人结合上皮细胞体外培养与冻存方法的改良

目的:改良人结合上皮(junctional epithelium,JE)细胞的分离培养及冻存方法。方法:取正畸拔除的牙周组织健康的前磨牙,沿龈缘剪去冠方1mm左右的沟内上皮,用无菌11号刀片紧贴牙面刮下、剪碎JE组织,简化培养操作步骤。取第2代JE细胞,加入冻存液,按设定的降温程序(从0℃开始,以每分钟下降1.5℃速度下降至-18℃,保持5min;再以每分钟下降20℃的速度降至-80℃,然后投入-196℃液氮罐)冻存细胞,40℃水浴复苏,进一步研究JE细胞的形态、增殖、鉴定及复苏后存活率等生物学活性。结果:不用Dispase冷消化,采用0.1%胰蛋白酶-0.02%EDTA消化,同样可成功地培养人JE细胞;JE细胞复苏后的存活率为(93.87±3.11)%,生长状况良好,与第2代细胞相似;免疫细胞化学染色显示,培养的JE细胞角蛋白-19(cytokeratin-19,CK19)表达强阳性,而波形丝蛋白(vimentin)表达也为阳性。结论:改良的JE分离培养与冻存方法可行,JE表型在体内和体外并非完全一致,可能与细胞生长的底物有关。     

长结合上皮和结合上皮生物学特性的比较

目的:体外培养长结合上皮(long junctional epithelium,LJE)细胞,研究其生物学特性并与结合上皮细胞(junctional epithelium,JE)进行比较。方法:LJE取自慢性牙周炎患者翻瓣手术切除的标本,无菌剪刀剪去沟内上皮,采用酶消化法和无血清的角化上皮细胞培养液培养。JE取自健康正畸牙或阻生牙,同法培养。采用细胞分析仪、流式细胞仪和激光共聚焦显微镜比较2种细胞在形态、大小、细胞活力、分子表达及增殖凋亡上的差异。采用SPSS11.5软件包对数据进行独立样本t检验。结果:LJE和JE细胞形态相似,LJE活力低于JE细胞但不存在统计差异(P0.05)。LJE细胞直径为(19.03±0.19)μm,体积为(4.29±0.19)×103fL;JE细胞直径为(17.16±0.95)μm,体积为(3.20±0.52)×103fL,2种细胞大小有显著差异(P0.05)。LJE细胞的倍增时间比JE细胞长。CK19在LJE中表达比JE细胞弱,而CK6、CK13在2种细胞中的表达相似。与对照组相比,LJE细胞的早期、晚期凋亡率分别是(4.62±2.16)%和(9.46±1.84)%,而JE细胞分别是(0.47±0.33)%和(3.84±0.98)%,2组细胞的凋亡率有显著差异(P0.05)。结论:LJE和JE细胞在大小、CK19表达、增殖、凋亡上存在差异,对进一步了解LJE的生物学特性和牙周炎损伤、修复过程有一定意义。     

表皮生长因子对结合上皮和牙龈上皮体外损伤修复的影响

目的:研究表皮生长因子(epidermal growth factor,EGF)对结合上皮(junctional epithelium,JE)和牙龈上皮(gingival epithelium,GE)体外损伤后细胞修复和增殖的影响.方法:采用RT-PCR和免疫细胞化学方法,分别检测表皮生长因子受体(epidermal growth factor receptor,ECFR)mRNA和蛋白在JE和GE中的表达;制备细胞爬片,待生长融合至单层时,于玻片中央作宽为3mm的损伤区,然后加入含有EGF(20ng/mL)的条件培养液继续培养,直接加新鲜培养液作为阴性对照.在损伤后第5、9和12天,分别作HE和增殖细胞核抗原(proliferating cell nucleal antigen,PCNA)染色,计算细胞修复率及PCNA阳性率,采用SAS6.12软件包进行统计学分析.结果:RT-PCR显示,JE表达EGFR mRNA,与GE之比为1:1.2;EGFR蛋白在两者表达均为阳性.在第9天,EGF促进GE和JE损伤后的细胞增殖,与阴性对照组比较,均有显著性差异(P<0.05),但GE和JE两组间无显著差别(P>0.05).另外,EGF也可以促进GE和JE损伤后的细胞修复,在第9和12天,GE的修复速度显著快于JE(P<0.05).结论:JE和GE细胞均存在EGFR,EGF对两者体外损伤后的细胞修复与增殖具有调节作用.     

