48h流式细胞仪测定0B周期。结果显示,10^-9mol／L 1α,25-二羟维生素D3作用24、48、72h均促进oB增殖（P〈0.05或P〈0.01）,抑制ALP活性（P〈0.01）;10^-8、10^-7mol／L作用24、48h,OB增殖率与对照组差异不显著（P〉0.05）,但24h时ALP活性均明显升高（P〈0.05或P〈0.01）,48h则抑制了ALP活性并使OB滞留在G2／M期（P〈0.05或P〈0.01）;72h时10^-7mol／L组OB增殖率极显著低于其余各组（P〈0.01）,并使ALP活性升高（P〈0.01）。表明低浓度1α,25-二羟维生素D3能促进OB增殖,抑制其分化;高浓度1α,25-二羟维生素D3能抑制OB增殖,促进其分化,并使细胞滞留在G2／M期。     

成骨生长肽对体外培养奶牛成骨细胞增殖分化的影响

体外培养奶牛成骨细胞,用10-12,10-11,10-10,10-9和10-8mol/L成骨生长肽干预5 d后,噻唑蓝比色法(MTT)检测细胞增殖,紫外分光光度法检测细胞上清液中碱性磷酸酶(ALP)。结果显示:10-12~10-8mol/L的成骨生长肽均能促进成骨细胞的增殖,并且呈现剂量依赖方式,10-12mol/L试验组即可促进成骨细胞增殖,与对照组比较差异不显著(P>0.05),10-11~10-8mol/L试验组与对照组比较均有极显著差异(P<0.01),以10-10mol/L试验组差异最为显著;成骨生长肽对细胞上清液中ALP活性起抑制作用,其中在10-10mol/L时抑制作用最为显著(P<0.01)。结果表明成骨生长肽对体外培养奶牛成骨细胞增殖作用明显,对碱性磷酸酶起轻微抑制作用而且呈双向性。     

骨碎补水提液对大鼠成骨细胞的影响

为观察骨碎补水提液对体外培养大鼠成骨细胞增殖、分化的影响,用体外培养新生SD大鼠颅盖骨分离的成骨细胞,将不同浓度的骨碎补水提液分别与第3代大鼠成骨细胞进行体外共同培养,采用噻唑蓝(MTT)法测定细胞增殖能力,对硝基苯磷酸盐法(PNPP法)测定细胞的碱性磷酸酶(ALP)活性.结果显示,与对照组相比,作用48 h、72 h,骨碎补水提液均能显著促进大鼠成骨细胞的增殖,且与时效有关;作用48 h ,10-3、10-2 mg/mL骨碎补水提液浓度组可显著升高细胞ALP活性(P<0.05).表明骨碎补水提液中存在较高活性的促大鼠成骨细胞增殖和分化的物质.     

Differentiating effect of pamidronate on canine osteo‐sarcoma cell lines in vitro

Introduction: Pamidronate has been traditionally used to manage pathologic osteoclastic disorders. In addition to its effects on osteoclasts, pamidronate has also been demonstrated to promote phenotypic maturation and inhibition of proliferation in osteoblasts. Canine osteosarcoma (OSA) consists of malignant, undifferentiated osteoblasts. The objective of this study was to determine if micromolar concentrations of pamidronate could induce malignant osteoblastic differentiation as evaluated by an increase in alkaline phosphatase (ALP) activity and/or osteocalcin (OC) production, two specific markers of normal osteoblastic activity. Methods: Two canine OSA cell lines (HMPOS and COS 31) were used for all experiments. Cells were incubated for 48 or 72 hours with various pamidronate concentrations (0, 0.1, 1, 5, 10, and 20 μM). After incubation, the supernatants were sampled and the relative amounts of viable cells were determined with a cell proliferation assay (Cell Titer 96^® AQ_uous, Promega). An ALP detection kit (Starbright^®, Sigma^®) was used to measure the ALP activity and an ELISA (Osteocalcin EIA kit, Biomedical Technologies) was used to determine the concentration of osteocalcin in the supernatants. The ALP and osteocalcin values were corrected for the amount of viable cells. Results: Pamidronate induced a dose‐dependent reduction in the number of viable COS 31 and HMPOS cells at both 48 and 72 hours. A dose‐dependent elevation in ALP activity from baseline was observed. At 20 μM, a 2.3‐fold increase was observed for HMPOS at 72 hours, while a 1.43‐fold increase was observed for COS 31 at 72 hours. Very low level (less than 2 ng/ml) of osteocalcin pre‐ and post‐pamidronate treatment was detected for both COS 31 and HMPOS. Conclusion: The data suggests that pamidronate increases alkaline phosphatase activity in canine OSA cells in a dose‐dependent manner. However, cytotoxic assays are needed in order to accurately characterize any concurrent decrease in the number of viable cells. The potential differentiating effect of pamidronate on malignant osteoblasts provides an additional argument for its use in the palliative treatment of OSA.     

