不同培养条件对骨髓肌细胞增殖及分化的影响

目的：研究不同培养条件对骨髓肌细胞增殖分化的影响。方法：以体外培养小鼠肌母细胞Ｃ２Ｃ１２为研究模型，对照组用含φ＝２％胎牛轿清的ＤＭＥ液，分别于更换培养液后２４、４８、７２ｈ，观察细胞形态变化。结果：对照组细胞形态实验前后无变化，实验组２４．４８ｈ时部分细胞出现死亡，７２ｈ存活细胞开始融合，肌管形成。结论：胎牛血清含量为φ＝２％的ＤＭＥＭ液有促进肌母细胞分化，从而进上步形成骨管的作用；含φ＝１０％     

小鼠骨骼肌细胞体外培养的增殖及分化关系

目的 :研究体外培养的小鼠骨骼肌细胞增殖及分化的关系 ,为临床肌肉损伤的治疗提供理论基础。方法 :采用细胞培养方法 ,对小鼠肌母细胞C2C12进行活细胞观察 ,采用MTT法研究骨骼肌细胞生长特性。结果 :C2C12小鼠肌母细胞 ,在对照组中不断增殖 ,表现为数量增加 ,而细胞的形态不发生改变。实验组在分化培养2 4h后部分细胞出现死亡 ,活细胞率下降 ,尤以 48h为著 ,72h后活细胞数量开始上升 ,存活的鼠肌母细胞开始出现分化现象 ,肌母细胞离开初始的细胞周期 ,而进入细胞分化阶段 ,形态上表现为细胞开始融合 ,形成多核肌管。结论 :体外培养的骨骼肌细胞的增殖和分化有非常密切的关系 ,细胞的增殖为分化做准备 ,分化时需要细胞退出增殖周期 ,活细胞率有所下降 ,肌管形成后活细胞基本保持不变。     

新生SD大鼠成肌细胞体外培养的实验研究

目的 :了解新生SD大鼠成肌细胞在体外培养条件下的生物学特性。方法 :新生 3d内SD大鼠幼崽 ,切取骨骼肌标本。应用Blau法经胰蛋白酶与胶原酶多步消化、分离成肌细胞。差速贴壁法纯化后 ,在 φ =2 0 %的胎牛血清生长培养基中培养 ,绘制生长曲线评价其增殖情况 ,φ =5 %的胎牛血清融合培养基培养 ,计算成肌细胞融合率评价其分化能力。通过光镜、免疫组织化学方法鉴定所得细胞。结果 :培养后成肌细胞免疫组织化学染色强阳性 ,能融合形成肌小管 ,证明为骨骼肌成肌细胞。在生长培养基中原代细胞倍增时间为 5d ,在融合培养基中肌小管融合率较高。结论 :多步酶消化法与差速贴壁法能获得足量、纯净的成肌细胞 ,所得细胞增殖力强 ,能表达骨骼肌特异收缩蛋白 ,融合培养基能促进其体外分化。     

HEMA对人乳牙牙髓干细胞分化潜能的影响

目的:探究HEMA对人乳牙牙髓干细胞向成牙本质细胞分化的影响。方法:将不同浓度的HEMA加入到SHED细胞中分别培养24 h、48 h和72 h后，MTT实验检测其毒性作用。将50 mg/mL抗坏血酸，10 mmol/L β-甘油磷酸钠和100 nmol/L地塞米松加入含10%胎牛血清的DMEM中制备矿化诱导液，无毒浓度HEMA(0.1 mmol/L和0.25 mmol/L)下，诱导SHED向成牙本质细胞分化，素红染色观察矿化结节的形成。结果:当HEMA浓度超过0.5 mmol/L,培养时间超过48 h时，HEMA对SHED细胞有毒性作用。长时间暴露于无毒浓度HEMA中，SHED细胞的分化潜能受到抑制。结论:HEMA可抑制SHED细胞向成牙本质细胞分化。     