人牙龈结合上皮和口腔龈上皮细胞的附着生长特性比较

目的 比较牙龈结合上皮(junctional epithelium,JE)与口腔龈上皮(oral epithelium,OE)细胞的生长附着特性,观察附着结构的形成过程,为牙周附着的形成提供实验依据.方法 人JE和OE取自正畸或阻生原因拔除的牙周健康牙,采用酶消化法和无血清角质细胞培养液分离培养细胞.将5×108个/L JE细胞1ml接种到放置于24孔板的预处理5mm×3mm×1mm牙骨质片上(每孔3片,共21片),于37℃ 5%CO2培养箱中复合培养,1～14d内取样本,透射电镜下观察附着结构的形成;同法以人OE细胞-牙骨质片复合培养作对照研究.结果 JE细胞形态多样,排列散乱,CKl9表达强阳性;OE细胞呈多边形或角形,"铺路石"状排列,仅少量OE细胞CK19阳性.JE细胞-牙骨质片复合培养1～3d,牙片上附着细胞少,9d时接触牙片的细胞膜局部出现少量电子致密沉积物,11～14d出现复层生长,与牙面接触处可见半桥粒样结构;而OE细胞-牙骨质片复合培养1～3 d牙骨质片上细胞较多,部分已伸展成扁平形,7 d时细胞膜和牙面间出现大量颗粒状电子致密沉积物,9 d时细胞呈复层生长,可见半桥粒样结构.结论 人JE细胞是一种不同于OE细胞的未成熟、低分化细胞,在同样条件下体外培养的JE细胞在牙片上的生长和附着均较OE细胞慢,其对牙面的附着强度值得深入研究.     

大鼠牙囊细胞体外培养与表型鉴定

目的：体外培养大鼠牙囊细胞（RDFCs）,鉴定其细胞来源及表型,为牙周组织再生的研究提供可靠的种子细胞来源。方法：选择出生后6-7d SD仔鼠,分离上、下颌第一、第二磨牙牙胚,取牙囊组织进行原代和传代培养。通过倒置显微镜观察细胞形态及生长情况;波形丝蛋白（Vimentin,VIM）和角蛋白（Cytokeratin,CK）鉴定其细胞来源;免疫组化染色技术检测细胞骨桥蛋白（osteopontin,OPN）、骨涎蛋白（bone sialoprotein,BSP）、Ⅰ型胶原（type Ⅰ collagen,CoL-Ⅰ）、Ⅲ型胶原（typeⅢ collagen,CoL-Ⅲ）及纤维连接蛋白（fibronectin,FN）的表达。结果：细胞形态呈多形性,有长梭形,纺锤形、不规则三角形等,波形丝蛋白染色阳性,角蛋白染色阴性。免疫组化染色显示OPN、BSP、CoL-Ⅰ、CoL-Ⅲ、FN在胞浆中呈不同程度的阳性表达。结论：大鼠牙囊细胞来源于外胚间充质,具有成纤维细胞的形态特征及成骨/成牙骨质细胞表型特征,可将牙囊细胞作为种子细胞用于牙周组织工程的再生研究。     

山羊颞下颌关节盘细胞体外培养研究

目的:研究山羊颞下颌关节盘纤维软骨细胞体外培养的生物学特性及其损伤修复的细胞学基础.方法:切取1 月龄山羊颞下颌关节盘, II型胶原酶消化获得关节盘纤维软骨细胞.逐日观察细胞的形态变化, 测定其生长曲线,甲苯胺蓝染色、I型胶原免疫组化染色鉴定.透射电镜观察细胞超微结构.结果:原代培养的关节盘纤维软骨细胞以长梭形为主, 部分多角形, 7 d即可传代.甲苯胺蓝染色阳性, I型胶原免疫组化染色胞质内可见棕黄色颗粒.超微结构显示细胞分为2 种,软骨细胞样细胞和成纤维细胞样细胞;线粒体、内质网发达.结论:体外分离培养的山羊颞下颌关节盘纤维软骨细胞具有较强的增殖能力,1～3代细胞可作为颞下颌关节盘组织工程的种子细胞.     

