60 Coγ射线照射对肺泡II型细胞和肺泡隔间质细胞的生物效应

目的:探讨60Coγ射线照射对肺泡II型细胞和肺泡隔间质细胞的生物效应.方法:原代分离肺内II型上皮细胞(AT-Ⅱ)和间质细胞包括巨噬细胞和成纤维细胞,分别进行0、3、5、7 Gy的γ射线照射,用细胞核嗜银染色观察照射对AT-Ⅱ增殖的影响;用酶谱分析检测照射后AT-Ⅱ和间质细胞培养上清中基质金属蛋白酶-2、-9(MMP-2,-9)的活性;用ELISA检测间质细胞培养上清中TGF-β1和IV型胶原含量.结果:AT-Ⅱ的核仁数量随照射剂量增加而增多,其中7 Gy组最高;AT-II培养上清中MMP-2、-9活性随照射剂量增加呈先增高后降低趋势,间质细胞上清中MMP-2、-9活性和TGF-β1水平逐渐升高,IV型胶原分泌水平呈先降低后升高趋势.结论:放射性肺损伤早期,AT-Ⅱ、巨噬细胞和成纤维细胞均参与肺组织无效性重建过程,与晚期肺纤维化启动有一定的内在联系.     

^60Coγ射线照射对肺泡Ⅱ型细胞和肺泡隔间质细胞的生物效应

目的：探讨^60Coγ射线照射对肺泡Ⅱ型细胞和肺泡隔间质细胞的生物效应。方法：原代分离肺内Ⅱ型上皮细胞（AT-Ⅱ）和间质细胞包括巨噬细胞和成纤维细胞，分别进行0、3、5、7Gy的γ射线照射，用细胞核嗜银染色观察照射对AT-Ⅱ增殖的影响；用酶谱分析检测照射后AT-Ⅱ和间质细胞培养上清中基质金属蛋白酶-2、-9（MMP-2，-9）的活性；用ELISA检测间质细胞培养上清中TGF-β1和Ⅳ型胶原含量。结果：AT-Ⅱ的核仁数量随照射剂量增加而增多，其中7Gy组最高；AT-Ⅱ培养上清中MMP-2、-9活性随照射剂量增加呈先增高后降低趋势，间质细胞上清中MMP-2、-9活性和TGF-β1水平逐渐升高，Ⅳ型胶原分泌水平呈先降低后升高趋势。结论：放射性肺损伤早期，AT-Ⅱ、巨噬细胞和成纤维细胞均参与肺组织无效性重建过程，与晚期肺纤维化启动有一定的内在联系。     

骨性关节炎软骨下骨成骨细胞的分离培养与增殖活性的观察

目的 探讨骨性关节炎软骨下骨成骨细胞的分离、培养、鉴定方法及生长特性.方法 复制改良Hulth兔膝关节不稳模型;采用Ⅰ型胶原酶消化联合组织块贴附法获得软骨下骨成骨细胞;应用倒置显微镜、Ⅰ型胶原免疫化学染色以及瑞氏-姬姆萨染色来进行形态学观察和生物学鉴定;利用软骨细胞与滑膜细胞分别于软骨下骨成骨细胞共培养,应用四甲基偶氮唑蓝检测软骨下骨成骨细胞的增殖活性;检测细胞的Ⅰ型胶原在基因水平的表达.结果 Ⅰ型胶原酶消化后在组织块贴附第11天有细胞开始从组织块周围爬出;Ⅰ型胶原酶免疫化学染色后可见胞浆内出现黄褐色颗粒,为阳性反应.瑞氏-姬姆萨染色显示细胞染成蓝紫色,胞核饱满,核仁清晰;四甲基偶氮唑蓝检测显示在3d时,滑膜细胞和软骨细胞对于软骨下骨成骨细胞的增殖有着明显的促进作用,在第6、10、14天时这种促进作用变得不明显,而软骨下骨成骨细胞的增殖活性超过2个共培养组.Ⅰ型胶原表达水平检测显示在第10天时,软骨细胞对于软骨下骨成骨细胞分泌Ⅰ型胶原的促进作用最强,而滑膜细胞对其的促进作用不明显.结论 酶消化联合组织块贴附法可获得理想的软骨下骨成骨细胞;利用Ⅰ型胶原酶免疫化学染色鉴别软骨下骨成骨细胞方法简便易行;利用软骨下骨成骨细胞与软骨细胞和滑膜细胞共培养的体系适用于骨性关节炎(0A)微环境的研究,且该体系可以模拟类OA软骨下骨成骨细胞、滑膜细胞和软骨细胞相互影响作用的进程.     