大鼠颌突外胚间充质细胞向骨骼肌细胞诱导分化和三维培养观察

目的：探讨体外诱导外胚间充质细胞向骨骼肌细胞分化的潜能，利用分化细胞构建组织工程骨骼肌模型。方法：取妊娠10．5d胎鼠颌突外胚间充质细胞，纯化后分别在含1、5、10mL／L体积浓度二甲基亚砜的DMEM／F12培养基中培养。相差显微镜下观察细胞的形态变化，成肌相关因子免疫组化鉴定细胞性质，将诱导后的细胞种植于胶原膜，并于培养3、6d后用光镜、扫描电镜观察细胞的生长情况。结果：培养3d后，外胚间充质细胞形态出现成肌样细胞变化，细胞变得大而扁平，培养5d后细胞出现管样结构。免疫组化鉴定显示：添加二甲基亚砜后，大部分细胞出现成肌相关因子的表达，其中5mL／L实验组效果最好，约75％的细胞转化为骨骼肌细胞。对照细胞形态未见明显改变，免疫组化鉴定未能诱导分化出骨骼肌细胞。种植于BAM膜后，细胞生长良好，并可形成复层结构。结论：二甲基亚砜可诱导外胚间充质细胞向骨骼肌细胞分化，最佳浓度为5mL／L；在BAM膜上分化细胞生长状态良好，说明该生物可降解膜是构建组织工程骨骼肌的适合材料。     

骨髓基质细胞矿化诱导条件优化的研究

目的:探讨骨髓基质细胞在不同矿化诱导条件下的生物学特性,优化最佳矿化条件。方法:采用普通矿化液诱导组、BMP诱导组和矿化液BMP联合诱导组,体外诱导兔骨髓基质细胞,以含10%胎牛血清的DMEM作为对照组。倒置相差显微镜观察细胞形态的改变,MTT法检测细胞增殖情况,组化方法检测ALP活性,免疫组织化学方法检测I型胶原合成情况。结果:与对照组相比,实验组骨髓基质细胞增殖力均有所抑制,而ALP活性增强,I型胶原呈强阳性表达。其中矿化液BMP联合诱导组诱导产生的成骨细胞表型最为显著。结论:联合应用矿化液和BMP能在较短的时间内有效诱导BMSC分化为成骨细胞。     

尼古丁对体外培养鼠磨牙牙胚发育的影响

目的:探讨尼古丁对体外鼠磨牙牙胚发育的影响.方法:采用RPMI-1640半固态培养基,在培养基中加入不同浓度的尼古丁,将15d胎龄的胎鼠的下颌第一磨牙牙胚分别置于各浓度组RPMI-1640半固态培养基表面培养4、8d,每48h更换培养基一次.结果:实验组与对照组相比较,主要表现在实验组发育中的牙胚体积缩小,成牙本质样细胞的分化程度不足、数量和速度均低于对照组,前期牙本质样基质形成减少或不能形成,并且呈时间、浓度依赖性.结论:尼古丁对体外培养胎鼠牙胚的形态、细胞的增殖和分化有抑制作用.     

人牙龈成纤维细胞诱导分化为骨骼肌细胞的探讨

目的探讨人牙龈成纤维细胞向骨骼肌细胞分化的潜能和可能性。方法①收集河北医科大学口腔医院口腔颌面外科下颌阻生齿拔出后切除的牙龈组织。采用组织块培养法,培养液选用含20％胎牛血清的低糖DMEM培养传代。采用波形蛋白（vimentin）和骨骼肌肌动蛋白（α-sarcometric actin）＆肌球蛋白（myosin）的免疫细胞化学、Masson特殊染色进行鉴定。②采用含10％胎牛血清DMEM／F12混合培养液,应用5-氮杂胞苷（azacytidine,5-aza）、成肌分化因子（myogenic differentiation factor,Myod）、胰岛素样生长因子-1（insulin—like growth factors-1,IGF-1）对人牙龈成纤维细胞诱导培养38天,采用vimentin和α-sarcometricacfin＆myosin的免疫细胞化学、Masson特殊染色进行鉴定。扫描电镜观察细胞形态学特征。结果①牙龈组织原代培养7-10天,细胞爬出,成纤维细胞呈梭形、胞体大,核大圆形。对波形蛋白（vimentin）呈阳性反应。②人牙龈成纤维细胞经诱导培养38天细胞后,形态变为长圆柱状或棒状,胞浆两端突起消失。对α-sarcometricactin＆myosin呈阳性表达,Masson染色胞浆着红色。呈现骨骼肌细胞的形态特征。结论人牙龈成纤维细胞能够诱导分化为骨骼肌细胞。     

