胎兔颅骨成骨样细胞体外培养模型的建立

目的：建立成骨细胞体外培养模型,为骨代谢和成骨机理研究提供细胞学基础。方法：用酶消化和组织块移行生长相结合方法,从胎兔颅骨中分离出成骨样细胞群进行原代及传代培养,应用组织学、酶细胞化学、免疫细胞化学和染色体染色对其生物学和遗传学特性进行鉴定。结果：胎兔颅骨成骨样细胞具有体内成骨细胞的形态特征、ALP活性及合成分泌BMP等的功能,并能在体外发生钙生,其遗传学性状稳定。结论：该模型具有体内成骨细胞的生物学和遗传学特性,可用于成骨细胞代谢和成骨作用的体外研究。     

人肝细胞生长因子基因转染成骨细胞移植治疗早期股骨头缺血性坏死的实验研究

目的 探讨人肝细胞生长因子(hHGF)基因转染成骨细胞移植促进股骨头的早期血管新生,增强坏死股骨头的局部骨修复能力.方法 36只新西兰兔随机分成对照组、移植转染hGHF基因组和未转染成骨细胞组.体外分离、培养胎兔颅骨中的成骨细胞,利用脂质体介导hHGF基因体外转染成骨细胞,同时建立兔激素性股骨头缺血坏死模型,然后将细胞通过髓芯减压后移植于坏死的股骨头内,组织形态学分析血管发生和骨修复情况.结果 胎兔颅骨中分离培养的成骨细胞表达Ⅰ型胶原和碱性磷酸酶,hHGF基因转染成骨细胞体外及体内检测均有hHGF蛋白的表达.转染hHGF基因的成骨细胞组和成骨细胞组移植坏死的股骨头内的新生骨小梁与单纯髓芯减压组比较有差异性,同时基因转染组的新生血管数较对照组差异有统计学意义(P＜0.01).结论 hHGF基因转染可促进缺血股骨头的血管新生,并增加骨形成.显著促进坏死骨修复.     

兔骨髓基质细胞体外成骨分化鉴定及与煅烧骨复合培养的实验研究

目的研究兔骨髓基质细胞体外向成骨细胞分化的生物学特性及其与牛煅烧骨体外复合培养的生物相容性.方法将体外培养1周的兔骨髓基质细胞向成骨细胞诱导分化,于1、2、4周时提取RNA,采用RT-PCR技术检测ALP和I型胶原mRNA表达,并对培养2周的细胞行VonKossa染色观察钙结节形成;另制备细胞-煅烧骨复合物,继续培养1、7、14 d后取出,扫描电镜观察及X射线衍射仪检测.结果体外诱导培养2、4周后,兔骨髓基质细胞成功表达ALT和I型胶原,VonKossa染色可见钙结节形成.扫描电镜及X射线衍射分析证实细胞在煅烧骨表面大量贴附成活,14 d后细胞铺满支架表面,合成并分泌胶原纤维及钙盐.结论兔骨髓基质细胞体外可成功向成骨细胞诱导分化,成骨特性表达佳;与牛煅烧骨体外复合培养显示良好的生物相容性.     

兔骨髓基质细胞的分离培养

目的 为骨组织工程寻找一种理想的种子细胞。方法 采取兔骨髓组织，应用梯度离心获取骨髓基质细胞，体外培养传代，通过光镜、透射电镜及成骨特性的鉴定，观察细胞的形态、生长特点及成骨特性。结果 培养的骨髓基质细胞形态多为三角形或梭形，生长增殖迅速，具有成骨能力，易于定向分化为成骨细胞。结论 自骨髓获得的骨髓基质细胞具有明显的增殖能力和成骨活性，可以做为骨组织工程中比较理想的种子细胞。     

