成骨细胞的

Objective: To study osteogenic capability of rabbit bone marrow stromal cells and biological characteristical identification in vitro in order to explore the appropriate methods of osteoblast culture in vitro and to select an idea source of seed cells for bone tissue engineer.

目的：研究兔骨髓基质细胞在体外培养条件下的成骨能力及生物学特性，探讨简便易行的成骨细胞体外培养方法，为组织工程选择理想的种子细胞来源。方法：取新西兰白兔骨髓液经离心后得骨髓单个核细胞，以1× 106／ml的细胞浓度进行培养，经传代培养，选择条件培养液培养(1640完全培养液中加入地塞米松10-8Smol／l，β-甘油磷酸钠10mmol／l，VitC 50μg／ml)作为骨髓基质细胞增殖和成骨的调节因子，通过倒置显微镜、HE染色、扫描电镜、碱磷酸酶(alkaline phosphatase，ALP)测定、Ⅰ型胶原免疫组化染色和四环素荧光钙染色等手段对获得的细胞进行生物学特性研究。

Methods We isolated and cultured the MSCs, then induced MSCs into osteoblast and checked the osteoblast by RT-PCR; the conditioned medium in different concentration was prepared containing diphosphonate or assemble flavone of drynaria rhizome; with the conditioned medium the osteoblast was cultured; then the growth curve was analyzed and the cytogene (collagenase col Ⅰ) col Ⅰ and ALP expression was detected.

使用诱导方法作用于骨髓基质干细胞，使之转化为成骨细胞；用RT-PCR检验成骨细胞；不同浓度的二磷酸盐或骨碎补总黄酮配制成条件培养基，作用于成骨细胞，并分析生长曲线，分析细胞基因碱性磷酸酶和胶原酶Ⅰ(collagenase col Ⅰ)表达情况。

In previous studies, Cyclooxygenase-2(COX-2) is believed to be linked with inflammations and tumors. But in recent reports, COX-2 is found to play important roles in osteoblastic mechanotransduction and in bone remodification and resorptions in some ways. It was found that mechanical stretches, such as hydrostatic forces and pulsating fluid flow, could increase the expressions of COX-2 mRNA and then prostaglandins, and activate many signal pathways in osteoblasts.

环氧合酶-2(cyclooxygenase-2，COX-2)以往被认为与炎症、肿瘤等有密切关系，近年研究发现它在骨骼重建、骨质吸收以及成骨细胞力学信号转导中发挥着重要作用，主要表现为机械牵拉、流体力、节律性液体流(pulsating fluid flow，PFF)等机械刺激均能引起成骨细胞COX-2高水平表达，进而引起前列腺素(prostaglandins，PGs)量的增加，激活成骨细胞内多条信号传导通路。

Methods: Human osteoblasts were cultured on flexible-bottomed plates and subjected to 8% elongation by stress unit at 6 cycles/min (i.e. 5-s elongation and 5-s relaxation) for 24 hours in the experimental groups. Control group did not exert any stress.

通过体外细胞加载系统对培养的人成骨细胞施加8%形变率、6周／min的周期性牵张力24 h，应用基因表达谱芯片技术对人成骨细胞中mRNA表达水平进行对比杂交分析，检测周期性牵张力加载组成骨细胞基因表达谱的变化。

In SMC group, relative stable of the both ends of fracture reduce unnessary repeated stimulation of injury, and at same time, axio-stress advantageous for preosteoblast and mesenchymal cell transmit to osteoblast and chondroblast. Material and design superiority of SMC form unique internal mechanical environment, and which benefit for bone healing. The study took deep investigation of SMC on fracture healing.

