新型多孔β-磷酸三钙作为骨组织工程支架材料的评价

背景: 新型多孔β-磷酸三钙是采用适当配方和独特工艺制成,其气孔率(75±10)%,球型孔>80%,微孔<20%,孔与孔的沟通率达100%,力学强度>2MPa.目的: 评价新型多孔β-磷酸三钙作为骨组织工程支架材料的应用效果.设计、时间及地点: 对比观察实验,于200-07/2006-03在南方医科大学组织工程实验室完成.材料: 6月龄新西兰大白兔12只,制备左侧桡骨1.5cm大段骨与骨膜缺损.多孔β-磷酸三钙为法国bio-lu公司产品.方法: 将兔骨髓间充质干细胞诱导为成骨细胞,与β-磷酸三钙复合培养,倒置相差显微镜和扫描电镜下观察细胞的生长情况,MTT法测定细胞增殖情况判断其细胞相容性.通过不同含量的β-磷酸三钙浸提液对细胞增殖的影响检验其细胞毒性.主要观察指标: 对β-磷酸三钙进行细胞相容性与细胞毒性检测.术后2,6,12周分别取材进行组织学检查,放射性核素骨扫描测定,X射线片检查,观察骨缺损部位的修复情况.结果: 新型多孔β-磷酸三钙细胞黏附性好,细胞毒性为0级.组织学、影像学和放射性核素骨扫描显示能够修复兔桡骨的大段骨缺损,且体内降解速率与骨的形成速率一致.结论: 新型多孔β-磷酸三钙是一种细胞相容性好的骨组织工程支架材料,修复兔桡骨大段骨缺损的效果良好.     

感觉、运动神经匀浆对兔成骨细胞增殖与成骨功能的影响

目的 研究兔周同感觉、运动神经匀浆对兔骨髓间充质干细胞来源的成骨细胞增殖与成骨能力的影响.探讨周围神经影响成骨的可能原因.方法 提取大隐神经、坐骨神经肌支分别作为感觉、运动神经组织,制备匀浆.体外培养兔骨髓间充质干细胞,常规成骨诱导后鉴定备用.以含感觉、运动神经匀浆的诱导培养基、成骨诱导培养基、无地米诱导培养基及对照培养基作用于干细胞来源的成骨细胞,观察其增殖、分化及矿化情况.结果 感觉神经组较其他各组显著促进成骨细胞增殖(P<0.01),成骨诱导组及对照组抑制细胞增殖(P≤0.001);感觉神经匀浆显著促进成骨细胞总蛋白及碱性磷酸酶(ALP)的合成(P<0.05),成骨诱导组及对照组ALP蛋白比活性显著高于运动神经组及无地米组(P<0.05);感觉神经组、成骨诱导组及对照组茜素红结合量显著高于无地米组及运动神经组(P<0.001),前三组之间无显著性差异.结论 感觉神经成分能显著促进成骨细胞的增殖、分化与矿化,运动神经成分未见促成骨作用.     

磁共振灌注成像监测组织工程骨血管化的研究

目的探讨磁共振灌注成像在组织工程骨血管化监测中的应用价值。方法成年恒河猴体内制备胫骨20mm的骨缺损，随机分为5组，A组：／β-磷酸三钙（β-TCP）＋骨髓基质干细胞（BMSCs）＋血管束；B组：β-TCP＋血管束；C组：β-TCP＋BMSCs；D组：β-TCP；E组：空白对照。术后4、8、12周行磁共振灌注成像检查并计算信号强度-时间（SI—T）曲线的最大线性斜率（SSmax）和基线值（SIbascline），拍摄恒河猴胫骨x线片并计算其透光度。结果术后4、8、12周A组的SSk值最高，术后8周与4周相比SSmax有较大幅度的提高（P〈0．01）。术后12周A组5个样本的SSmax与X线片透光度呈负相关（r=-1．0，P〈0．01）。结论SI-T曲线的SSmax能准确反映组织工程骨的血管化情况，磁共振灌注成像检查具有无创、无辐射、高灵敏度和定量分析的优点。     

