过量氟诱导成釉细胞钙超载及细胞凋亡的实验研究

目的 研究过量氟对体外培养大鼠成釉细胞内钙超载及细胞凋亡的影响。方法 取大鼠成釉细胞系HAT-7细胞,分别加入不同浓度（0、0.4、0.8、1.6、3.2、6.4 mmol·L^-1）的氟化钠培养液,培养48 h后,采用Cell Counting Kit 8（CCK-8）试剂盒检测各组细胞的活性,流式细胞术分析氟对细胞凋亡的影响,激光扫描共聚焦显微镜、Western blot试验和实时荧光定量聚合酶链反应技术检测过量氟诱导大鼠成釉细胞内Ca²⁺浓度和钙网蛋白表达的变化。结果 氟化钠浓度高于1.6 mmol·L^-1时,可抑制成釉细胞的活性,成釉细胞内Ca²⁺浓度升高,钙网蛋白表达上调,细胞早期凋亡数量增加,并且随着浓度的增加,细胞凋亡的数量也随之增加。结论 过量氟可引起成釉细胞内钙超载,诱导成釉细胞凋亡。     

氟对体外培养大鼠成釉细胞活性和细胞凋亡的影响

目的 观察氟对体外培养的大鼠成釉细胞活性的影响,为探讨氟斑牙的形成机制提供依据。方法 取对数生长期的大鼠成釉细胞,在培养液中加入浓度为0、0.4、0.8、1.6、3.2、6.4 mmol/L的 NaF溶液,培养24、48、72 h后,采用CCK-8检测各组细胞的活性。荧光显微镜下观察成釉细胞核形态的变化,流式细胞术分析氟对细胞凋亡的影响。采用SPSS13.0软件包对数据进行统计学分析。结果 ①NaF浓度为0.4、0.8 mmol/L时,对成釉细胞有促增殖作用;NaF浓度为1.6、3.2、6.4 mmol/L时,对成釉细胞的活性有抑制作用,随着NaF浓度的增加,对细胞的抑制作用也逐渐增强,并且这种双向调节作用呈时间依赖性。②NaF浓度为0.4、0.8 mmol/L时,鲜见核破碎;NaF浓度为1.6、3.2 mmol/L时,存在核破碎,并且随着浓度的提高,核破碎的数量随之增加,即1.6 mmol/L浓度的NaF可引起成釉细胞凋亡,随着浓度的增加,细胞凋亡的数量随之增加。结论 ①氟对体外培养成釉细胞的增殖具有双向调节作用,即低浓度促进,高浓度抑制。②浓度超过1.6 mmol/L时,NaF诱导成釉细胞凋亡。     

氟对大鼠成釉细胞GRP-78和caspase-12表达的影响

目的研究GRP-78和caspase-12在氟致大鼠成釉细胞内质网应激和细胞凋亡中的作用,探讨氟所介导的内质网应激是否导致细胞凋亡。方法应用CCK8和流式细胞术观察不同浓度的氟对成釉细胞活性及凋亡数量的影响,以实时定量PCR和Western印迹法检测氟对内质网伴侣分子GRP-78和caspase-12基因和蛋白表达的影响,应用SPSS13.0软件包对数据进行统计学分析。结果随着氟浓度的不断增加,大鼠成釉细胞活性呈逐渐下降趋势,流式细胞术同样显示发生凋亡的细胞数量逐渐上升;实时定量RT-PCR和Western印迹结果显示,GRP-78和caspase-12的表达量随着氟浓度增加而增加。结论过量氟介导了大鼠成釉细胞内质网应激,并且通过内质网应激,引起细胞凋亡。     