人牙龈结合上皮、口腔龈上皮及沟内上皮中细胞角蛋白的表达

目的 检测人牙龈上皮的细胞角蛋白(CK)谱，探讨结合上皮(JE)与口腔龈上皮(OE)、沟内上皮(SE)的不同。方法 5例人磨牙及前磨牙牙龈颊舌向石蜡切片，采用免疫组化SP法行CK5／6、7、8／18、10／13、16、17、19、20染色。结果 CK7、17在3种上皮中均无表达；CK5／6、20在3种上皮的基底上层(尤其是近表层)为弱阳性及阳性表达，基底层无表达；CK10／13、16在JE全层均有表达，在OE和SE仅限于基底上层，其中CK10／13为强阳性，CKl6为弱阳性及阳性；CK19在JE全层强阳性，在OE和SE仅限于基底层，JE和SE分界明显；CK8／18与CK19相似，只是表达较弱，近基底层的基底上层也有少量表达。结论JE是一种不同于OE和SE的特殊低分化上皮，CK19可作为JE的特征性标记物区别于OE和SE，CK10／13、16可用于体外培养的上述3种细胞的鉴别。     

体外培养大鼠颌下腺细胞的生物学特性研究

目的 :研究体外培养颌下腺细胞生物学特性。方法 :应用胰酶消化法进行颌下腺细胞的分离纯化 ,然后进行颌下腺细胞原代和传代培养。绘制培养细胞的生长曲线 ;MTT法检测培养细胞活力大小。透射电镜 (TEM )观察结合免疫组化检测鉴定细胞来源及性质。结果 :整个颌下腺细胞生长期间为呈多样性的上皮细胞 ,细胞可传 4～ 5代 ,成活 3 0～ 40d。随培养时间延长 ,各组细胞数量都有不同程度的增加 ,比较培养 72h原代、第 2代与第 4代细胞吸光度值 ,原代、第 2代与第 4代相比较有显著性差异 (P <0 .0 5 )。超微结构观察及免疫组化染色培养细胞表现为腺上皮细胞的特征。结论 :原代和传代培养第 1代、第 2代颌下腺细胞活性较强 ,可在进一步的实验得以应用     

高度浓缩生长因子对MC3T3-E1成骨细胞生物学性能的影响

目的：观察高度浓缩生长因子（concentrated growth factor,CGF）对成骨细胞生物学性能的影响。方法：将MC3T3-E1细胞在CGF环境下进行培养,并设立空白对照组。扫描电镜观察成骨细胞在CGF表面的附着;培养1、4、7 d后,检测细胞增殖情况以及碱性磷酸酶（ALP）活性;茜素红染色观察矿化结节和成骨相关基因Runx2的表达。CGF浸出液培养细胞24 h后,以鬼笔环肽染色观察细胞骨架的形态变化。采用 SPSS 21.0软件包对数据进行统计学分析。结果：CCK-8比色显示,培养1、4、7 d,在相同时间点,实验组与对照组相比,CGF细胞增殖活性显著增强（P< 0.05）。ALP活性检测显示,培养1 d后,实验组与对照组无显著差异（P>0.05）;培养4、7 d后,实验组与对照组相比,ALP活性具有显著差异（P<0.05)。茜素红染色显示,CGF组钙化结节数量增多且面积较大。鬼笔环肽染色和DAPI染色可见,CGF组中细胞数量增多,细胞铺展面增大,肌动蛋白形态更为清晰。CGF可促进Runx2 mRNA表达（P<0.05）。结论：CGF可促进MC3T3-E1细胞的增殖、分化以及Runx2的表达。     