可注射壳聚糖-β-磷酸三钙复合富血小板血浆的骨修复作用

目的 以新型可注射生物材料壳聚糖-β-磷酸三钙(β-TCP)为支架,负载富血小板血浆(PRP)构建成复合体,观察其体内成骨效应.方法 18只中国青山羊随机分为三组,每组6只,建立双侧胫骨平台下腔穴型骨缺损模型.空白组:骨缺损部不植入任何牛物材料;单纯材料组:单纯植入可注射生物材料壳聚糖-β-TCP;PRP组:植人复合PRP的壳聚糖-B-TCP.于术后第4、8周取出骨缺损区标本进行大体、绀织学切片观察,图像分析骨缺损区域骨组织的牛成比例. 结果大体标本显示4、8 周时PRP组骨缺损部硬度均强于空白组、单纯材料组.组织切片显爪空白组到8 周时骨缺损区域仍未修复,仅在骨缺损边缘部分区域有少苗类骨质形成,骨缺损区域多为纤维组织填允;单纯材料组骨缺损边缘区域有少量类骨质形成,骨缺损区域多为小的点片状新牛骨组织,中央区域到8周时无明显新生骨组织形成;PRP组骨缺损边缘区域类骨质较单纯材料组增多,骨缺损中间部多为点片状新牛骨助织,骨缺损中央区同中间部相似,见点片状新生骨组织.术后4周空白组、单纯材料组、PRP组的成骨面积百分比分别为(8.79±3.63)%、(14.49±3.72)%、(24.18±5.38)%,8周时分别为(15.41±4.21)%、(25.36±5.37)%、(30.71 ±4.39)%,4周、8周PRP组骨修复效果均优于其他两组(P＜0.05). 结论负载PRP的壳聚糖-β-TCP具有良好的骨修复效果,PRP在体内早期对骨组织修复有促进作用.     

不同浓度5-溴脱氧尿嘧啶核苷标记山羊骨髓基质干细胞的体外研究

Objective To explore the feasibility of labeling and tracing in vitro goat bone marrow mesenchymal stem cells (BMSCs) by bromodeoxyuridine (BrdU) on the basis of investigation of its optimal concentration, incubating time and cytotoxicity. Methods A healthy goat, aged 10 months old, male, weighing 32 kg, was used in this study. Bone marrow was aspirated. BMSCs were isolated and cultured using the adherence method in vitro. The fourth passage of BMSCs (P4) were incubated with BrdU at 5, 10, 15, 20 μmol/L as 5, 10, 15, 20 μmol/L BMSC groups. Cells were not labeled by BrdU as negative control. The following parameters were measured: induction, differentiation and determination of goat BMSCs; the optimal mass concentration and incubation time of 5-BrdU labeling; cell positive rate at 12, 24, 48 and 72 hours in each group using immunofluoreseenee; the cell survival rate after various concentrations of BrdU ladling by trypan-blue exclusion. Results The morphology of the primary and passage goat BMSCs was fusiform in shape. Goat BMSCs could differentiate into osteoblasts and chondrocytes following induction. BMSC nucleus showed green fluorescence under fluorescence microscope after being labeled by BrdU. The mean labeling rate increased with the increase in the concentration and incubation time of BrdU, and reached to (93.32± 3.25)% after incubation in 15 μmol/L, BrdU for 48 hours. There were no significant differences between 15 μmol/L BrdU for 72 hours, 20 μmol/L BrdU for 48 hours and 72 hours (P > 0.05), or between the other groups or time points (P < 0.05). The labeling rate of the blank control group was 0. The cell survival rate was all above 90% (P > 0.05). Conclusions BrdU can be used as a labeling marker for goat BMSCs. When the concentration is 15 μmol/L and the incubation time is 48 hours, the optimal labeling effect can be achieved. Goat BMSCs labeled with BrdU is of high efficiency and safety.     

灌注培养对骨髓基质干细胞在大段材料上增殖与分布的影响

Objective To study the role of perfusion bioreactor in proliferation and distribution of rat bone marrow stromal cells (BMSCs) in a large-scale scaffold. Methods SD rat BMSCs transfected with enhanced green fluorescent protein (eGFP) (eGFP-BMSCs) were planted in large-scale porous β-tricalcium phosphate (β-TCP) scaffolds. In the dynamic perfusion culture group, the scaffold with eGFP-BMSCs was continuously cultured in our self-designed three-dimensional perfusion bioreactor for 7, 14 and 28 days. In the static culture group, the scaffold was put into a medium reservoir without perfusion for 7, 14 and 28 days.Proliferation and distribution of the cells in the scaffold were examined by scanning electronic microscopy (SEM), fluorescence microscopy (FM), measuring daily glucose consumption, and counting eGFP-BMSCs in each layer. Results SEM and FM showed that eGFP-BMSCs distributed and proliferated throughout the scaffold in dynamic perfusion culture, but distributed and proliferated only in the peripheral pores of the scaffold in static culture. The daily glucose consumption in both groups increased with time. Cell proliferation reached the plateau phase after culture for 14 days in the static culture group, but after culture for 21 days in the perfusion culture group. The rate and margin of increase were much more evident in the perfusion culture group. On day 28, glucose consumption in the perfusion culture group was 36. 33 ± 3. 14 mg/d, 3. 7 times as large as that in the static culture group (9. 82 ± 1. 33 mg/d). The eGFP-BMSCs counting revealed there were no significant differences in cell number between layers of the scaffold on days 7 and 28 d ( P > 0. 05), but there were significant differences on day 14 in the perfusion culture group ( P < 0. 05); while in the static culture group, there were significant differences in cell number between layers of the scaffold ( P < 0. 05),with most of the cells assembled at the bottom of the scaffold.Conclusion Our self-designed three-dimensional perfusion bioreactor may help eGFP-BMSCs proliferate and distribute uniformly in a large-scale porous scaffold.     