过量氟对大鼠HAT-7细胞系自噬的影响

目的:研究过量氟对大鼠HAT-7细胞系自噬的影响.方法:取大鼠HAT-7细胞,分别加入不同浓度的氟化钠培养液,培养48 h后,透射电子显微镜检测过量氟对大鼠成釉细胞自噬泡的影响,Western免疫印迹和定量反转录聚合酶链反应技术检测过量氟诱导大鼠成釉细胞内微管相关蛋白轻链(LC3)和Beclin 1表达的变化.选择40只Wistar大鼠,随机分为A、B、C、D 4组,进行免疫组织化学实验,检测饮水氟对大鼠自噬分子表达的影响.采用SPSS 13.0软件包对数据进行统计学分析.结果:实验组自噬泡明显增多;Western免疫印迹及定量反转录聚合酶链反应结果显示,随着氟浓度的增加,HAT-7细胞自噬相关基因LC3和Beclin 1表达增加.回归分析结果显示,氟化钠与LC3及Beclin 1的表达有线性关系;大鼠体内免疫组织化学染色结果显示,实验组LC3以及Beclin 1为棕褐色,呈阳性表达.结论:过量氟诱导大鼠HAT-7细胞系自噬.     

内质网应激分子在慢性氟中毒大鼠成釉细胞中的表达

目的 研究不同浓度的氟化物对大鼠成釉细胞内质网应激分子表达的作用,探讨氟牙症形成的机制.方法 选择30只Wistar大鼠,随机分成A、B、C3组,并分别饮用氟浓度为0、75、150mg/L的自来水,8周后处死动物,并制备下颌切牙切片,通过HE染色、免疫组化、透射电镜、TUNEL检测等实验方法,观察3组大鼠成釉细胞内质网应激分子的表达及细胞凋亡情况.结果 随着氟浓度的升高,内质网应激分子GRP78 A组为阳性表达,B、C组为强阳性表达（F=27.42,P＜0.05）;XBP-1 A组为弱阳性表达,B组为阳性表达,C组为强阳性表达（F=139.7,P＜0.05）;Caspase-12 A组为弱阳性表达,B组为阳性表达,C组为强阳性表达（F=43.91,P＜0.05）;CHOP A组为阴性表达,B组为阳性表达,C组为强阳性表达（F=19.61,P＜0.05）;TUNEL检测显示氟浓度为150mg/L组成釉细胞凋亡数量显著高于75mg/L组和自来水组（F=124.02,P＜0.05）.利用MetaMorph显微图像分析软件对结果进行分析,用spss13.0软件包进行统计处理.结论 大鼠饮用高浓度的氟化水可激活内质网应激分子,导致成釉细胞产生内质网应激,并最终诱导细胞发生凋亡.     

不同浓度氟化物对体外培养成釉细胞活性的影响

目的建立大鼠成釉细胞原代培养技术,观察不同浓度氟化钠对成釉细胞活性的影响,为研究氟斑牙的形成提供依据。方法取10¹5 d的Wistar大鼠磨牙牙胚组织进行原代培养,通过酶消化法,分离培养成釉细胞。加入不同浓度(0、0.4、0.8、1.6、3.2、6.4 mmol/L)的氟化钠作用于成釉细胞,分别培养24、48、72 h后,采用CCK-8法检测各组细胞的活性情况。结果①当氟化物浓度为0.4、0.8 mmol/L时,对成釉细胞的增殖有促进作用,且随着时间的增加而增强。②当氟化物浓度为1.6、3.2、6.4 mmol/L时,对成釉细胞的增殖有抑制作用,随着氟化钠浓度的增加,对细胞的抑制作用也逐渐增强,并且抑制作用随着时间的延长愈发明显。结论不同浓度的氟化物对体外培养成釉细胞的活性具有促进和抑制双向作用。     

高氟对成釉细胞内氯离子浓度及pH值影响的研究

目的：检测高氟对成釉细胞内氯离子浓度以及pH值的影响。方法：2mmol／LNaF处理成釉细胞样细胞系LS8 30min,采用新型氯离子荧光探针MQAE以及LysoSensorTMDND-167分别显示细胞内氯离子浓度和pH值的变化;利用活细胞工作站实时监测加氟30min后细胞内氯离子浓度和pH值的变化。结果：MQAE呈现绿色荧光,其辉度值与细胞内的氯离子呈反比;加氟细胞30min后,实验组细胞平均绿色荧光强度较对照组增高;活细胞检测显示,对照组蓝色荧光和绿色荧光均少许下降,而实验组细胞双染后两种荧光均有所增强。结论：氟离子能影响细胞内氯离子浓度从而引起细胞内DH佰的降低而导致细胞的酸化。     

