软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究

Objective To explore the effects of co-culture of chondrocytes and bone mesenchymal stem cells (BMSCs) on constructing engineered cartilages, and confirm the most suitable ratio of chondrocytes to BMSCs. Methods Chondrocytes and BMSCs were isolated from articulars cartilages of rabbit (1 month old)cells (40 μl 4×107/ml) were seeded into a poly(lactic-co-glycolic acid) (PLGA) scaffold and cultured statically for 2 days. They were transferred into the cyclic pressures system, and cultured under cyclic pressures for 3 weeks. The engineered cartilages were harvested and examined by gross observation, histological staining, immunohistochemisty of collagen Ⅱ, the content of glyeosaminoglycans, GAGs, DNA and the percentage of collagen Ⅱ dyeing area. Results The engineered cartilages of the co-cultured groups grew bigger than those of the chondrocytes alone group, and their surfaces were smooth and glossy. The distributions of cartilaginous extracellular matrices in the co-cultured groups were more homogenous than those of the chondrocytes alone group.gen Ⅱ dyeing area of the co-cultured groups were higher than those of the chondrocytes alone group. The conConclusion Co-culture of chondrocytes and BMSCs could improve the quality of engineered cartilages. The     

不同持续时间的周期性压力对构建组织工程软骨的影响

Objective To determine the best duration for exerting the cyclic pressure under which the tissue-engineered cartilage is constructed. Methods Free chondrocytes isolated from rabbit articular cartilage were seeded into polylactic acid-co-glycolic acid(PLGA) scaffolds after expansion in vitro, and ran-domized into 4 groups. In Groups 1 to 3, chondrocytes were cultured under daily cyclic pressure (0 ～ 200 kPa, 0.1Hz) for 4 hours, 8 hours, 12 hours respectively; Group 4 was a control in which no pressure was exerted. In each group, after 2 weeks of culture, the tissue engineered cartilages were observed in vitro and assessed by his-tological staining of liE. Next, the content of DNA and the secretion of type Ⅱ collagen and GAG in cartilages were detected quantitatively. Results Under the daily cyclic pressure (0 ～200 kPa, 0.1 Hz), the scaf-fold-chondrocytes complex in the group of 8 hours got the largest volume, smooth, lucidus, and elastic surface, the most queuing chondrocytes, and the highest content of type Ⅱ collagen and GAG (P < 0.01). Conclusions Since chondrecytes are baro-senstive, the metabolism of chondrocytes can be affected by the time of cyclic pressure. Under the effect of 0 ～200 kPa, 0.1Hz, the daily cyclic pressure of 8 hours may be optimal for chondrocytes to multiply and synthesize extracellular matrixes such as type Ⅱ collagen and GAG.     

携带hIGF-Ⅰ基因的腺病毒载体构建

目的 探讨构建携带hIG-Ⅰ基因腺病毒载体的可行性.方法 从pcDNA 3.1-hIGF-Ⅰ质粒中克隆出hIGF-Ⅰ基因的编码序列,酶切、连接后,插入腺病毒系统中的穿梭载体pAdshuttle-CMV,获得重组质粒pAdshuttle-CMV-hIGF-Ⅰ,将其转化含有腺病毒骨架载体pAdeasy-1的大肠杆菌BJ5183,进行同源重组,获得重组子pAdeasy-1-IGF-Ⅰ.鉴定后,将pAdeasy-1-IGF-Ⅰ转染人胚肾细胞(Ad293细胞),获得含hIGF-Ⅰ基因的重组腺病毒(rAd-hIGF-Ⅰ).再鉴定后,Ad293细胞反复感染冻融扩增腺病毒,50%组织培养感染量法(TCID50)检测病毒滴度.将病毒颗粒感染绿猴肾成纤维细胞(COS-7细胞),荧光显微镜观测绿色荧光蛋白(GFP)的表达,RT-PCR分析hIGF-Ⅰ基因在mRNA水平的表达,Western blot分析hIGF-Ⅰ在蛋白水平的表达.结果 成功构建出含有hIGF-Ⅰ基因的腺病毒载体rAd-hIGF-Ⅰ,转染COS-7细胞后发现绿色荧光蛋白表达,同时RT-PCR扩增出318 bp的目的 条带,Western blot得到7.6kDa大小的同的蛋白,证明了腺病毒载体rAd-hIGF-Ⅰ能成功转染COS-7细胞,hIGF-Ⅰ基因能在其中成功表达.结论 成功构建出含有hIGF-Ⅰ基因的腺病毒载体rAd-hIGF-Ⅰ,并且证实其能对COS-7细胞进行有效转染,为组织工程软骨的进一步改良提供了高效的基因载体.     

