外源性Ⅰ型胶原对人胚骨膜成骨细胞生物学特性的影响

目的　研究 型胶原对人胚骨膜来源成骨细胞生物学特性的影响 ,为 型胶原在组织工程人工骨中的应用提供依据。方法　在不同浓度 型胶原涂层上培养成骨细胞 ,用细胞计数法研究成骨细胞粘附情况 ,3 H- Td R掺入实验观察成骨细胞增殖能力 ,通过检测成骨细胞胶原、骨钙素和碱性磷酸酶的合成情况 ,以不涂层 型胶原为对照组 ,比较骨膜来源成骨细胞成骨能力的改变。结果　 型胶原对成骨细胞发挥以下作用 :1增加粘附细胞数量 ,且在 2 5 μg/ m l浓度达最大效应 ;2减弱成骨细胞增殖能力 ,且以 12 .5 μg/ ml以上浓度作用显著 ( P<0 .0 5 ) ;3轻度减弱成骨细胞 型胶原合成能力 ,以 2 5 μg/ ml以上浓度作用显著 ( P<0 .0 5 ) ;4增加骨钙素合成 ,以6 .2 5 μg/ m l以上浓度作用显著 ( P<0 .0 5 ) ,2 5 μg/ m l浓度达最大效应 ;5增加成骨细胞碱性磷酸酶活性 ,以 12 .5 μg/ml以上浓度作用显著 ( P<0 .0 5 )。结论　 型胶原能促进成骨细胞的粘附与分化 ;支架材料上复合 型胶原涂层可增强成骨细胞的成骨能力 ; 型胶原最佳复合浓度为 2 5 μg/ ml     

表面修饰对纳米晶胶原基骨细胞相容性的影响

目的:探索纤维蛋白(fibrin,FB)修饰的纳米晶胶原基骨(nanoHydroxyapatite/collagen,nHAC)支架材料上成骨诱导的骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)黏附、增殖及分化的情况。方法:实验分为两组:实验组,纤维蛋白修饰的纳米晶胶原基骨(FB-nHAC);对照组,单纯的纳米晶胶原基骨(nHAC)。将SD大鼠骨髓间充质干细胞定向诱导为成骨细胞,种植于支架材料上,体外复合培养。通过检测支架材料的细胞黏附率、不同时间点(3,7,10,14 d)支架材料中细胞数、碱性磷酸酶表达量以及扫描电镜观察细胞在支架材料上的生长状况,比较分析不同支架材料与细胞生物相容性差异。结果:大鼠MSCs经诱导培养14 d后,I型胶原免疫荧光染色为阳性;实验组支架材料的细胞黏附率显著高于对照组(P<0.05);且相同时间点实验组支架材料中的细胞数及碱性磷酸酶表达量也明显高于对照组(P<0.05);电镜观察发现两组材料上均有细胞生长,但实验组的细胞生长状况明显好于对照组。结论:表面修饰纤维蛋白后的nHAC支架材料具有更好的细胞黏附、增殖及促成骨分化能力。     

体外诱导条件下大鼠骨髓基质细胞骨钙素和Ⅰ型胶原mRNA的表达

目的 研究体外培养大鼠骨髓基质细胞诱导条件下，成骨分化标志骨钙素和Ⅰ型胶原mRNA水平的表达。方法 采用成骨诱导剂（含地塞米松10^-8mol/L,β甘油磷酸钠10mmol/L和抗坏敌国酸AA50μg/ml)对培养状态下的不同代大鼠骨髓基质细胞进行成骨诱导，提取RNA，采用RT-PCR方法，以β-肌动蛋白cDNA为内参照，检测骨钙素和Ⅰ型胶原mRNA的表达。结果 与对照组相比，接受成骨诱导剂作用的大鼠骨髓基质细胞随诱导时间的延长骨钙素和Ⅰ型胶原mRNA出现高表达，且维生素D为诱导骨钙素mRNA表达的必需成分。结论 体外培养的大鼠骨髓基质细胞保持成骨未分化状态，无骨钙素和Ⅰ型胶原mRNA的表达，诱导后可向成骨分化，骨钙素和Ⅰ型胶原mRNA表达升高。     