钙离子对人成骨细胞迁移与成骨分化的影响

目的 研究不同浓度Ca ²⁺对人成骨细胞迁移与成骨分化的影响,探讨促进迁移与成骨合适的Ca ²⁺浓度及相关机制。 方法 设置Ca ²⁺浓度,Transwell检测成骨细胞迁移;CCK-8法评估成骨细胞增殖;逆转录聚合酶链反应（RT-PCR）检测细胞成骨分化相关基因的表达;茜素红染色检测成骨分化生成的矿化结节。钙敏感受体（CaSR）拮抗剂拮抗后,观察Ca ²⁺对人成骨细胞迁移与成骨分化的影响。 结果 在迁移实验中,2、4、6 mmol·L ^-1的Ca ²⁺在3个时间点（8、16、24 h）都能明显地促进人成骨细胞的迁移,10 mmol·L ^-1的Ca ²⁺在8 h时明显抑制迁移。2~10 mmol·L ^-1 Ca ²⁺能促进人成骨细胞的增殖、成骨分化与矿化,8、10 mmol·L ^-1的Ca ²⁺诱导的矿化作用更明显。CaSR拮抗降低Ca ²⁺诱导的人成骨细胞迁移与成骨分化作用。 结论 低浓度Ca ²⁺有利于人成骨细胞迁移,高浓度Ca ²⁺有利于人成骨细胞分化,4、6 mmol·L ^-1的Ca ²⁺能较明显地同时诱导人成骨细胞迁移与成骨分化,Ca ²⁺-CaSR通路参与相应的信号传导。     

尼古丁抑制成牙本质细胞增殖及作用机制的研究

目的观察尼古丁对成牙本质细胞增殖的抑制作用，并检测其对成牙本质细胞中Ca2+浓度的影响，以探讨尼古丁抑制成牙本质细胞的分子机制。方法体外培养成牙本质细胞系MDPC- 23细胞, 按每毫升2×104个接种，随机分为实验组和对照组，对照组不加任何刺激，实验组施加质量浓度为100 μg/mL尼古丁，并于8 h后加入浓度为10 μmol/L BrdU进行细胞周期标记，刺激24 h后固定细胞，行免疫荧光抗BrdU染色，碘化丙锭（PI）复染胞核，荧光显微镜下计数细胞总数与BrdU阳性细胞数，计算S期阳性细胞率并进行统计学分析。培养成牙本质细胞于特制培养皿中，施加质量浓度为100 μg/mL尼古丁刺激，激光共聚焦显微镜下检测成牙本质细胞中Ca2+浓度的动态变化。结果实验组、对照组S期阳性细胞率分别为36.3%、48.2%，实验组显著低于对照组。尼古丁刺激后，成牙本质细胞中Ca2+浓度迅速升高，在较高水平维持一段时间后缓慢下降。结论尼古丁可抑制成牙本质细胞增殖，这种作用同尼古丁升高成牙本质细胞中Ca2+浓度有关。     

维甲酸对口腔上皮细胞内游离钙的调节

目的:探讨维甲酸(RA)对细胞内钙水平的调节作用。方法:将培养的第二代口腔上皮细胞以2×104/cm2接种于激光共聚焦测试培养皿,细胞贴壁后,分别在Ca2 浓度为0.09mmol/L和1.2mmol/L的角朊细胞培养基(K-SFM)中继续培养12h。先分别加入钙离子指示剂Fluo-3/AM负载后,再加入10-7M的维甲酸在不同时段激光共聚焦显微镜下观察细胞内钙离子分布变化并进行定量分析。结果:经含维甲酸处理的高钙环境生长的细胞内钙含量明显减少,而维甲酸对正常K-SFM中生长的细胞内钙含量无明显影响。结论:维甲酸调节上皮细胞分化的机制可能是通过减少细胞的游离钙水平而发挥作用。     