IL—6及IFNs对大鼠成骨细胞Ⅰ型胶原及Ⅰ型胶原酶mRNA表达的影响

采用原位杂交和碱性磷酸酶积分相结合的方法,分析了IL-6对分离培养的大鼠成骨细胞Ⅰ型原胶原。αl链mRNA和Ⅰ型胶原酶mRNA表达的影响以及α和7干扰素对上述两种mRNA表达的调节。结果表明:成骨细胸可自发分泌IL-6,同时表达一定量的Ⅰ型原胶原。αL链和Ⅰ型胶原酶mRNA,外源性IL-6则能以剂量依赖方式抑制Ⅰ型原胶原αl链mRNA表达并促进Ⅰ型胶原酶mRNA表达(P均<0.01),?干扰素可下调成骨细胞IL-6分配,而影响Ⅰ型原胶原e1链mRNA和Ⅰ型胶原mRNA表达。o干扰素调节上述两种mRNA的机制有待进一步阐明。     

生物活性玻璃对成骨细胞Ⅰ型胶原和骨钙素基因表达的影响

背景：在成骨细胞培养液中加入生物活性玻璃离子,对成骨细胞的细胞因子分泌是正相关还是负相关？ 目的：观察生物活性玻璃对成骨细胞Ⅰ型胶原和骨钙素基因表达的影响,以进一步探讨生物活性玻璃促进成骨细胞增殖的可能机制。 方法：以DMEM完全培养液作为对照组培养液,以加入生物活性玻璃离子液的DMEM完全培养液作为实验组培养液,取第2代乳鼠颅骨源性成骨细胞,用两种培养液分别培养4 d进行实验观察。 结果与结论：鼠成骨细胞2种生长因子的表达量实验组与对照组之比为：Ⅰ型胶原=3.376、骨钙素=2.687,两者与内参基因相对量两两比较差异有显著性意义(P < 0.05)。生物活性玻璃促进成骨细胞对Ⅰ型胶原、骨钙素的基因表达,有利于骨修复过程中成骨细胞周围基质的形成和矿化,从而促进骨愈合。 关键词：生物活性玻璃;成骨细胞;细胞因子;Ⅰ型胶原;骨钙素 doi:10.3969/j.issn.1673-8225.2012.08.002     

大剂量γ线照射对小鼠免疫功能近期、远期的影响

目的 :观察大剂量γ线照射对小鼠免疫功能近期、远期的影响。方法 :将 2 2 5只清洁级C5 7小鼠 ,随机分为 0、6、9、12、15和 2 0Gy 6个剂量组 ,经γ线全身 1次照射后 ,于照后 1～2 8d和 3～ 12月活杀取材 ,用原位末端标记 (TUNEL)和May Grunwald Giemsa(MGG)染色 ,检测细胞凋亡并观察其与照射剂量的关系。用流式细胞仪检测外周血T细胞亚群的变化。结果 :(1)照后早期 (1～ 14 )d外周血淋巴细胞的凋亡率即出现明显升高 ,随着照射剂量的增加凋亡率的升高更为显著 ;而T细胞亚群经不同剂量照射后持续下降 ,剂量为 2 0Gy时降至最低 ,其中以CD8 T细胞对辐射的敏感性最高 ,因而推测早期的严重损伤是急性辐射免疫损伤的重要特点之一。 (2 )照射后 1月淋巴细胞的凋亡率降低 ,T细胞及其亚群也逐渐恢复。然而直至照后 6～ 12月 ,无论是淋巴细胞的凋亡率还是CD3 T细胞及CD8 T细胞亚群 ,均未恢复到对照的水平 ,提示大剂量辐射对机体免疫系统的损伤呈现较重的远期效应。 (3)本实验还发现 ,12≤Gy照射后 ,外周血淋巴细胞的凋亡率与照射剂量呈明显的剂量效应关系 ,15～ 2 0Gy照射后未观察到明显的量效关系。结论 :照射后早期外周血淋巴细胞的大量凋亡 ,可能是导致T细胞亚群的百分率急剧下降和后期免疫功能受损的重要原     