葡萄糖、胰岛素对大鼠骨髓间充质干细胞向成骨细胞分化的影响

目的 观察不同浓度葡萄糖及胰岛素(INs)对骨髓间充质干细胞(BMSC)向成骨细胞(OB)分化能力的影响,探讨葡萄糖及INs对骨代谢的作用机制.方法 采用体外细胞培养技术自大鼠股骨和胫骨中分离BMSC进行纯化,培养,然后在成骨培养基中诱导BMSC分化为OB,并用不同浓度的葡萄糖(5.6、25、50 mmol/L)及加或不加INs(0.6 μg/ml)干预诱导过程,光镜下观察茜素红染色分化后的OB钙结节的细胞比例.Realtime PCR测定OB的标记物碱性磷酸酶(ALP)、骨钙素(BGP)及转录因子Runx2 mRNA表达.结果 随着糖浓度升高,茜素红染色红色钙结节数目明显减少,PCR结果显示ALP,BGP及Runx2 mRNA表达也明显降低,差异有统计学意义(P<0.05).提示随着糖浓度增加成骨分化逐渐减少,在同等糖浓度下,加入INs干预组,茜素红染色阳性细胞计数明显增加,PCR结果ALP,BGP及Runx2 mRNA表达明显升高(P<0.01),提示INs可提高OB分化.结论 葡萄糖呈量效性地抑制BMSC向OB分化,这可能为糖尿病性骨质疏松形成的重要机制之一.INs可促进BMSC向OB分化,并可改善高糖对BMSC向OB分化的抑制作用.     

Ⅰ型胶原—整合素α2β1系统对成骨细胞生物学特性的调控

为进一步探讨Ⅰ型胶原及其受体系统是否为成骨细胞功能活动所必需,使用Ⅰ型胶原和整合素α２β１的一抗阻断Ⅰ型胶原－整合素α２β１系统,以细胞计数法比较成骨细胞的增殖能力,流式细胞仪分析成骨细胞的凋亡情况,ＲＴ－ＰＣＲ技术研究Ⅰ型胶原、整合素α２β１及骨钙素的ｍＲＮＡ表达情况。结果发现阻断Ⅰ型胶原－整合素α２β１系统后,成骨细胞的增殖能力减弱,凋亡率增高,Ⅰ型胶原、整合素α２β１及骨钙素的ｍＲＮＡ表达减     

基因沉默组织型转谷氨酰胺酶抑制SaOS-2细胞成骨分化

目的 研究组织型转谷氨酰胺酶（tissue transglutaminase, TG2）是否参与人SaOS-2细胞系成骨分化过程。方法 使用携带短发夹RNA（short hairpin RNA, shRNA）的慢病毒转染SaOS-2细胞以敲减TG2表达，以SaOS-2细胞及转染了含阴性对照shRNA病毒的SaOS-2作为对照组，分别进行体外成骨诱导培养，并进行以下检测：1）诱导14 d后各组矿化情况（茜素红染色），2）诱导4 d、7 d后碱性磷酸酶活性及I型胶原、骨钙素、骨形态发生蛋白-2（BMP-2）的mRNA表达，并与诱导前的表达水平相比较。结果 SaOS-2细胞组及转染阴性对照shRNA组在体外成骨诱导过程中I型胶原、骨钙素、BMP-2的mRNA表达和ALP活性逐渐增加，14 d时形成明显矿化结节，而TG2敲减后的SaOS-2细胞在诱导14 d时矿化水平显著低于对照组，诱导7 d时ALP活性及I型胶原、骨钙素、BMP-2的mRNA表达水平显著低于对照组。结论 组织型转谷氨酰胺酶参与SaOS-2细胞体外成骨分化及矿化。     

New markers of bone and collagen turnover in children and adults with growth hormone deficiency.