腺病毒介导的hIGF-Ⅰ基因转染对构建组织工程软骨的影响

目的 探讨腺病毒介导的人胰岛素样生长因子I(hIGF-Ⅰ)基因转染对构建组织工程软骨的影响.方法 分离新两兰大白兔关节软骨细胞,培养到第一代,用荧光标记的Ad/hrGFPhlGF-Ⅰ转染,然后接种在PLGA支架上(转染组);未转染的软骨细胞接种在PLGA支架上(对照组),在体外培养2周.利用荧光显微镜、Western blot、RT-PCR、免疫组织化学等方法鉴定hIGF-Ⅰ基因的有效转染,并观察其对构建的组织工=程软骨细胞外基质的影响.结果 Ad/hrGFP-hlGF-Ⅰ能对兔关节软骨细胞进行有效转染,RT-PCR显示构建的组织工程软骨Ⅰ、Ⅱ型胶原、聚合素的表达高于对照组;定昔检测显示胶原和GAG的含量高于对照组(P＜0.01).结论 腺病毒介导的hIGFⅠ基因转染能成功转染兔关节软骨细胞,用它构建组织工程软骨能显著促进软骨细胞外基质的合成.     

ERK2的CC区域在HeLa细胞中的定位作用

目的 探索细胞外调节蛋白激酶2(ERK2)的coiled-coil(CA3)区域在表皮生长因子(EGF)所诱导的HeLa细胞中的定位作用.方法 用定点突变试剂盒删除ERK2的盘绕结构[ERK2(del CC)],免疫共沉淀的方法检测ERK2(del CC)在EGF刺激的条件下和G蛋白耦联受体激酶相互作用分子1(GIT1)的相互关系,免疫荧光染色检测ERK2(del CC)在细胞内的位置以及和GIT1的相互关系.结果 成功删除ERK2的CA3区域;免疫共沉淀结果显示,删除ERK2的CC区域显著抑制了ERK2和GIT1在EGF刺激条件下的相互结合;免疫荧光染色结果显示,删除ERK2的CC区域显著抑制了磷酸化的ERK2在细胞局部黏附内的定位.结论 在EGF的刺激条件下,增加了ERK2和GIT1的相互结合,ERK2的CC区域是与GIT1相互结合的重要区域.删除ERK2的CC区域不仅显著抑制了ERK2和GIT1的相互结合,而且明显降低ERK2在细胞局部黏附内的定位.     

软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究

Objective To explore the effects of co-culture of chondrocytes and bone mesenchymal stem cells (BMSCs) on constructing engineered cartilages, and confirm the most suitable ratio of chondrocytes to BMSCs. Methods Chondrocytes and BMSCs were isolated from articulars cartilages of rabbit (1 month old)cells (40 μl 4×107/ml) were seeded into a poly(lactic-co-glycolic acid) (PLGA) scaffold and cultured statically for 2 days. They were transferred into the cyclic pressures system, and cultured under cyclic pressures for 3 weeks. The engineered cartilages were harvested and examined by gross observation, histological staining, immunohistochemisty of collagen Ⅱ, the content of glyeosaminoglycans, GAGs, DNA and the percentage of collagen Ⅱ dyeing area. Results The engineered cartilages of the co-cultured groups grew bigger than those of the chondrocytes alone group, and their surfaces were smooth and glossy. The distributions of cartilaginous extracellular matrices in the co-cultured groups were more homogenous than those of the chondrocytes alone group.gen Ⅱ dyeing area of the co-cultured groups were higher than those of the chondrocytes alone group. The conConclusion Co-culture of chondrocytes and BMSCs could improve the quality of engineered cartilages. The     

髓内钉固定对肺血流动力学影响的实验研究

目的 探讨骨折后内钉固定及手术时机的选择对肺血流动力学的影响。方法 选择成年雌性杂种犬２４只,随机分成３组,每组８只,模拟股骨干骨折。第一组于骨折后６ｈ做髓内钉固定,为６ｈ组;第二组于骨折后４８ｈ做髓内钉固定,为４８ｈ组;另一组骨折后不做髓内钉固定,为对照组。分别测定各组不同时间的髓内压、肺动脉压,并进行血气分析。然后处死动物,取肺组织做病理学检查,应用ＩＡ－３００１影像分析系统测定每个视野中脂肪     

