培养破骨细胞扫描电镜样本制备方法的探讨

目的:探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法:实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果:破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论:牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

环孢素A抑制骨水泥颗粒诱导破骨细胞形成及功能

目的:探讨环孢素A对颗粒诱导破骨细胞形成及功能的影响。方法:取SD仔鼠双侧股骨和胫骨的骨髓,以不含血清的α-MEM培养液洗涤并收集骨髓细胞,再将细胞重悬于含10%胎牛血清及10~(-8)mol/L1,25-(OH)_2D_3的α-MEM培养液中,细胞计数后配成1.5×10~7/ml的细胞悬液,加入聚甲基丙烯酸甲酯(PMMA)颗粒和不同浓度的环孢素A(10~((-8)mol/L、10~(-7)mol/L、10~(-6) mol/L)于24孔培养板进行培养,并设置阳性对照组(只加PMMA颗粒)和阴性对照组(PMMA颗粒和CsA均不加),每组均有4孔放置骨磨片1片进行培养。培养2周后,行抗酒石酸(TRAP)染色检测破骨细胞形成;骨磨片行甲苯胺蓝染色观察。结果:PM- MA颗粒能够诱导大量TRAP染色阳性的破骨细胞形成,骨磨片有吸收陷窝形成;用环孢素A(10~(-8)mol/L、10~(-7)mol/L)和PMMA颗粒共同培养下TRAP染色阳性的破骨细胞形成数量明显减少,环孢素A浓度达到10~(-6)mol/L时无TRAP染色阳性的破骨细胞形成;环孢素A浓度在(10~(-8)mol/L、10~(-7)mol/L)时骨磨片有吸收陷窝形成,但少于阳性对照组,在10~(-6)mol/L时骨磨片则无吸收陷窝的形成。结论:环孢素A对PMMA颗粒诱导的破骨细胞的形成有着明显的抑制作用,且呈剂量依赖性。     

乳腺癌体外骨转移瘤3D模型的建立

目的:构建乳腺癌体外骨转移瘤3D模型。方法:新生CD-1小鼠的颅骨单独孵育为正常组,新生CD-1小鼠的颅骨与MDA-MB-231细胞低氧共孵育四天为模型组。通过扫描电镜(SEM)鉴定骨转移瘤模型,中性红染色法鉴定骨组织中的破骨细胞,硝酸银复染法观察骨的溶解,结晶紫染色法观察骨转移瘤模型中肿瘤细胞的生长。结果:正常组骨表面光滑完整;模型组骨组织表面黏附大量肿瘤细胞,破骨细胞活性增强,产生严重的溶骨,表面出现骨陷窝,骨纤维发生断裂。结论:成功建立了乳腺癌骨转移瘤体外3D模型,该模型能够模拟体内骨转移瘤微环境。     

脱细胞牛颈静脉内皮细胞黏附实验研究

目的:探讨去细胞处理后牛颈静脉血管片表面改性方法,增加内皮细胞在材料表面的黏附生长.方法:选取三种表面预衬组分:纤维连接蛋白(Fibronectin)单纯组,纤维连接蛋白+Ⅳ型胶原+明胶混合组,磷酸缓冲液空白组,采用物理包裹法.剪取多步骤去垢剂-酶消化法脱细胞处理的牛颈静脉片,无茵下静态培养7天,标本血管片表面细胞记数,切片染色,扫描电镜检测,比较不同组分预衬对细胞黏附生长的影响.结果:混合组在选取时间点表面细胞数目最多,单纯组次之.第7天HE染色结果显示混合组细胞连接成片,呈单层排布;单纯组表面细胞少量细胞黏附;对照组基本无细胞.扫描电镜显示:单纯组血管片表面细胞稀疏,细胞间无桥接,与下方的纤维结构联结不明显,数目较少;混合预衬组铺连成片,细胞间长梭形有桥接和突起,与去细胞的血管片纤维之间形成连接.结论:去细胞处理的牛颈静脉血管片可生长内皮细胞.经过纤维连接蛋白单独预衬处理的血管片表面,可增强内皮细胞黏附和贴壁生长,三组分混合预衬增强细胞黏附性及贴壁生长方面效果更好.     

雪旺氏细胞与同种异体骨支架的体外共培养研究

目的:探讨雪旺细胞(Schwann’s cells,SCs)在同种异体骨支架上的生物相容性,体外构建组织工程骨神经化模型。方法:利用新鲜人体骨骼制备同种异体骨支架材料,检测其物理性能;采用优化方法提取新生SD大鼠坐骨、臂丛神经培养SCs,实验分为三维培养实验组(SCs+同种异体骨)、二维培养对照组(SCs+胶原玻片),S-100抗体免疫荧光染色鉴定SCs纯度;细胞计数法检测两组细胞增殖特点;细胞接种后第3、7天取样,扫描电镜观察。结果:同种异体骨支架具有良好的三维孔隙结构,适宜细胞贴附生长;S-100免疫荧光染色证实SCs纯度95%;扫描电镜检测显示两组SCs均可正常粘附增殖,细胞间排布规律相似,培养早期实验组SCs胞体更加细长,伪足更加明显,随着培养时间的延长表现出较强的迁移能力;细胞增殖检测:两组SCs生长曲线特征基本一致,支架材料对SCs无毒性作用。结论:同种异体骨支架SCs具有良好的生物相容性,其三维立体多孔结构有利于SCs的粘附与迁移,初步构建了体外组织工程骨神经化模型。     