Serum bone Gla protein (BGP), a marker of osteoblastic function, serum carboxyterminal cross-linked telopeptide of type I collagen (ICTP), a marker of bone resorption, and serum aminoterminal propeptide of type III procollagen (PIIINP) levels, an index of collagen synthesis, were determined in seven children and eight adults with congenital growth hormone deficiency (GHD). In children with GHD, serum BGP (mean +/- s.e.: 12.9 +/- 0.7 ng/ml), ICTP (8.3 +/- 1.3 ng/ml) and PIINP (3.5 +/- 0.5 ng/ml) levels were significantly lower (P < 0.001) than those recorded in normal children (BGP 18.9 +/- 0.8 ng/ml, ICTP 14.4 +/- 0.5 ng/ml and PIIINP 6.7 +/- 0.7 ng/ml). Total alkaline phosphatase (184.7 +/- 13.4 IU/l) and bone alkaline phosphatase (77.8 +/- 4.1 IU/l) levels were also significantly lower (P < 0.0001) than in controls (338.1 +/- 14.9 IU/l and 181.0 +/- 7.8 IU/l, respectively). Serum BGP, ICTP and PIIINP levels were not significantly correlated with height velocity values. In adults with GHD, mean BGP levels (3.8 +/- 0.3 ng/ml) were significantly lower (P < 0.0001) than those recorded in normals (5.4 +/- 0.1 ng/ml). On the contrary, serum ICTP levels were similar to those found in controls (patients: 4.7 +/- 0.8 ng/ml vs normals: 4.1 +/- 0.3 ng/ml), suggesting the presence of a normal resorption activity associated with a reduced osteoblastic function. This finding was also confirmed by the presence of reduced bone alkaline phosphatase levels (GHD: 44.9 +/- 6.9 IU/I vs controls: 58.3 +/- 2.0 IU/I; P<0.02), while the less specific total alkaline phosphatase levels (119.5 +/- 14.8 IU/I) were similar to those recorded in normal subjects (122.3 +/- 4.0 IU/I). Serum PIIINP levels (3.7 +/- 0.6 ng/ml) were similar to those recorded in normals (3.2 +/- 0.2 ng/ml), suggesting that in adulthood the collagen turnover is not negatively influenced by the chronic GHD. No significant correlations were found between BGP/ICTP/PIIINP and IGF-I levels. In conclusion, our data show that in children with GHD the lack of GH insulin-like growth factor-I (IGF-I) effects on bone and collagen turnover is associated with a significant reduction of bone turnover (low bone formation plus low bone resoRption) and collagen synthesis. On the contrary, adult GHD seems to exert less relevant effects on bone and collagen turnover, probably due to the fact that in adult life further hormones or local factors might partially counteract the negative consequences of chronic GH-IGF-I deficiency.     

Serum sclerostin levels associated with lumbar spine bone mineral density and bone turnover markers in patients with postmenopausal osteoporosis

Background Sclerostin, expressed exclusively by osteocytes, is a negative regulator of bone formation. To gain insights into the action of sclerostin in postmenopausal osteoporosis, we evaluated serum sclerostin levels in postmenopausal women and investigated its possible associations with bone turnover markers in patients with postmenopausal osteoporosis.

Methods We detected serum sclerostin, and measured lumbar spine bone mineral density in 650 Chinese postmenopausal women. We also assessed serum levels of β-isomerized C-terminal crosslinking of type I collagen, intact N-terminal propeptide of type I collagen, N-mid fragment of osteocalcin, 25-hydroxyvitamin D, and estradiol.

Results Serum sclerostin levels were lower in postmenopausal osteoporotic women compared with non-osteoporotic postmenopausal women ((38.79±7.43) vs. (52.86±6.69) pmol/L, P <0.001). Serum sclerostin was positively correlated with lumbar spine bone mineral density (r=0.391, P <0.001) and weakly negatively correlated with β-isomerized C-terminal crosslinking of type I collagen, intact N-terminal propeptide of type I collagen, N-mid fragment of osteocalcin (r= –0.225, P <0.001; r= –0.091, P=0.046; r= –0.108, P=0.018; respectively) in postmenopausal osteoporosis. There was no significant association of serum sclerostin with age, body mass index, 25-hydroxyvitamin D, and estradiol (r= –0.004, P=0.926; r=0.067, P=0.143; r=0.063, P=0.165; r= –0.045, P=0.324; respectively).

Conclusion Sclerostin may be involved in the pathogenesis of postmenopausal osteoporosis and may play a role in bone turnover.