应用牙龈角质细胞构建组织工程化牙龈的实验研究

目的：建立体外连续培养的人牙龈角质细胞系，探讨构建人工复合牙龈的方法，为种植体周围软组织改善提供帮助。方法：体外无血清培养人牙龈角质细胞．探索细胞体外生长的动力学规律。将角质细胞与脱细胞真皮基质复合，构建组织工程化牙龈。结果：体外连续传代培养人牙龈角质细胞，可传4～6代，存活30～50d；培养细胞具有角质细胞的典型形态和染色特征。第1、2代细胞群体倍增时间最短，贴壁率、克隆形成率最高。角质细胞可以接种于脱细胞真皮基质上，形成组织工程化牙龈。结论：体外培养扩增人的牙龈角质细胞，第2、3代较适合用于组织工程化牙龈的构建。脱细胞真皮基质可作为组织工程化牙龈的支架材料。     

Histological study of lectin binding in regenerated rat junctional epithelium

We investigated the expression of carbohydrate residues in regenerated junctional epithelium (JE) cells histopathologically with lectin staining to clarify the mechanisms responsible for the changes in their expression in JE cells derived from residual oral epithelium. Curettage and root planing procedures were performed on the buccal gingival sulci of rat first lower molars, and JE and connective tissues were completely removed. The mandibles were resected after 12 h and 1, 2, 3, 4, 5, 7, 10 and 14 days, fixed with paraformaldehyde, decalcified with EDTA and embedded in paraffin. Serial sections were stained histochemically with four kinds of lectins (PNA, DBA, GS I, UEA I) to clarify the expression patterns of carbohydrate residues in regenerating epithelium. No binding of PNA or DBA was observed even when the regenerating epithelium was attached to the root surface, and they showed the same negative reactions as the basal cells of oral gingival epithelium (OGE). Positive reactions were, however, observed on the more stratified regenerating epithelium along the root surface. Positive reactions with GS I and negative reactions with UEA I were observed throughout the regeneration process, and these were the same as those observed in the basal cells of OGE. Therefore, we concluded that the basal cells and regenerated epithelium derived from OGE expressed the same carbohydrate residues. Futher-more, the expression of carbohydrate residues, one of the characteristics of JE, was related not only to the attachment to the tooth surface but also to changes of cell shape and cytoskeleton with stratification along the root surface.     

Junctional epithelium in rats is characterized by slow cell proliferation

BACKGROUND: The integrity of junctional epithelium (JE) and a firm epithelial adhesion to the tooth surface are maintained by the balance between cell proliferation and cell death. Maintaining the JE structure is essential for the protection of periodontal connective tissues against oral microbes. In this study, the proliferative activity and the expression of caspase 3, a cysteine protease associated with cell death, were studied in rat JE and other epithelial structures during molar tooth development. METHODS: Fourteen rats aged 10 to 70 days were injected with bromodeoxyuridine (BrdU). Samples of first and second molars were selected for immunohistochemical staining. BrdU incorporation was studied in oral epithelium (OE) covering the erupting tooth, reduced enamel epithelium (REE), and gingival epithelium (GE), sulcular epithelium (SE), and JE. Samples were also subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA) and caspase 3. RESULTS: The basal cells of the GE were actively proliferating, but in the JE, only a few cells were positive for BrdU or PCNA immunostaining. Some outer REE cells were proliferating during tooth eruption. Caspase 3 expression was in specific areas of REE after completion of amelogenesis. CONCLUSIONS: Results showed slow proliferative activity in the rat JE. However, specific studies on cellular turnover and cell migration are needed to understand tissue homeostasis in this area.     

Localization of ODAM,PCNA, and CK14 in regenerating junctional epithelium during orthodontic tooth movement in rats

Objective:To identify the regenerating junctional epithelium (JE) during orthodontic tooth movement in rats.Materials and Methods:Closed-coil springs were used to create a 20 g mesial force to the maxillary first molars. On days 1, 3, 7, 10, and 14 after force application, histologic changes in JE were examined by immunohistochemistry using proliferating cell nuclear antigen (PCNA), odontogenic ameloblast-associated protein (ODAM), and cytokeratin 14 (CK14).Results:On day 1, JE was destroyed and lost attachment to the tooth surface. Cell division activity was rarely observed in JE, and ODAM localization was weakly detected in damaged JE. By day 3, regenerating JE had not fully recovered. High cell proliferation activity and CK14 expression started to appear in most basal cells of JE. ODAM expression was reduced and appeared in a small area. By day 7, JE had almost recovered. Cell proliferation activity was still observed in several basal cells of JE, and ODAM expression was detected among JE cells. CK14 was hardly observed in JE except in the basal cells. By days 10 and 14, regenerated JE appeared. ODAM, PCNA, and CK14 expression was similar to that of the control.Conclusions:Damaged JE might recover rapidly during orthodontic tooth movement because basal cells of the remaining JE, which show higher proliferation activity, are involved in JE regeneration. Reduced ODAM expression during proliferation of JE cells may increase again after JE regeneration is complete. Therefore, ODAM may be associated with the normal function of JE.     