17β-estradiol combined with testosterone promotes chicken osteoblast proliferation and differentiation by accelerating the cell cycle and inhibiting apoptosis <Emphasis Type="Italic">in vitro</Emphasis>

Medullary bone is a unique tissue in the long bones cavities of lay hens, and plays an important role as a calcium reservoir for egg-shell formation. Medullary bone formation requires the synergistic action of estrogen and androgen on osteoblasts during the early stage of sexual maturity. The objective of the current study was to investigate the effects of 17β-estradiol, testosterone, and the combination on the proliferation, alkaline phosphatase (ALP) activity, apoptosis, the cell cycle of chicken osteoblasts in vitro. The proliferation of osteoblasts was examined with the MTT assay. Apoptosis and the cell cycle were assessed with flow cytometry. Either 17β-estradiol (200 pg ml⁻¹) or testosterone (100 pg ml⁻¹) or the combination (100 pg ml⁻¹ each) significantly enhanced osteoblast proliferation and ALP activity, accelerated the osteoblast cell cycle, and stimulated osteoblast DNA synthesis in a period of 24 h. 17β-estradiol, used alone or with testosterone, inhibited chicken osteoblast apoptosis; However, testosterone alone induced cell apoptosis. In conclusion, 17β-estradiol combined with testosterone promoted osteoblast proliferation and ALP activity, accelerated the osteoblast cell cycle, inhibited osteoblast apoptosis.     

The stimulatory effect of insulin-like growth factor-1 on the proliferation, differentiation, and mineralisation of osteoblastic cells from Holstein cattle

This study investigated the effect of exogenous insulin-like growth factor (IGF)-1 on the proliferation and differentiation of osteoblastic cells from Chinese Holstein cattle and the resultant bone nodule formation and mineralisation in vitro. The osteoblastic cells were isolated and cultured, then identified using Giemsa and alkaline phosphatase (ALP) staining methods. The effect of different concentrations of IGF-1 on cell growth was assessed by MTT assay. The ALP activity and osteocalcin (OC) concentration in the osteoblastic cells were measured by a colorimetric assay and a radioimmmunoassay, respectively. Calcium nodules were observed using alizarin red S stain, while the content of matrix calcium was determined by atomic absorption spectrophotometry. Cell proliferation in the cultures was stimulated by IGF-1 at concentrations ranging from 1 to 200 ng/mL, with the maximum effect observed at 100 ng/mL. This effect was observed from day 1 and peaked at day 5, but decreased at day 7. At concentrations of 10 ng/mL and 100 ng/mL, IGF-1 significantly induced ALP activity, OC level, matrix calcium content, and nodule formation of the osteoblastic cells by 20–180% (P < 0.05 or P < 0.01), compared to controls. The results suggested that IGF-1 is an anabolic agent for the proliferation, differentiation, mineralisation and calcium content of dairy cow osteoblasts, and could therefore act as a potential treatment for the metabolic bone diseases in these animals.     

不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。     

Subclinical characteristics of the wasting pig syndrome

The plasma concentrations of cortisol and corticosteroid-binding globulin and the adrenal synthesis capacity of cortisol were analysed in 10-week-old healthy and age-matched wasting or unthrifty pigs. Crypt cell multiplication, villus height and intestinal mucosal alkaline phosphatase (ALP) activity were also investigated. Furthermore, the effect of amperozide, a psychotropic drug with specific effects on emotional behaviour, was analysed for its effect on plasma ALP activity and villus height. Although the wasting pigs exhibited an increased cortisol synthesis capacity, there was a decreased plasma concentration of cortisol in these pigs. Furthermore, the plasma cortisol binding capacity was found to be significantly lowered in wasting pigs. There was also a reduced crypt cell proliferation, a reduced villus height and a decreased ALP activity in the ileal mucosa. Treatment with amperozide resulted in a normalisation of plasma ALP activity in unthrifty pigs, indicating a stimulation of body growth. The results indicate that the growth depression of wasting pigs is most probably a chronic stress syndrome caused by the inability of these animals to cope with the events following weaning and mixing.     