缺氧大鼠成骨细胞增殖、分化及基因表达

背景：研究表明,低氧会引起骨折延迟愈合或不愈合,骨密度减低,使骨质疏松、骨折等疾病的发病率升高。成骨细胞是骨形成、生长和发育主要的功能细胞。 目的：观察缺氧对体外培养成骨细胞增殖、分化及基因表达的影响。 方法：选用新生Wistar大鼠颅盖骨,使用胰酶-胶原酶序贯消化法获取成骨细胞,进行体外传代培养及鉴定。在缺氧培养下应用MTT法测定成骨细胞增殖率,对硝基苯磷酸盐法测定成骨细胞碱性磷酸酶活性,反转录-聚合酶链反应法测定成骨细胞内骨钙素及Ⅰ型胶原的表达。 结果与结论：缺氧具有抑制成骨细胞增殖,降低碱性磷酸酶活性及下调大鼠成骨细胞中Ⅰ型胶原α1、骨钙素基因表达的作用,随缺氧时间的增加作用更加明显。提示缺氧可通过抑制成骨细胞增殖、分化成熟及下调Ⅰ型胶原α1、骨钙素基因表达而降低成骨能力,从而促进骨质疏松的发生。     

骨髓间充质干细胞成骨方向诱导过程中的基因表达

背景:目前骨髓间充质干细胞向成骨细胞分化演变中的基因表达模式尚不明确。目的:观察骨髓间充质干细胞向成骨细胞诱导分化过程中碱性磷酸酶、骨桥蛋白、Ⅰ型胶原、碱性成纤维细胞生长因子和骨钙素基因的表达情况,验证骨髓间充质干细胞是否向成骨细胞成熟分化。方法:抽取2月龄新西兰大白兔股骨骨髓,全骨髓贴壁法培养获得骨髓间充质干细胞,用矿化诱导培养基(DMEM/F12、地塞米松1×10-8mmol/L、β-磷酸甘油钠0.01mol/L、维生素C0.05g/L)进行成骨诱导培养,用反转录-聚合酶链反应方法检测诱导培养后第一二代骨髓间充质干细胞碱性磷酸酶、骨桥蛋白、Ⅰ型胶原、碱性成纤维细胞生长因子和骨钙素基因的表达情况,并对该骨髓间充质干细胞表面抗原CD44进行鉴定。结果与结论:经矿化诱导培养基诱导培养后,第一二代骨髓间充质干细胞阶段性表达碱性磷酸酶、骨桥蛋白、Ⅰ型胶原、碱性成纤维细胞生长因子和骨钙素基因;第1代骨髓间充质干细胞抗原CD44阳性率达44.4%。提示兔骨髓间充质干细胞在体外矿化诱导培养中逐渐向成骨细胞分化,分别于诱导后第一二代细胞中阶段性顺序表达成骨细胞特异性基因碱性磷酸酶、骨桥蛋白、Ⅰ型胶原、碱性成纤维细胞生长因子和骨钙素,该细胞已具备成骨细胞特征,为揭示骨髓间充质干细胞向成骨细胞分化过程中基因表达机制提供了实验依据。     

γ射线对人淋巴细胞cx43和ANLN基因转录表达的影响

目的 研究γ射线对人外周血淋巴细胞 cx43和ANLN基因转录表达的影响.方法 对数生长期的淋巴细胞,分别给予1、2、3、4、5、6 Gy的60Coγ射线照射,照射后12 h,以及2 Gy照射后4、8、12、24、36、48、72 h,分别提取总RNA,反转录成cDNA.利用实时荧光定量PCR技术,检测各组cx43和ANLN基因表达改变.结果 人外周血淋巴细胞cx43 mRNA表达水平在2 Gy照射后4、8、12 h明显增高,分别为对照组(未照射组)的6.74、9.06、7.22倍(P＜0.05);24～72 h,其表达水平与对照组相比没有明显变化.1、2、3、4、5、6 Gy剂量照射后12 h,cx43 mRNA表达水平显著增高(P＜0.05).ANLN mRNA表达水平在2 Gy不同时间点及1～5 Gy照射后12 h,表达降低(P＜0.05),6 Gy照射后12 h其表达开始升高,为对照组的6.08倍(P＜0.05).结论 γ射线照射2 Gy不同时间点及不同剂量照射后12 h,cx43基因表达上调,ANLN 基因表达下调.1～3 Gy剂量照射后12 h,cx43 mRNA表达在此范围内有时间和剂量的依赖性.cx43可能会发展为核事故受照射人员的分子生物学剂量标记物.     