bcr-abl P210和abl基因实时荧光定量PCR检测共用质粒标准品的制备及应用

 目的 建立一个荧光实时定量PCR（RQ-PCR）共用质粒标准品,用于同时检测bcr-abl P210白血病融合基因和abl内参基因。方法 分离K562细胞RNA,反转录为cDNA,以此为模板,设计引物进行PCR,PCR产物经琼脂糖电泳、胶回收,T-A载体克隆后,转化JM109工程菌,抽提质粒、测序、测定拷贝／μl,冻存备用。以RQ-PCR和定性巢式PCR进行验证,检测23例CML患者的骨髓标本,并同bcr-abl基因荧光原位杂交（FISH）结果比较。结果 获得了预期的模板质粒,以欧洲抗癌协会推荐的引物探针RQ-PCR检测bcr-abl P210和abl基因,浓度相同时,Ct值相同;反复检测日内差和日间差均<5 %。23例标本按FISH结果≥10%（n=8）、0.5 %～10 %（n=6）和阴性（n=9）分组,RQ-PCR结果分为0.492±0.263、0.023±0.033和（4.33±3.84）×10-4;FISH阴性组和其余两组间P值均＜0.001,三组间P值均＜0.05。结论 该研究建立了bcr-abl P210基因和abl基因共用的质粒标准品,定量准确,性质稳定,能简化操作,满足临床检测和科研的要求。     

eGFP标记肿瘤细胞小鼠模型的建立与应用

目的〖HT5"SS〗： 以携带绿色荧光蛋白基因（eGFP）的重组逆转录病毒（RV）标记肿瘤细胞,建立小鼠体内长期、系统肿瘤细胞研究模型,并初步探讨其应用。〖HT5W〗方法〖HT5"SS〗： 制备高滴度eGFPRV,感染小鼠白血病P388细胞,有限稀释法获得eGFP＋的单细胞克隆;在体外和DBA/2小鼠体内,分别以流式细胞术、荧光倒置显微镜、冰冻切片和普通病理切片、半固体集落培养和PCR等方法,观察小鼠体内eGFP标记肿瘤细胞的效率、定位和时效。〖HT5W〗结果 〖HT5"SS〗： eGFP基因导入率达80.2%,经克隆筛选后eGFP＋率>99.2%。P388eGFP细胞体外传代3个月后eGFP＋率为95.2%,体内序贯接种(56.3±1.25)d后eGFP+率为(93.3±0.50)% (n=3)。eGFP基因导入未影响P388细胞的体内外生物学特性。多聚甲醛灌注固定法和液氮速冻法可有效保存组织中的荧光,灌注固定法制备的标本适用于多种病理和组化染色观察;外周血、肝、脾、脑等脏器中eGFP＋细胞的分布、增殖,可用流式细胞仪和荧光显微镜等动态、定量观察,并可通过外周血的荧光细胞变化动态观察肿瘤细胞对治疗的反应;无菌获得的eGFP＋细胞可用于体外培养或体内序贯移植;PCR等技术可提高微量肿瘤细胞检测的敏感性。〖HT5W〗结论〖HT5"SS〗： 成功建立的模型可在体内外以多种方法长期、动态、敏感地追踪观察荧光标记的肿瘤细胞,可广泛应用于肿瘤和细胞组织工程等领域。     

血管束植入组织工程骨修复兔股骨缺损对血管内皮生长因子表达的影响

目的 探讨血管束植入组织工程骨修复兔股骨缺损对局部血管内皮生长因了(VEGF)表达的影响.方法 将兔自体骨髓基质下细胞经诱导后与β-磷酸三钙材料复合,植入制备的兔股骨缺损处,其中实验组在材料侧槽中植入股骨的动静脉血管束,对照组则单纯植入组织工稗骨,分别于术后2、4、8、12周通过形态学检测新生骨量,免疫组织化学方法检测新生骨中VEGF的表达量.结果 随着时间进展,各组成骨量逐渐增加,差异有统计学意义(P<0.05),并且从第4周开始实验组成骨量高于对照组,差异有统计学意义(P<0.05);符时问点实验组新生骨中VEGF的表达最均高于对照组,差异有统汁学意义(P<0.05),且4周时达到最人值.结论 血管束植入组织工程骨可明显增加新生骨中VEGF的表达并能促进骨缺损的修复.