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma‐bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration.     

硒对体外培养小鼠成骨细胞形态、增殖和活力的影响

本文旨在探讨不同浓度亚硒酸钠溶液对体外培养的小鼠成骨细胞的形态、增殖及活力的影响。以出生24h的昆明小鼠颅骨为材料,通过酶消化法来分离成骨细胞,并分别接种于不同浓度的亚硒酸钠培养液(0.00、0.05、0.1、0.2、0.4、0.6、0.75、1.0 mg/L)中,倒置显微镜下观察成骨细胞的形态变化,采用MTT法测定细胞增殖率,结晶紫染色法测定细胞活力。48 h内,浓度高于0.4 mg/L时亚硒酸钠抑制细胞增殖且降低细胞活力,而低于0.2mg/L时亚硒酸钠可促进细胞增殖,增强了细胞活力。结果显示硒对成骨细胞的增殖和活力呈双向调节,硒作用呈浓度剂量依赖性。     

不同桑叶提取物对小鼠脾脏淋巴细胞增殖能力的影响

试验旨在比较不同桑叶提取物对小鼠脾脏淋巴细胞增殖作用的影响。用不同浓度乙醇醇沉桑叶水提物,制备桑叶粗多糖MLP-1、MLP-2、MLP-3、MLP-4、MLP-5,采用苯酚硫酸法测定各提取物多糖含量。以5种桑叶粗多糖和桑叶水提物（MLAE）为试验药物,采用MTT法比较多糖含量分别在15.625、31.25、62.5、125、250 μg/mL浓度时各提取物单独刺激及协同LPS、PHA共同刺激小鼠脾脏B、T淋巴细增殖的变化。结果显示,多糖浓度在15.625~250 μg/mL范围内,各提取物无论单独还是协同LPS和PHA时均能促进小鼠脾脏B、T淋巴细胞的增殖,其中,MLP-1多糖含量在低浓度时能显著刺激脾脏B淋巴细胞增殖（P<0.05）;MLP-3和MLP-5在低浓度时表现出较强的刺激T淋巴细胞增殖的能力;MLAE协同LPS在大部分浓度点的脾脏B淋巴细胞的增殖能力均显著高于细胞对照组和LPS对照组,可显著提升体液免疫功能（P<0.05）。     

复合植物精油对脂多糖刺激仔猪肝脏的保护作用

试验旨在研究复合植物精油(OCT)对脂多糖(LPS)刺激仔猪肝脏的保护作用。选取18头仔猪,随机分为对照组、LPS组和OCT+LPS组,每组6个重复。对照组与LPS组饲喂基础日粮,OCT+LPS组在基础日粮中添加50 mg/kg OCT。试验期21 d。于试验第21天,LPS组与OCT+LPS组仔猪腹腔注射LPS,对照组注射等量的生理盐水,3 h后采血,6 h后屠宰。结果表明:与对照组相比,LPS刺激提高了血浆谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、谷酰转肽酶(GGT)、肝脏诱导型一氧化氮合酶(i-NOS)的活力(P0.05),肝脏热休克蛋白70(HSP70)基因的相对表达量显著提高(P0.05);仔猪肝脏表皮生长因子(EGF)、白细胞介素10(IL-10)、胰岛素样因子(IGF-1)、丝氨酸蛋白激酶(mTOR)基因的相对表达量显著降低(P0.05)。在日粮中添加50 mg/kg的OCT,可缓解LPS导致的仔猪血浆ALT、AST、GGT活力的升高以及肝脏i-NOS活力的升高(P0.05),且有缓解LPS导致的仔猪肝脏MPO活力升高的趋势(P0.1),另外可缓解LPS导致的仔猪肝脏HSP70基因相对表达量的升高以及EGF、IL-10、IGF-1、mTOR等基因相对表达量的降低(P0.05)。综上可知,日粮中添加50 mg/kg的OCT可缓解LPS刺激引起的肝脏炎症反应,提高仔猪肝脏细胞的生长和增殖,进而缓解LPS刺激导致的仔猪肝脏氧化应激损伤。