二磷酸盐对骨水泥微粒刺激单核细胞分泌肿瘤坏死因子的影响

目的：探讨二磷酸盐类药物－阿可达（帕米磷酸钠）对人工关节无菌性松动的影响。方法：随机选择准备行人工关节置换术患者17例。采集外周血,分离单核细胞,每例标本分成5份：A组：仅单核细胞,为对照组;B组：单核细胞＋骨水泥微粒;C组：单核细胞＋骨水泥微粒＋阿可达（5μg/ml);D组：单核细胞＋骨水泥微粒＋阿可达（10μg/ml);E组：单核细胞＋骨水泥微粒＋阿可达（20μg/ml)。培养48h后,放免法测定细胞上清中肿瘤坏死因子（TNF）含量。结果：单核细胞＋骨水泥微粒组的TNF含量明显高于对照组（P＜0．01）;单核细胞＋骨水泥微粒＋阿可达组的TNF含量明显低于单核细胞＋骨水泥微粒组（P＜0．01）。而不同浓度阿可达组之间的差异无统计学意义（P＞0．05）。结论：骨水尼微粒可刺激单核细胞分泌ＴＮＦ,而阿可达能有效地抑制单核细胞的这种分泌。二磷酸盐能防治人工关节置换术后由ＴＮＦ等溶骨性因子引起的人工关节周围的骨溶解,由此二磷酸盐有望成为防治人工关节松动的有效药物。     

不同持续时间的周期性压力对构建组织工程软骨的影响

Objective To determine the best duration for exerting the cyclic pressure under which the tissue-engineered cartilage is constructed. Methods Free chondrocytes isolated from rabbit articular cartilage were seeded into polylactic acid-co-glycolic acid(PLGA) scaffolds after expansion in vitro, and ran-domized into 4 groups. In Groups 1 to 3, chondrocytes were cultured under daily cyclic pressure (0 ～ 200 kPa, 0.1Hz) for 4 hours, 8 hours, 12 hours respectively; Group 4 was a control in which no pressure was exerted. In each group, after 2 weeks of culture, the tissue engineered cartilages were observed in vitro and assessed by his-tological staining of liE. Next, the content of DNA and the secretion of type Ⅱ collagen and GAG in cartilages were detected quantitatively. Results Under the daily cyclic pressure (0 ～200 kPa, 0.1 Hz), the scaf-fold-chondrocytes complex in the group of 8 hours got the largest volume, smooth, lucidus, and elastic surface, the most queuing chondrocytes, and the highest content of type Ⅱ collagen and GAG (P < 0.01). Conclusions Since chondrecytes are baro-senstive, the metabolism of chondrocytes can be affected by the time of cyclic pressure. Under the effect of 0 ～200 kPa, 0.1Hz, the daily cyclic pressure of 8 hours may be optimal for chondrocytes to multiply and synthesize extracellular matrixes such as type Ⅱ collagen and GAG.     

肿瘤坏死因子与人工关节无菌性松动关系的探讨：附7例报告

目的　探讨人工关节无菌性松动的病因。方法　选择 7例松动人工髋关节病例 ,翻修手术时取松动关节周围的界膜组织 ;选择 1 0例骨折内固定患者 ,拆除内固定时取内固定物周围瘢痕组织。标本作组织学检查 ,并作肿瘤坏死因子(TNF)测定。结果　松动人工髋关节周围的界膜组织中含大量组织细胞和纤维母细胞 ;免疫组化染色显示 ,组织细胞标记抗体、溶菌酶和 α1 -抗胰蛋白酶染色阳性。而骨折内固定物周围的瘢痕组织主要为纤维成分。松动人工髋关节周围界膜组织中的TNF浓度明显高于骨折内固定物周围的瘢痕组织 (P<0 .0 1 )。结论　在生物性因素方面 ,磨损微粒是松动发生的关键性因素 ,它可刺激组织细胞分泌 TNF等溶骨性因子 ,这些溶骨性因子直接或间接地激活破骨细胞 ,从而引起人工关节周围骨吸收、骨溶解 ,最终导致人工关节无菌性松动。