人骨膜细胞生物学特性的实验研究

目的 探讨人骨膜细胞体外培养的生物学特性. 方法 以胫骨骨膜组织为材料,采用组织块法分离细胞,完全培养基培养,通过倒置显微镜观察细胞形态学,锥虫蓝染色计数法检侧细胞增殖能力;流式细胞学分析细胞表面抗原.随机分为3组,成骨实验组和成软骨实验组分别加入不同的定向培养剂,对照组加入完全培养基,采用碱性磷酸酶染色、Von Kossa染色、甲苯胺蓝染色、Ⅱ型胶原免疫组化染色检测各组细胞的成骨和成软骨分化指标. 结果 骨膜细胞在体外培养条件下呈贴壁生长,细胞生长曲线证实骨膜细胞传至第9代仍保持良好的增殖能力,流式细胞仪检测证实细胞表面抗原CD90及CD105呈阳性;组织化学染色检测证实成骨实验组分化后细胞碱性磷酸酶及钙结节呈阳性,成软骨实验组分化后细胞蛋白聚糖及Ⅱ型胶原呈阳性,对照组各项指标均生阴性.结论 骨膜细胞体外培养细胞增殖能力强,具有间充质干细胞的特性和良好的成骨和成软骨分化潜能,其定向诱导分化的成骨细胞和软骨细胞均具有各自明显的细胞功能表达.     

TGF-β和BMP对胎兔颅骨成骨样细胞增殖和分化相互作用的实验研究

目的研究转化生长因子β(TGF-β)和骨形态发生蛋白(BMP)对成骨细胞增殖和分化的影响及其相互作用。方法取胎兔颅骨成骨样细胞进行体外培养，使用不同方法，加入TGF-β与BMP，在不同时间点检测其成骨样细胞增殖和分化的相互作用。结果TGF-β增加成骨样细胞DNA含量，抑制ALP活性、骨钙素的产生和矿化骨基质的形成。而BMP的作用相反。联合应用时，TGF-β减弱了BMP对成骨样细胞骨钙素产生和ALP活性的增强作用，BMP降低了TGF-β对成骨样细胞DNA合成的促进作用。在序惯性给药时，早期(9d)用BMP治疗并不影响随后加入的TGF-β对成骨样细胞ALP活性和基质钙化的抑制作用。而早期(10d)用TGF-β治疗却诱导了随后加入的BMP对成骨样细胞分化的促进作用。结论TGF-β促进成骨细胞增殖，BMP促进成骨细胞分化，它们在各自的不同时期发挥对成骨样细胞增殖和分化的影响。     

成骨细胞体外培养模型的研究进展

多种细胞参与骨的形成、重建和修复过程,其中成骨细胞的作用尤为重要,它不仅调节骨的矿化,还间接调节骨吸收功能,它的数量和功能变化直接影响多种骨骼疾病的发生、发展和预后。作为骨组织中最重要的力学感受细胞和成骨效应细胞,它的功能及调节机制已成为骨生物学研究的重中之重。目前用于研究成骨细胞生物学特性的细胞模型种类繁多,而每种模型都有其自身特点,在选择实验对象时需要对其生物学特性进行综合评估。本文将针对成骨细胞不同种属来源的原代细胞和细胞系作一简要概述。     

TGF-β对胎兔颅骨成骨样细胞体外成骨作用

目的 :探讨TGF β对胎兔颅骨成骨样细胞体外成骨作用。方法 :显微相差动态观察、细胞增殖计数、ALP活性测定及骨结节计数观察TGF β体外成骨作用。结果 :TGF β对该细胞的增殖 ,低剂量有促进作用 ,高剂量有抑制作用。TGF β(在 3～ 7d)对ALP活性有促进作用 ,并呈一定的时效和量效关系。TGF β低剂量对骨结节形成有促进作用 ,高剂量时为强有力的抑制作用 ,与剂量有相关关系。结论 :TGF β对胎兔颅骨成骨样细胞体外成骨表现为双相作用。     

c-Fos、c-Jun在兔成骨样细胞内的表达

用组织块移植法对兔颅骨细胞进行分离培养。相差显微镜观察，组织化学染色，免疫组织化学染色，mRNA原位杂交及体外钙化检测表明该细胞群符合成骨样细胞的特点。在此基础上用免疫组织化学SP法观察了c-F0s，c-Jun在成骨样细胞内的表达及其与骨钙素合成的关系。结果显示c-Fos、c-Jun从第一代到第十一代成骨样细胞内均有表述，且早期表述较多，可位于胞核、核膜及胞浆内。第六代以后的细胞仅胞浆着色。在c-Fos、c-Jun高表达时骨钙素合成减少c-Fos、c-Jun低表达时骨钙素合成增加，提示c-Fos、c-Jun对骨钙素具有负调控作用。本实验结果为c-Fos、c-Jun参与骨代谢提供了直接证据，同时也为观测成骨细胞生长分化及其影响固素提供了有价值的指标．有助于促进牙周炎牙槽骨骨吸收机理及其它骨代谢相关研究。     