纳米铁核素靶向治疗肿瘤的辐射效应研究

目的:综合考察纳米铁核素(59Fe)的照射剂量率和照射时间对杀死肿瘤细胞的影响,为低剂量率的β粒子放射性核素靶向疗法提供指导。方法:采用的三个肿瘤细胞系为:SKBR-3乳腺癌、U-118MG神经胶质瘤和A-431宫颈癌;实验模型为:在每一个样品中取103个肿瘤细胞,用低剂量率的β粒子照射;初始剂量率为:0.1 Gy/h～0.8 Gy/h;持续照射时间为:1天、3天或者7天。结果:分别用0.2 Gy/h～0.3 Gy/h和0.4 Gy/h～0.6 Gy/h的剂量率连续照射肿瘤细胞7天和3天,能够将所有肿瘤细胞样品中的细胞杀死。细胞的总辐射剂量为30 Gy～40 Gy。而用0.8 Gy/h的剂量率照射24小时后,仅SKBR-3细胞被杀死,其它肿瘤细胞系的所有细胞都能自动修复。结论:在低剂量率的β粒子放射性核素靶向疗法中,该实验结果为照射剂量率和照射时间的确定提供了指导。     

Temporal pattern of stimulation of osteoblast-associated genes during mechanically-induced osteogenesis in vivo: early responses of osteocalcin and type I collagen.

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.     

Temporal Pattern of Stimulation of Osteoblast-Associated Genes During Mechanically-Induced Osteogenesis In Vivo: Early Responses of Osteocalcin and Type I Collagen

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.     

Mature osteoblasts in human non-union fractures express collagen type III.

AIMS: High levels of collagen type III are biochemically detectable in biopsies of non-uniting fractures, and in the serum of patients suffering from this condition. The aim of this study was to determine whether the expression of collagen type III was limited to fibrous tissue in non-unions, or whether some was present in bone. METHODS: Biopsies from normally healing human fractures and non-unions were examined using in situ hybridisation and immunohistochemistry. RESULTS: The mesenchymal cell population, which includes fibroblast and osteoblast precursors, expressed mRNA for collagen type III. However, mature osteoblasts on the surface of woven bone varied profoundly between normally healing fractures (in which they were negative or occasionally weakly positive) and non-unions (in which they were strongly positive). Areas of woven bone that had osteoblasts positive for collagen type III mRNA also immunostained positively for the protein. CONCLUSIONS: This study shows that non-union fracture callus osteoblasts on the surfaces of woven bone exhibit an unusual phenotype: they express collagen type III, a molecule characteristic of an earlier stage of osteoblast differentiation, which is not expressed by osteoblasts on woven bone surfaces of bone that develops normally. This finding may be useful in developing an early clinical test for impending non-union.     

Synergistic effect of titanium alloy and collagen type I on cell adhesion, proliferation and differentiation of osteoblast-like cells

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors.     

纳米淡水珍珠粉对成骨相关基因表达的影响

背景:珍珠中高含量的钙离子可以促进钙盐沉积,抑制破骨细胞的骨吸收活性,促进骨再生,且其含有的水溶性蛋白具有骨诱导作用,可促进成骨细胞的分化。目的:观察纳米淡水珍珠粉对成骨细胞成骨相关基因表达的影响。方法:取第3代小鼠成骨细胞MC3T3-E1细胞,分别与纳米淡水珍珠粉(实验组)、纳米羟基磷灰石(对照组)共培养,以单独培养的细胞为阴性对照。培养7 d后,采用RT-PCR实验检测各组Runx2、骨桥蛋白、Ⅰ型胶原m RNA的表达。结果与结论:(1)实验组、对照组Runx2与骨桥蛋白mRNA表达量高于阴性对照组(P <0.05),并且实验组Runx2与骨桥蛋白m RNA表达量高于对照组(P <0.05);(2)实验组Ⅰ型胶原m RNA表达量高于对照组、阴性对照组(P <0.05),对照组与阴性对照组Ⅰ型胶原m RNA表达量比较差异无显著性意义(P> 0.05);(3)结果表明,纳米淡水珍珠粉较纳米羟基磷灰石更能显著促进成骨相关基因Runx2、骨桥蛋白、Ⅰ型胶原m RNA的表达。