机械损伤大鼠结合上皮后的组织修复观察

目的：观察两种机械方式破坏大鼠结合上皮（JE）后的组织修复过程。方法：12只健康sD大鼠分别用探针、龈下刮治器损伤左、右下前牙JE,于2、7、14、21d处死大鼠,HE、PCNA染色,镜下观察计数。结果：第2天大量炎细胞浸润,上皮PCNA阳性细胞散在分布;第7天炎症减轻,上皮PCNA阳性率开始增加,增殖并部分和牙面贴附;14d纤维组织增殖明显,上皮增厚;21dJE基本修复。结论：龈下刮治对牙周损伤比探损伤重;结缔组织的快速增殖可能会阻止UE形成。     

Cytokeratin profile of the junctional epithelium in partially erupted teeth

This study uses cytokeratins (CK) as markers to investigate the phenotype of the junctional epithelium (JE) in partially erupted human teeth. The gingival samples, which were clinically healthy, were carefully dissected from the teeth. Cryostat sections were cut for histological staining, immunofluorescence microscopy and gel electrophoresis. Cytokeratins were extracted after microdis-section. The basal and suprabasal epithelial cell markers, cytokeratins 4, 5, 13, 14 and 19 were detected with specific monoclonal antibodies. They showed that the junctional epithelium in erupting teeth has a complex topography. The cytokeratin immunohistochemical profile distinguished between the primary junctional epithelium (CK 5, 14 and 19 in basal and suprabasal cells and CK 13 faintly stained throughout the suprabasal layers) and the adjacent epithelium that had the same cytokeratin profile as the sulcular epithelium (CK 5, 14 and 19 in basal cells and CK 4 and 13 intensively stained in the suprabasal cells). Extraction, two-dimensional electrophoresis and western blotting showed that this transitional JE during eruption also contained CK 6, 16 and perhaps CK 4. Thus, the JE in erupting teeth shows patterns of CK distribution that are very similar to that of developing oral epithelia.     

Immunohistochemical distribution of keratin proteins in clinically healthy human gingival epithelia

Abstract – In clinically healthy/subclinically inflamed biopsies of marginal gingiva, the immunohistochemical distribution of keratin proteins was studied in junctional (JE), sulcular (SE), oral gingival (OGE) and in a few samples of alveolar mucosal epithelium (AE) by means of various mouse monoclonal anti-keratin antibodies in an indirect fluorescence technique. All regions stained in a nearly similar way with AE3 (keratins 1–8, all cells) and BE 14 (keratin 5, basal and supra/parabasal cells). AE8-staining (keratin 13, supra/parabasal and spinous cells) was primarily confined to the stratified, nonkeratinized epithelia SE and AE, but also a variable part of JE and less frequently OGE were positive. The parakeratinized OGE was distinct in showing a homogeneous staining with AE2 (keratins 1/2, 10) and AE5 (keratin 3) throughout spinous cell layers. These antibodies did not stain JE and AE whereas SE stained in a scattered way with AE5 and sometimes also with AE2. The latter finding might indicate initial keratinization at molecular level. The JE was distinct in retaining basal characteristics throughout the epithelium with PKK2 (keratin 7, 16, 17, 19) and BE14 (keratin 5) although some initial suprabasal maturation, as observed with AE8, cannot be excluded. Differences in keratin staining of gingival epithelia and the AE was found with respect to AE 1 -reactivity (keratins 10, 14–16, 19) which was suprabasal in JE, SE and OGE but basal in AE.