体外培养成骨细胞研究新进展

成骨细胞是骨形成细胞,对骨组织的生长发育、骨代谢平衡、骨量维持和损伤修复起关键作用.一旦成骨细胞生成不足或功能降低,不能及时补充由于破骨细胞活动所导致的骨吸收、骨量丢失,将直接导致骨形成减少.随着细胞培养技术的发展,人们已经从许多动物的颅骨、骨髓基质和骨膜中成功分离培养出了具有典型成骨细胞特征的细胞株,研究表明培养后的成骨细胞在体内仍具有良好的成骨能力,在不同环境下可以形成骨组织.将分离培养的成骨细胞大量增殖后回植于体内,具有取材方便,成骨能力好等优点,且不需考虑免疫反应和传播疾病的危险.     

An osteo-inductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes

Urea and guanidine extracts of demineralized beef and rabbit bone matrix were assayed both in vivo and in vitro. One month following intramuscular implantation into mouse thighs, these extracts induced ectopic cartilage and bone. Seven days following continuous in vitro exposure to the same extracts, stage 24 chick limb bud mesenchymal cells in cultures had differentiated into greater numbers of chondrocytes than controls. These results suggest the feasibility of using limb bud mesenchymal cell cultures as an in vitro assay for bone matrix derived, extractable bioactive factors which effect the conversion of mesenchymal cells into chondrocytes as a requisite step in in vivo osteogenesis.     

建立成骨与破骨细胞共育体系的一种方法

目的：建立成骨细胞和破骨细胞共育的体外模型，为研究骨生理和病理提供新的方法。方法：从胎鼠和4周鼠中分离成骨和破骨细胞，并按照10：1、100：1的比例混合培养，应用碱性磷酸酶(AKP)、抗酒石酸(TRAP)、甲苯胺蓝染色法，鉴定成骨及破骨细胞，并测量碱性磷酸酶的含量，从而建立一个较稳定的共育体系。结果：破骨细胞和成骨细胞达到了相互接触培养，且随着破骨细胞数量的不同，碱性磷酸酶的含量会有不同的变化。结论：该模型可用骨形成和吸收代谢的相关研究。     

An in vitro study of electrical osteogenesis using direct and pulsating currents

Although many investigators have electrically stimulated osteogenesis in vivo, this phenomenon has not been reported in vitro. In this work direct and pulsating currents were applied to in vitro fetal rat tibiae. The results indicated that electric current did cause osteogenesis in vitro, precipitates of calcium compounds formed in the cathode region, and direct current was more effective in stimulating bone growth than pulsed current delivering the same total charge (coulombs).     

兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性

目的：探索新西兰白兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性。方法：取兔阴蒂海绵体，采用酶消化法进行平滑肌细胞体外培养；在倒置显微镜下观察培养细胞的生长状况、形态特征及贴壁过程；用计数法测定细胞贴壁率；用MTT法描绘原代培养细胞的生长曲线；利用逆转录聚合酶链反应(RT—PCR)方法检测平滑肌细胞特征性标记物α-actin表达。结果：培养的兔阴蒂海绵体平滑肌细胞具有典型的平滑肌细胞形态特征，为梭形，呈长轴平行排列，具有明显的方向性；体外贴壁快，生长迅速，体外培养的阴蒂海绵体平滑肌细胞在合适的传代条件和比例下能够生存并保持其稳定的生物学特性。结论：体外培养的兔阴蒂海绵体平滑肌细胞模型可用于进一步研究阴蒂组织细胞学、分子生物学机制以及受体介导的平滑肌收缩信号转导机制及药理学作用等。     