微弧氧化和碱处理技术在多孔钽修复兔颅骨缺损中的应用

目的观察微弧氧化和碱处理对多孔钽表面性状、生物相容性和成骨能力的影响。方法微弧氧化和碱处理多孔钽片后,扫描电镜观察表面微孔数量、表面钙磷沉积和接触角。植入钽片修复兔颅骨缺损模型,在4周和12周观察骨愈合情况。结果扫描电镜显示处理组表面有更多的微孔和钙磷沉积以及更小的接触角(P<0.05)。植入多孔钽片后,所有动物均生长良好,伤口愈合佳。CT观察多孔钽片和周围骨组织耦合良好;钙黄绿素标记检测显示12周时有新生骨长入多空钽材料内部;扫描电镜观察发现4周时多空钽材料内部有新生血管,12周时有骨小梁长入材料内部。结论微弧氧化和碱处理能改变多孔钽材料表面形状,处理后多孔钽片具有良好的生物相容性和成骨能力。     

珍珠质自然涂层钛种植体表面对MC3T3E1成骨样细胞的作用

为了研究珍珠质自然涂层钛种植体表面的体外生物相容性,将珍珠质自然涂层的钛片与MC3T3E1成骨样细胞复合培养以观察细胞的生长、增殖和分化.分别以羟基磷灰石涂层钛片和没有涂层的纯钛片作为对照组,以MC3T3E1细胞单纯培养作为空白组,分别培养3天,5天和7天,通过倒置相差显微镜和扫描电镜观察细胞生长情况,流式细胞技术检测细胞增殖活性,金氏比色法检测碱性磷酸酶(ALP)活性以及蛋白质印迹(Western blotting)法测定转化生长因子-β1(TGF-β1)表达水平.结果发现,细胞在珍珠质周围能形成良好附着,在其表面生长丰满.细胞培养第3天,第5天和第7天时,珍珠质表面的细胞增殖指数分别为(35.9±2.5)%、(69.7±3.3)%和(58.2±2.6)%,ALP活性分别为(6.123±2.917)U/g、(17.486±1.986)U/g和(23.987±1.372)U/g.第5天和7天时,实验组的细胞增殖指数、ALP活性和TGF-β1表达水平显著高于对照组和空白组(P<0.05).珍珠质自然涂层钛表面有利于MC3T3E1细胞的生长、增殖和分化,表明了珍珠质涂层能提高种植体表面的生物相容性,有可能会促进种植后的骨整合.     

石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料研究

目的：通过建立兔股骨缺损的动物实验模型,对采用等温化学气相沉积法和等离子喷涂技术所制备的石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料进行骨植入实验的的生物相容性进行评价,探索该复合材料作为植入机体骨组织的可行性依据。方法：采用骨科钻在实验动物股骨髁上钻孔的方法建立骨缺损的动物实验模型,将待研究比较的实验材料分别植入实验动物的股骨髁内,持续观察8周,在术后第2、4、8周时应用X线照片、组织学染色和扫描电镜技术,分别观察所研究材料在机体内对骨缺损愈合及其对机体的影响,进行组间比较和相关性分析。结果：石墨化炭/炭复合材料＋羟基磷灰石涂层（C/C＋HA）复合骨植入材料的骨植入实验生物相容性良好,材料与骨组织结合牢固,界面中成骨细胞生长明显,且炭颗粒脱落现象少,未见炎症细胞浸润。植入动物体内的材料在植入期未引起机体局部的炎症浸润反应且表面脱落的碳颗粒在机体组织中也未引起局部严重的炎症反应。在实验动物植入材料后的连续8周观察期中,组织学观察显示：表面涂有HA的炭/炭复合材料对骨组织形态改建上表现良好,其与骨组织接界处所形成的纤维结缔组织膜层厚度明显比未涂HA的材料要小,与骨组织结合更为紧密和牢固;碳颗粒出现脱落游离的现象明显减少。结论：在炭/炭复合材料表面涂以HA生物涂层对骨的形态改建和促进骨小梁生长等方面具有良好的作用,是一种具有发展潜力的骨修复材料。     

牛煅烧骨的表征及其对成骨细胞的作用

以天然牛骨为原料,经过加工处理,分别在1120和1250℃烧结得到两组牛煅烧骨样品。测定了材料的物理性能,讨论了烧结温度对材料孔隙率及其结构等的影响。将两组煅烧骨样品分别与成骨前体细胞在体外复合培养,通过对细胞生长和分化情况的观察和测定,对两组多孔材料与成骨细胞的亲和性作出了评价。结果表明牛煅烧骨的主要成分为羟基磷灰石,保留了天然骨的网状孔隙结构,成骨细胞在两组材料上均能正常生长和分化,而且在1120℃烧结的样品具有更好的成骨细胞亲和性,说明牛煅烧骨是一种有发展前途和应用前景的骨修复材料和骨组织工程支架材料。     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