上述结果证实：SMC内固定可对骨折端施加主动的、持续的轴向应力刺激，形成沿长骨骨干轴向的成骨导向力值，配合环抱翼的把持作用，形成了区别于CP、IN的独特的稳定、有效的机械内环境，骨折端的相对稳定减少了不必要的损伤再刺激，同时持续轴向应力有利于成骨祖细胞、间充质细胞向成骨细胞和成软骨细胞转化、促进了成骨诱导过程的发生与发展，加快了骨折愈合的修复与重建。

Results We can induce MSCs into osteoblasts which can express gene col Ⅰ and ALP. Through analysis growth curve, we demostrateed the diphosphonate or assemble flavone of drynaria rhizome with 10^(-4)~10^(-6)mmolL^(-1) had the inhibition on the osteomast; it with 10^(-8)mmolL^(-1) had a promotion on the osteoblast; it with 10^(-10)~10^(-12)mmolL^(-1) had a restraint on osteoblast. Conclusion Through gene analysis, we found the diphosphonate or assemble flavone of drynaria rhizome can have the promotion on the osteoblast.

结果 骨髓基质细胞经过4周诱导可转化为表达colⅠ和ALP的成骨细胞，经过生长曲线分析，二磷酸盐和骨碎补总黄酮10^(-4)~10^(-6)mmolL^(-1)时，对诱导后的成骨细胞有抑制作用；10^(-8)molL^(-1)有促进作用；10^(-10)~10^(-12)mmolL^(-1)不明显，其中10^(-8)mmolL^(-1)作用最强；基因分析后，发现二磷酸盐和骨碎补总黄酮促进成骨细胞表达colⅠ和ALP，且两者合用效果大于单独用药。

Microscope examination:in group A,at 2 weeks,a few inflamatory cells appeared in the holes with many osteoblasts and ostoid appeared around the drilled holes.At 8 weeks,marrow formed in the drilled holes in group B,at 2 weeks,there were a large amount of osteoblasts in the drilled holes and some ostoid formed around thc drilled holes.At 4 weeks,the drilled holes were full of trabeculae.At 8 weeks,the trabeculae matured with the marrow appeared in the intertrabecular space.

组织学结果：2周时A组钻孔区出现少许炎症细胞，边缘出现较多成骨细胞并有骨组织形成，至8周时，钻孔区内形成骨髓组织，只在边缘形成骨小梁结构。2周时B组钻孔区有大量的成骨细胞，边缘有较多骨组织形成，4周时钻孔区内充满重生骨小梁结构，8周时钻孔区内骨小梁成熟，小梁有骨髓组织填充。

Immunohistochemistrical section: observe the VEGF protein expression in different cells, the positive ratio detected by a morphometrical analysis system was used as the amount of the VEGF protein expression.

免疫组化染色切片：观察不同给药时间股骨头内VEGF的表达情况；高倍视野下（400倍）沿骨小梁表面计数50个成骨细胞中的阳性细胞数，VEGF成骨细胞阳性率=阳性成骨细胞数/50×100％，3次取平均值；计数每个高倍视野软骨细胞中的阳性细胞数，VEGF软骨细胞阳性率=阳性软骨细胞数/软骨细胞总数×100％，5次取平均值。

The tibiae of the rats were kept for the samples of decalcification, IGF-1 in bone tissue was measured by immunohistochemistry and Enzyme linked-immuno-sorbent assay was used to detect the TGF- P 1, IL-6, IL-1 P and TNF- a in serum.

结果 成骨细胞呈线状排列于骨小梁的边缘，细胞因子IGF-1表达于成骨细胞胞浆，骨细胞、软骨细胞及骨基质中也存在IGF-1。不同部位的成骨细胞IGF-1的表达量并不一致，卵巢切除及PTH注射对不同部位成骨细胞IGF-1表达的影响也不一样。

The change of cytoskeletal architecture of osteoblasts with Phalloidin strain actin filaments was visualized by confocal microscopy The results showed: 1 the secretion of estrogen were depressed and bone mass were decressed and biomechanic properties of bone were impaired in osteoporotic rats at 3 months after ovariectomy .

在1 000μstrain力学刺激下，成骨细胞integrinα2，β1，β3的表达量出现上调；在4 000μstrain力学刺激下，成骨细胞integrinα2，β1，β3的表达量明显上调，成骨细胞合成骨基质的能力与增殖活性增加。

It has been put forward that there exists single Ball point and double Ball points on the symmetrical connecting-rod curves of equilateral mechanisms.

从鲍尔点的形成原理出发，分析对称连杆曲线上鲍尔点的产生条件，提出等边机构的对称连杆曲线上有单鲍尔点和双鲍尔点。

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