Bilogic characteristics of fibroblast cells cultured from the knee ligaments

OBJECTIVE: To culture fibroblast cells from the knee ligaments and to study the biological characteristics of these cells. METHODS: Cells of the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) from New Zealand white rabbit were cultured in vitro. Cellular growth and expression of the collagen were analyzed. Moreover, an in vitro wound closure model was established and the healing of the ACL and the MCL cells was compared. RESULTS: Maximal growth for all these cells were obtained with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Ham's F12 media were not suitable to maintain these cells. Morphology of both ACL and MCL cells from New Zealand white rabbit was alike in vitro, but the MCL cells grew faster than the ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage type I to type III produced by ACL cells was higher than that produced by MCL cells. Wound closure assay showed that at 36 hours after injury, cell-free zones created in the ACL cultures were occupied partially by the ACL cells; in contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. CONCLUSIONS: Although the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen as well as the healing in vitro were significantly different. These differences in intrinsic properties of the two types of cells in vitro might contribute to the differential healing potentials of these ligaments in vivo.     

完全脱蛋白骨复合rhBMP2构建人工骨及其体内异位成骨

目的: 探讨异种完全脱蛋白骨复合基因重组骨形态发生蛋白(rhBMP2) 在体外构建人工骨的可行性,并对其体内异位成骨进行评估。方法: 体外将兔骨髓基质细胞进行成骨诱导, 然后接种复合rhBMP2的小牛完全脱蛋白骨, 构建组织工程骨, 植入自体大腿肌袋内, 术后4、8周处死动物分别进行常规组织学检查、碱性磷酸酶活性测定、折弯力学测试, 观测这种组织工程骨在体内的异位成骨作用。结果: 这种方法构建和组织工程骨在体内异位成骨效应显著且抗折能力较强。结论: 异种完全脱蛋白骨复合rhBMP2可作为骨组织工程支架材料, 这种组织工程骨在体内的成骨能力随植入时间的延长而增加。     

Transplantation of human fetal brain cells into ischemic lesions of adult gerbil hippocampus.

OBJECT: The goal of this study was to establish whether transplanted cells derived from fetal human brain can survive in an ischemic lesion. METHODS: Sixteen adult male Mongolian gerbils underwent transient bilateral common carotid artery occlusion. One week later, cell suspensions prepared from fetal human brain were injected using stereotactic guidance into the CA1 region of the hippocampus on one side. On the contralateral side injection of the cell suspension medium only was performed. One week after transplantation, the animals were perfusion fixed and their brains were processed for histological studies as well as expression of neuron and glia-specific antigens. Data from ischemic animals were compared with eight nonischemic gerbils that served as sham-operated controls. Last, the in vivo data were correlated with observations made from matching in vitro cultures of the fetal brain cell suspension. The in vivo data indicated that transplanted human fetus-derived brain cells survived in ischemic lesions of gerbil hippocampus after 1 week, provided that the host animal underwent adequate immunosuppression and the transplanted cells were not incorporated into the scar caused by the transplantation procedure. Unlike their in vivo counterparts, after 1 week, most cultured fetal brain cells expressed either neuron- or astrocyte-specific antigens. CONCLUSIONS: This work demonstrates that xenotransplanted fetal human brain cells are able to survive in an ischemic lesion in a rodent model. These data might be useful for future neural transplantation studies of treatments for cerebrovascular ischemia in humans.     

5-溴脱氧尿嘧啶核苷标记示踪脂肪成体干细胞体内成骨分化的研究

目的 观察经成骨诱导后的脂肪成体干细胞(ADASCs)在体内的成骨分化行为.方法 分离培养兔ADASCs,用5-溴脱氧尿嘧啶核苷(BrdU)标记,并通过免疫组织化学方法检查标记结果.将标记的ADASCs体外成骨诱导培养2周后与脱钙骨基质(DBM)复合,共培养7d后植入自体肌袋内.术后8周取材,固定、脱钙、包埋后切片染色,镜下观察.结果 大量ADASCs被标记,标记率达(82.3±8.6)%;体外标记后的ADASCs可以向成骨方向分化;切片苏木素-伊红(HE)染色示体内有新生骨形成,切片BrdU免疫组织化学染色示新生骨处有阳性细胞.结论 ADASCs经体外成骨诱导后植入体内能够继续维持成骨分化,并进一步形成新生骨.     