目的采用倒置显微镜、扫描电镜（scanning electron microscopy,SEM）、荧光显微镜和激光共聚焦显微镜（（laser scanning confocal microscopy,LSCM））技术对大鼠颌下腺细胞（rat submandibular gland cells,RSMGs）与丝素-壳聚糖（silk fibroin-chitosan,SFCs）的体外复合培养进行形态学观察。为观测、评估种子细胞在三维支架的内部生长情况提供技术支持。方法取0～8 d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体（CK8）及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源。选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜（5×5×2）mm作为支架材料构建组织工程化涎腺样结构。将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况。结果倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行。SEM可以较精确的展示细胞支架复合生长的表面超微结构。经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞。荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内。LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息。结论四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测。LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值。     

Comparison of tissue-cultured bovine endothelial cells from aorta and sphenous vein

Summary Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial. This work was supproted by Grants HL-1330 and HL-17269 from NIH.     

Comparison of tissue-cultured bovine endothelial cells from aorta and saphenous vein

Endothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoy's 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.     

Culture of murine mastocytes: heterogeneity of the ultrastructural aspects

In cultures of normal mouse hematopoietic cells containing mast cell growth factor develop cells with many features of mast cells. These cells seem heterogeneous with respect to size, cell surface, granules maturity and morphology of nucleus using transmission and scanning electron microscopy. Alcian blue-safranin staining shows that most of the proteoglycan synthesized by cultured mast cells is weakly sulfated and non heparin-mucopolysaccharides. These results support the view that cultured mast cells resemble to mucosal mast cells, and are clearly different from serosal mast cells.     

Resorption of vital or devitalized bone by isolated osteoclasts in vitro

Summary The maintainance of resorptive capability towards vital or devitalized bone in osteoclasts isolated from the medullary bone of laying hens and cultured for five days in vitro has been investigated morphologically with the aid of light and transmission electron microscopy. Devitalized bone particles ranging in size from 50 to 100 m, added to cultures of osteoclasts, were rapidly surrounded by the osteoclasts which, in transmission electron microscopy, showed ruffled borders and clear zones at the surfaces of contact with bone — features typical of resorptive activity. Alternatively osteoclasts were added onto the endosteal surfaces of vital or devitalized diaphyses of quail femurs after removal of the endosteal and periosteal cell layers. The results indicated that, when the vital or devitalized bone surfaces were devoid of cells, the osteoclasts adhered and resorbed bone (as confirmed by transmission electron microscopy). When vital bone of quail was cultured for 24 h before the addition of osteoclasts a new cell layer was formed; it enveloped all bone surfaces and precluded the access of osteoclasts to bone. The role of these lining cells, ultrastructurally indistinguishable from resting osteoblasts, is discussed.     

分步消化法原代培养鼠阴道黏膜上皮细胞的研究

目的探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。方法取大鼠阴道全层组织,经Dispase酶和胰酶分步消化后,接种于无血清角化细胞培养液中连续培养,观察细胞形态、体外生长特性和超微结构,绘制生长曲线,免疫组化鉴定。结果原代细胞培养24-36 h后开始贴壁,7-10d约80%融合,呈铺路石样外观,可连续传5-6代;扫描电镜下细胞表面可见微绒毛嵴;角蛋白染色阳性,细胞纯度98%;第五代细胞为正常二倍体核型。结论该方法培养的阴道上皮细胞增殖状态良好,细胞纯度高,扩增迅速,可在较短时间内获得大量细胞用于组织工程学研究。     

人胃癌细胞株裸鼠皮下移植瘤模型的建立及其生物学特性

目的初步建立不同浓度下制备人胃癌细胞株BGC-823裸鼠皮下移植瘤模型,观察其生物学特性。方法培养人胃癌细胞系BGC-823细胞,并分别以四个浓度皮下注射于裸鼠腋下每只0.2 mL。根据BGC-823细胞注射浓度将40只裸鼠随机分为四组：组一5×107个活细胞/mL（n=10）;组二1×107个活细胞/mL（n=10）;组三1×106个活细胞/mL（n=10）;组四1×105个活细胞/mL（n=10）。观察各组裸鼠摄食、活动情况、精神状态、死亡率、成瘤时间、成瘤率、肿瘤生长情况,采用免疫组化法检测肿瘤微血管密度。结果各组裸鼠摄食、精神情况正常,无死亡现象。除组四1×105个活细胞/mL浓度组成瘤率为0%外,其余各组成瘤率均为100%,瘤体出现时间在3～7 d,肿瘤血管密度MVD平均为：（123.26±31.57）个/mm2。结论初步建立了人胃癌裸鼠皮下移植瘤模型及建立此细胞系模型的最低浓度。为胃癌的进一步研究奠定了基础。     

Gold labeling of cell monolayers grown on dextran beads

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these monolayers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 microns, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.     

Membrane molecules involved in adhesion properties of cultured sertoli cells

Membrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody-induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS-PAGE), M_r 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.