Role of injured endothelial cells in the recruitment of human pulp cells

In restorative dentistry, deep cavity preparation may lead to partial destruction of the odontoblastic layer. However, newly formed odontoblast-like cells can replace the necrotic odontoblasts and secrete a reparative dentine matrix. While growth factors such as transforming growth factor beta1 (TGFbeta1) and bone morphogenetic proteins (BMP-2 and BMP-4) seem to be involved in the proliferation and differentiation of pulp cells, little is known about the migration of the newly proliferating stem cells to the injury site. Our hypothesis was that endothelial cell injury may be involved in directing these cells towards the injury site. For this study, human pulp fibroblasts and L929 cells were fluorescence-labeled by transduction with the Enhanced Green Fluorescent Protein (EGFP). Similarly, human umbilical vein endothelial cells (HUVEC) were labeled with the Discosoma Red Fluorescent Protein-2 (DsRed2). Cell migration was then studied in an insert cell culture system. The HUVEC cells were cultured in the lower compartment while the human pulp fibroblasts or L929 were in the upper compartment. After artificial injury to the HUVEC cells, only human pulp fibroblasts migrated to the lower compartment. At early time periods (4 days), migrating cells were randomly localized on the HUVEC layer. However, after 14 and 20 days, they were perfectly aligned along the injury site. In the absence of injury, no migration was observed. These results suggest that, the endothelial injury is involved in the recruitment of odontoblast-like cells at the injury site.     

Mast cells of the human gingiva

By way of degranulation, the mast cells release a number of biologically active substances into the connective tissue. The present study is concerned with the relation of the gingival mast cells to the pathogenesis of gingivitis. Following fixation in Newcomer's fluid and non-aqueous staining at pH 0.5 in acridine orange¹, topographically defined zones of sections of normal and inflamed marginal gingiva, histologically classified with regard to degree of inflammation, of 56 different individuals have been studied in the fluorescence microscope. The human gingiva was found to be comparatively rich in mast cells. Three main morphological variants were observed and their topographical distribution within the tissue have been described. Marked differences in stainability between mast cells of different areas of the connective tissue have been recorded, and correlated to the state of inflammation, In spite of individual variations in mast cell density, definite patterns of frequency and distribution were observed. The number of mast cells appeared inversely correlated to the density and distribution of the inflammatory cellular exudate within the pocket area of the connective tissue. Consequently, normal gingivae generally contained more mast cells per tissue unit than the moderately inflamed tissue, which, in turn, contained more than the severely inflamed gingivae. Exceptions were found in some moderately inflamed, fibrous gingiva with evidence of strong fibroblastic activity, where there was an increased number of mast cells. On basis of the distribution, frequency and stainability of the mast cells of the gingiva, it is suggested that the mast cells of the regions adjacent to the tooth are subject to an enzymatic degranulation as elicited by products elaborated by the gingival bacterial plaque or possibly by local antigen-antibody interactions. Substances released by degranulation may then act as mediators during the course of the inflammatory process as well as contribute to the local resistance against injury.     

Mast cells of the human gingiva

Comparative histochemical studies with specific regard to the demonstration of mast cells were carried out on 21 different chronically inflamed marginal gingival specimens. For the demonstration of sulphated acid mucopolysaccharides two metachromatic stains were used-toluidine blue (pH 1.0) and acridine orange (pH0.5)-, as well as one orthochromatic stain- astra blue (pH 0.2–0.3). In addition the alcian blue-safranin sequence was employed to distinguish between weakly and strongly sulphated mucopolysaccharides. Trypsin-like esterase activity was demonstrated by the EACNAS-GBC technique. The metachromatic stains used stain mature mast cells specifically, while the non-metachromatic techniques stain immature mast cells as well. The chemical backgrounds for the different staining reactions are discussed. The chronically inflamed human gingivae were found to be rich in both mature and immature mast cells. The great majority of the immature mast cells were found within the pocket area of the connective tissue. Here, the number of mature mast cells was significantly smaller than in the oral area. The distribution of the different mast cell variants indicate a relation between mast cells and diffusible products from the gingival bacterial plaque.     

