转化生长因子-β1和胰岛素对人鼻中隔软骨细胞增殖和分化影响的研究

目的 了解转化生长因子-β1(transforminggrowth factor-beta 1,TGF-β1)和胰岛素对人鼻中隔软骨细胞增殖和分化的影响。方法 体外培养人鼻中隔软骨细胞,采用四甲基偶氮唑蓝(MTT)代谢和35S-Na2SO4掺入的检测比较不同浓度TGF-β1和胰岛素对人鼻中隔软骨细胞的增殖以及软骨基质蛋白多糖合成的影响,观察TGF-β1和胰岛素对软骨细胞表型的影响。结果在15％血清的培养条件下,TGF-β1和胰岛素均能显著促进软骨细胞增殖,且在各自一定的浓度范围内呈量效关系,联合作用效果累加;TGF-β1、TGF-β1和胰岛素联合作用促使软骨基质蛋白多糖的合成量明显提高;TGF-β1使传代软骨细胞去分化提前。结论一定浓度的TGF-β1、胰岛素和TGF-β1 胰岛素对体外培养的人鼻中隔软骨细胞具有显著的促增殖作用。     

Characterization of Demineralized Bone Matrix-Induced Osteogenesis in Rat Calvarial Bone Defects: III. Gene and Protein Expression

Our previous studies of rat cranial defect repairs after the implantation of demineralized bone matrix (DBM) have demonstrated that healing occurs initially and principally by the direct induction and proliferation of osteoblasts derived principally from resident mesenchymal stem cells of the dura, and to a lesser extent by resident mesenchymal stem cells of the connective tissues beneath the skin flap. A small amount of cartilage is also synthesized after the direct process of ossification occurs. To further confirm the molecular phenotypes of the repair cells in rat cranial defects, the present study evaluated mRNA expression and synthesis of collagens I, II, and X and osteocalcin in the DBM-induced repair tissue by Northern blot analyses, autoradiography after in vivo ³H-proline labeling of collagen, and immunohistochemistry. The results demonstrated that osteocalcin mRNA appeared in small amounts by day 4 and continued to increase over the experimental period. Much lesser quantities of collagen types II and X mRNAs appeared by day 6 and day 8, respectively. Collagen type I mRNA was present at all times examined but its expression significantly increased by day 5. Autoradiographic and immunohistochemical studies showed that type II collagen was not detected whereas type I collagen was synthesized on days 3–5. The data provide definitive molecular evidence confirming that the initial and by far the major pathway of cranial defects repair induced by implantation of DBM is by the direct induction of resident mesenchymal stem cells to osteoblasts and the direct formation of bone, which is spatially and temporarily distinct from the later formation of cartilage. Received: 30 November 1999 / Accepted: 21 March 2000     

自制NECM修复兔耳廓软骨缺损的试验研究

目的本研究的目的在于评价自制同种异体天然细胞外基质(Nature Extracellular Matrix,NECM)在修复兔耳廓软骨缺损中的价值,从而在体外研究的基础上进一步证实我们采用的去污剂-酶四步法制作的NECM在组织工程学中的可行性.方法选用10只新西兰大白兔,于其双耳廓制造1.0×.0cm大小的软骨缺损后,分别作以下研究:①灰兔NECM修复兔耳廓缺损.②灰兔NECM复合软骨膜修复兔耳廓缺损;③不植任何材料的空白对照.术后4、6、12、24周取材并行组织学检查.结果NECM植入后4、6周有轻重不一的炎症反应,第12周炎症反应消失,NECM周边无包裹现象.第12周时NECM复合软骨膜组有软骨膜细胞长入NECM,此时的软骨膜细胞多处于增殖期,24周时软骨膜细胞继续增殖分化细胞逐渐长入NECM深部,且部分软骨膜细胞发育成熟.单纯NECM修复组,第6周出现纤维结缔组织长入NECM,12、24周纤维结缔组织逐渐长入NECM深部,并逐渐取代NECM.结论①NECM可诱导软骨膜细胞增殖分化生长,修复软骨缺损:②NECM具有较好的组织亲和性和相容性;③软骨膜细胞可以作为NECM再生软骨的种子细胞的来源;④软骨膜可以阻止纤维结缔组织侵入修复区NECM.     

藻酸包埋异体关节软骨细胞修复兔膝关节软骨缺损

目的 探讨异体关节软骨细胞作为软骨种子细胞修复关节软骨缺损的可行性。方法 无菌取成年新西兰大白兔四肢关节软骨，酶消化法分离细胞，条件培养基体外培养扩增，藻酸钙凝胶包埋培养1周，植入兔膝关节股骨髁间软骨缺损，对照组缺损旷置。术后3、6个月分别取材，观察缺损修复情况；切片特染和免疫组化观察修复组织病理，并Wakitnni评分评估修复质量。结果 7／8缺损为成熟透明软骨组织修复，1／8为纤维软骨修复；Wakitani平均评1．75分；空白对照组评7．65分。实验组3个月组标本未见淋巴细胞或白细胞浸润，6个月组标本未见明显退变；所有标本均未见藻酸残留。结论 用藻酸包埋异体关节软骨细胞修复兔膝关节软骨缺损的方法是可行的，异体关节软骨细胞在适当的处理后可成为关节软骨组织工程种子细胞。     

Developmental defects in zebrafish for classification of EGF pathway inhibitors

One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway.     

基于三维打印的钙化层重建生物活性支架制备及其性能研究

目的初步构建基于软骨钙化层损伤重建的有机—无机复合组织工程支架,探究掺镁硅灰石含量与支架抗压性能之间的关系。方法利用质量分数分别为1%、3%、5%的高生物活性钙镁硅酸盐超细颗粒复合Ⅰ型胶原—透明质酸钠进行三维打印,经海藻酸钠—氯化钙气雾交联成型,电镜下观察表面孔隙、孔径、无机相分布,万能材料试验机测试抗压性能,并计算支架孔隙率。结果支架表面平均孔径(212.3±34.2)μm,平均孔隙率(48.3±5.9)%,不同质量分数的高生物活性钙镁硅酸盐超细颗粒复合Ⅰ型胶原—透明质酸钠支架压缩模量差异无统计学意义( P>0.05),平均压缩模量(7.2±1.2)MPa,介于软骨和软骨下骨之间。 结论利用三维打印技术成功构建出多孔钙化层仿生重建支架,可为今后研制多层次复合支架治疗骨—软骨损伤奠定基础。