Advanced molecular profiling in vivo detects novel function of dickkopf-3 in the regulation of bone formation.

A bioinformatics-based analysis of endochondral bone formation model detected several genes upregulated in this process. Among these genes the dickkopf homolog 3 (Dkk3) was upregulated and further studies showed that its expression affects in vitro and in vivo osteogenesis. This study indicates a possible role of Dkk3 in regulating bone formation. INTRODUCTION: Endochondral bone formation is a complex biological process involving numerous chondrogenic, osteogenic, and angiogenic proteins, only some of which have been well studied. Additional key genes may have important roles as well. We hypothesized that to identify key genes and signaling pathways crucial for bone formation, a comprehensive gene discovery strategy should be applied to an established in vivo model of osteogenesis. MATERIALS AND METHODS: We used in vivo implanted C3H10T1/2 cells that had been genetically engineered to express human bone morphogenetic protein-2 (BMP2) in a tetracycline-regulated system that controls osteogenic differentiation. Oligonucleotide microarray data from the implants (n = 4 repeats) was analyzed using coupled two-way clustering (CTWC) and statistical methods. For studying the effects of dickkopf homolog 3 (Dkk3) in chondrogenesis and osteogenesis, C3H10T1/2 mesenchymal progenitors were used. RESULTS: The CTWC revealed temporal expression of Dkk3 with other chondrogenesis-, osteogenesis-, and Wnt-related genes. Quantitative RT-PCR confirmed the expression of Dkk3 in the implants. C3H10T1/2 cells that expressed Dkk3 in the presence of BMP2 displayed lower levels of alkaline phosphatase and collagen I mRNA expression than control C3H10T1/2 cells that did not express Dkk3. Interestingly, the levels of collagen II mRNA expression, Alcian blue staining, and glucose aminoglycans (GAGs) production were not influenced by Dkk3 expression. In vivo microCT and bioluminescence imaging revealed that co-expression of Dkk3 and BMP2 by implanted C3H10T1/2 cells induced the formation of significantly lower quantities of bone than cells expressing only BMP2. CONCLUSIONS: A bioinformatics analysis enabled the identification of Dkk3 as a pivotal gene with a novel function in endochondral bone formation. Our results showed that Dkk3 might have inhibitory effects on osteogenesis, but no effect on chondrogenesis, indicating that Dkk3 plays a regulatory role in endochondral bone formation. Further mechanistic studies are required to reveal the mechanism of action of Dkk3 in endochondral bone formation.     

家兔作为胎龄期骨髓间充质干细胞动物模型的实验研究

目的建立以家兔为对象的胎龄期BMSC研究动物模型。方法通过人工授精方法,获得孕期3周的孕兔4只,行剖腹产术取出胎兔20只,冲取胎兔骨髓,以贴壁培养法进行体外扩增培养。测量第3代胎兔BMSC的生长曲线,克隆形成率,并进行成骨、成脂及成软骨诱导分化。另外,将原代细胞冻存30 d后复苏,测量其第3代生长曲线,对冻存前后增殖能力的变化进行观察。扫描电镜观察胎兔BMSC与β-TCP形成细胞材料复合物的体外形态。将BMSCs-β-TCP复合物植入裸鼠皮下,于术后1、3、6个月分别取材,行HE、VG、Masson染色观察。结果胎兔来源的BMSC于倒置相差显微镜下观察,细胞饱满均匀,呈梭形或倒三角形状;传代后各代细胞形态未发生明显变化,生长曲线相差不大;成骨、成脂以及成软骨诱导观察到钙结节、脂肪空泡、黏多糖。冻存30 d后复苏,其第3代生长曲线与冻存前相比未见明显变化。电镜下观察,与β-TCP复合7 d后,细胞能均匀紧密贴合于材料上,伸展良好分布均匀。植入裸鼠皮不同时间点取材行HE、VG、Masson染色,均显示有新生骨组织生成。结论以家兔为研究动物模型,可以在胎龄期获取并分离得到BMSC,并可在异位构建组织工程骨,是间充质干细胞动物研究的新选择。