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In conclusion ,the results of this study showed that the inducing agent HMBA could effectively inhibit the proliferation activity of the human ostosarcoma MG-63 cells,cause the arrest of G0/G1 phase, reverse the malignant phenotype characters,upregulate expression of the osteoblat-like phenotype markers including collagen,osteocalcin,osteonectin , facilitated the formation of bone nodule .

本研究的结果表明HMBA能够有效抑制人成骨肉瘤MG-63细胞增殖活动,改变肿瘤增殖核抗原表达,将细胞周期阻滞在G0/G1期,逆转人成骨肉瘤细胞恶性表型特征,在促进终末分化标志物Ⅰ型胶原纤维蛋白、骨钙蛋白、骨粘素的表达同时,促进骨结节生成,从而说明HMBA能够诱导人成骨肉瘤MG-63细胞终末分化,并且Rg1、BPO-L、Rg1+BPO-L以及Rg1+HMBA等均具有终末诱导分化能力。

Effects of Adv-hBMP2 gene modify on the bone formation of tissue-engineered bone substitute. BMSCs of canine transferred by Adv-BMP2 gene expressed ALP in vitro and induced ectopic bone formation in nude mice. BMSCs adhered on the surface of FDB after 4hrs, expanded in 24hrs and proliferated in polylayer in 4d. FDB, combined with BMSCs transferred by Adv-BMP2 gene, induced bone formation in the subcutis of nude mice in 6 weeks.

结果:1.BMP-2基因修饰对组织工程化骨成骨的影响 BMSCs经Adv-BMP2基因转染后ALP染色强阳性、并在裸鼠肌肉形成异位骨组织;转基因的BMSCs与冻干骨复合后24小时即完全贴附生长,4天后复层生长并分泌大量胶原,植入裸鼠背部皮下6周后可见大量成骨,很少吸收,而单纯冻干骨或冻干骨复合BMSCs基本不成骨。2。

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。

Methods The embryo calvarial periosteum tissue was taken and, human osteoblasts were obtained by enzyme-assimilating methods. The morphological change, growth feature and osteogentic capability of osteoblasts were observel during culture in vitro, drew the growth curve was graphed and the cells was identified by alkaline phosphatase dye. At the same time, the morphology and bioactivity of 3 to 5 th-generation osbeoblasts and anabiotic cells were studied comparatively.

取人胚胎颅骨骨膜,采用酶消化法获取成骨细胞体外培养并传代,观察细胞形态,生物特性,绘制生长曲线,并经碱性磷酸酶染色鉴定成骨细胞以及比较冻存前3~5代与冻存后成骨细胞的特点。

Identification of these cells: Over 90% cells isolated from human fetal calvaria were stained positively for both alkaline phosphatase and Alizarin red.

采用酶消化后植块的改良植块培养法可在较短时间内获得大量成骨细胞,这些细胞具有典型的成骨细胞形态和功能,是一种较好的分离培养人胚成骨细胞的方法。

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。

The procedure could provide firm internal fixation,improve activity of osteoblasts in fractural sites.accelerate fracture healing.