Mast cells of the human gingiva

One-side gingivitis was induced in 21 individuals by local abolition of tooth cleansing for 15–17 days. Adjoining sections of biopsies from the non-cleansing side as well as from the control side with good tooth cleansing were stained with toluidine blue (pH 1.0), acridine orange (pH 0.5), astra blue (pH 0.2—0.3), alcian blue-safranin and the EACNAS-GBC technique in order to demonstrate mast cells specifically. The clinically normal gingiva was found to be rich in mast cells. In the inflamed tissue, the number was increased, especially in the pocket area of the connective tissue. The difference was due to an increase of both mature and, particularly, of immature mast cells. In those cases, where the inflammatory infiltrates were heavy, however, the number of mast cells was reduced in the area of cellular infiltration. The results indicate that products from the bacterial plaque at the gingival margin may directly or indirectly induce proliferation of mast cells in the adjacent connective tissue. Above a certain degree of stress, however the mast cells may respond by degranulation and release their active substances, which by contributing to the inflammatory reaction may enhance the local resistance.     

Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro

Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells' expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.     

生长期兔髁突纤维软骨细胞和骨骺透明软骨细胞生物学特性的比较研究

目的 建立软骨细胞体外培养模型,研究纤维软骨和透明软骨的生物学差异。 方法 分离4周龄雌性兔双侧髁突软骨和膝关节骨骺软骨,采用胰酶和胶原酶联合消化的方法收集软骨细胞,分别进行体外培养并连续传代至P10。使用倒置显微镜观察细胞形态;采用阿利新蓝和Ⅱ型胶原免疫组化染色的方法分别对髁突纤维软骨细胞和骨骺透明软骨细胞进行鉴定;用细胞计数的方法绘制生长曲线;运用荧光定量PCR和Western印迹技术对软骨特异性指标Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan进行检测,并使用SPSS 13.0软件包对数据进行统计学分析。 结果 倒置显微镜观察示,髁突纤维软骨细胞和骨骺透明软骨细胞形态均会随细胞传代而发生改变,且P2代以后改变明显;基因和蛋白水平的检测均证实P2代后软骨细胞去分化明显。阿利新蓝和Ⅱ型胶原免疫组化染色鉴定结果均为阳性,对照组为阴性。实时定量荧光PCR和Western印迹结果证明：在纤维软骨中,Ⅰ型胶原的表达量高于透明软骨;而Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan的表达量却明显低于透明软骨。 结论 使用本方法培养软骨细胞简单有效;髁突纤维软骨细胞和骨骺透明软骨细胞体外培养均存在去分化现象,且两者Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原、SOX9和Aggrecan的表达存在显著差异,生物学特性并不相同。     

小鼠骨髓细胞和脾细胞诱导形成破骨细胞潜能的比较

目的在体外破骨细胞分化因子(RANKL)和巨噬细胞集落刺激因子(M-CSF)联合应用的情况下,比较小鼠骨髓细胞和脾细胞形成破骨细胞的能力。方法选用小鼠M-CSF依赖性非附着性骨髓细胞和脾细胞,以不同的细胞密度在含有25ng/mlsM-CSF和30ng/mlsRANKL的(-MEM培养液中培养5、9天后,计数形成的抗酒石酸酸性磷酸酶染色(TRAP)阳性多核细胞的数目和骨吸收面积。结果脾细胞形成的破骨样细胞与骨髓形成的细胞形态与功能均无明显差异,但所需的细胞密度为骨髓细胞的10~20倍。结论在特殊情况下,脾细胞可替代骨髓细胞进行体外破骨细胞实验,但培养条件应适当调整。     

牙髓培养细胞特性与牙髓干细胞定位

目的 比较人牙根髓与冠髓培养细胞的特性，探讨牙髓干细胞在牙髓中的定位。方法：用组织块法分别培养人根髓、冠髓细胞，观察并比较两者的培养成功率、细胞贴壁率、细胞活性、增殖特性、细胞形态以及细胞诱导矿化能力，以探讨牙髓干细胞在牙髓组织中的定位。结果：培养的根髓细胞比冠髓细胞具有较高的成功率和贴壁率、较强的细胞活性以及相同的增殖活性，根髓细胞比冠髓细胞的形态更具有原始性，根髓比冠髓更易诱导矿化。结论：牙髓干细胞可能存在于全部牙髓之中，在根髓中比冠髓中具有更高的密度．