上述方法可提供早期坚强的内固定,补充成骨基质和成骨前质细胞,改善成骨能力。

The biomechanical tests showed that two kinds of artificial bones had not significant difference on compressive strength and Young\'s modulus(P>0.05),while the flexural strength of nano-nacre artificial bone was less than the control group(P<0.05).3.The results of CCK-8 showed that the difference were not significant in each group,the proliferation of osteoblast reached the peak at the 5th day;7 days after being co-cultured,the total protein content of study group was higher than control group and blank group(P<0.05),while the difference between control group and blank group was not significantP>0.05The difference of alkaline phosphatase activities among three groups was not significant(P>0.05The SEM view showed that osteoblast attached and grew well in two kinds of artificial bone.4.X-ray photography showed that two kinds of powder started to degrade in 2 weeks;this phenomenon became more appear in 4 weeks,nano-nacre powder degraded faster than micron-nacre powder,while the hole shadow was easy to be found;in 8 weeks,all the femoral holes recovered and returned to normal bone mineral density in all groups.Analysis of tetracycline fluorescent double marks in the hard tissue grinding slices indicated that new bone grew fastest around the bone defect area in study group,while most slowly in blank groupP<0.05 SEM(scanning electron microscope observation showed that nano-nacre powder degraded more quickly.The same result can be found through the demineralized sections morphometric analysis,and both of the composite artificial bones made from those two kinds of nacre powder had the good connection with the adjacent tissue in rats body without apparent inflammatory response.5.X-ray photography showed that rabbit\'s bone defects healed faster in study group since NNAB implanted than in control group since MNAB implanted.At 24 weeks after operation,bone density in radial defects had nearly accessed to the normal area,while lower in control group,and turned up nonunion in blank group;The checking of BMD showed that results in study group were higher than those in control group at 8,16 and 24 week(P<0.05), and the difference between the BMD values in study group at 24 week and those in blank group was not significant(P>0.05).The gross specimens showed satisfactory histocompatibility both in study group and in control group,with bone tissue growing from two sides into the center of implanted materials; Normal slices in HE stain and hard tissue grinding slices in Stevenel\'s blue/Van Geison\'s picro-fuchsin stain showed that the bone growth tendency was better in study group than that in control group,and the medullary cavity had been penetrated to the implanted materials in study group at 24 week;Analysis of tetracycline fluorescent double marks in the hard tissue grinding slices indicated that new bone in both groups grew fastest 8 weeks after surgery,while slow down at 16 week.

纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨分别与成骨细胞共培养后,其各时间点CCK-8法检测值与空白对照无显著差异(P>0.05),成骨细胞均在第5天达到增殖高峰期;培养7天后,实验组细胞蛋白含量高于对照组及空白组(P<0.05),后两者之间则无显著差异P>0.05碱性磷酸酶活性在三组间均无显著差异(P>0.05电镜下可见成骨细胞在两种人工骨上都有良好生长贴附能力。4.X-ray显示两种粉体在大鼠股骨骨洞植入第2周时都开始出现了降解,第4周时更为明显,纳米珍珠层粉较之微米珍珠层粉降解更快,而空白对照组骨洞阴影仍可见,至8周时,则所有组骨洞均己闭合修复,X-ray下已不可见原钻孔痕迹,恢复正常骨质密度;硬组织磨片四环素荧光双标记结果显示纳米珍珠层粉植入组较其余两组在骨缺损区周围新骨生长速度更快,空白组速度最慢P<0.05电镜观察及常规脱钙切片亦可见到纳米粉体降解较快;由以上两种原材料制得的纳米珍珠层/消旋聚乳酸复合人工骨与微米珍珠层/消旋聚乳酸复合人工骨在大鼠体内均与周围组织结合良好,无明显炎症反应。5.X-ray显示纳米珍珠层/消旋聚乳酸复合人工骨植入兔桡骨缺损区后其骨愈合速度较对照组微米珍珠层/消旋聚乳酸复合人工骨植入的快,至植入术后24周,实验组骨缺损区接近正常骨密度,对照组骨缺损区密度较低,空白组则呈现骨不连状态;骨密度测量结果显示术后8周、16周、24周实验组的骨密度值高于对照组(P<0.05,24周实验组的骨密度值与术前所测得的正常值无显著性差异P>0.05动物取材大体所见均显示组织相容性良好,骨组织逐渐由植入材料两端向中央生长;常规切片HE染色及硬组织磨片Stevenel\'s blue/Van Geison\'s picro-fuchsin联合染色均可见实验组骨缺损区长势优于对照组,至术后24周,实验组骨髓腔与材料已呈相交通状;硬组织磨片荧光显微镜下观察,两组材料在术后8周处于骨生长最快速时期,16周时速度开始减慢,术后4、8、16周时实验组的新骨生长速度均较对照组的快

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。

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周时钻孔区内充满新生骨小梁结构,8周时钻孔区内骨小梁成熟,小梁有骨髓组织填充。[结论]骨髓基质干细胞对兔股骨头缺血性坏死有良好的修复作用。

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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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