人涎腺上皮细胞体外培养及其生物学特性的初步研究

目的建立体外培养人涎腺上皮细胞的方法,观察其体外生长的生物学特性。方法应用组织块培养法,用无血清培养基(SFM),无血清1∶1DMEM/F12以及含25ml/L胎牛血清的1∶1DMEM/F123种不同的培养基对10例人涎腺标本进行体外原代和传代培养。用倒置显微镜观察培养细胞体外生长的形态特征。用细胞计数法观察细胞生长情况。用HE染色、PAS染色、细胞角蛋白、Vimentin免疫组化染色对培养细胞进行形态学检查和鉴定。结果10例人涎腺标本在体外原代培养均有细胞长出,培养的细胞为上皮样。用SFM培养的细胞增殖较旺盛,可传代培养5次,用其它2种培养基培养的细胞仅可传代1次。细胞角蛋白染色阳性,Vi-mentin染色阴性,PAS染色阳性。结论应用组织块培养的方法可以获得原代和传代培养的人涎腺上皮细胞,SFM较1∶1DMEM/F12更适宜涎腺上皮细胞生长。     

血清饥饿及融合培养对人牙髓细胞周期同步化和矿化活性的影响

目的:比较血清饥饿及融合培养对人牙髓细胞周期同步化及矿化活性的影响。方法:将人牙髓细胞分别培养至80%及100%融合后,使用含0.5%胎牛血清的培养基继续培养细胞24、48、72 h,使用流式细胞仪检测牙髓细胞的细胞周期。以100%融合培养后饥饿48 h的人牙髓细胞作为实验组,80%融合培养的细胞作为对照组,在基因水平检测碱性磷酸酶、Ⅰ型胶原、骨钙素的表达;在蛋白水平检测碱性磷酸酶活性。采用SPSS13.0软件包对数据进行统计学分析。结果:在人牙髓细胞达到100%融合后,经血清饥饿48 h的G0/G1期细胞比100%融合培养组以及血清饥饿组多（P<0.05）。在基因水平,实验组Ⅰ型胶原、骨钙素的表达与对照组无统计学差异, 但是能促进碱性磷酸酶的表达（P<0.05）,同时在蛋白水平也刺激了人牙髓细胞碱性磷酸酶的分泌（P<0.05）。结论:100%融合培养联合血清饥饿法使更多的人牙髓细胞周期同步于G0/G1期,能更好地促进人牙髓细胞矿化。     

人体脂肪基质细胞成肌潜能研究

目的　研究分离的人体脂肪基质细胞经过诱导培养后向骨骼肌细胞分化的潜能。方法　通过脂肪抽吸 术获取健康成人脂肪,机械分割后用Ⅰ型胶原酶消化,得到脂肪基质细胞。将脂肪基质细胞在BGJb培养基中原代 培养10 d,在DMEM/F12培养基中进行成肌诱导培养20 d,获得细胞爬片。细胞爬片经4%多聚甲醛固定后,用甲 苯胺蓝、Mallory磷钨酸苏木素染色观察细胞形态;Myosin单克隆抗体免疫细胞化学染色观察肌球蛋白表达情况。 结果　经过成肌诱导培养的细胞与常规培养的脂肪基质细胞在形态上有明显的差异,表现为细胞体积增大,单个 核的细胞融合形成多核的肌管样结构,胞浆内细胞器发达,肌浆网扩张并形成肌原纤维骨骼肌特异性的Myosin表 达阳性。未经诱导的脂肪基质细胞在相同时间点无此现象。结论　人类脂肪基质细胞中存在着成体干细胞,在成 肌诱导培养条件下具有向骨骼肌细胞分化的潜能。     

茶多酚对内毒素作用下人牙周膜细胞分泌和表达Toll样受体4的影响

目的:观察茶多酚(tea polyphenol,TP)对内毒素(lipopolysaccharide,LPS)作用下人牙周膜细胞(periodontal ligament cells, PDLCs)分泌和表达Toll样受体4(toll-like receptor 4,TLR4)的影响。方法:体外分离及培养PDLCs,实验组分别为LPS和不同质量浓度的TP的不同组合,对照组为仅含1% FBS的DMEM培养液。培养24 h、48 h和72 h后,通过酶联免疫吸附测定法检测TLR4的分泌量,荧光实时定量PCR法检测TLR4的表达。结果:100 mg/L LPS组PDLCs TLR4分泌量显著高于其它各组(P<0.05),加入TP进行干预后实验组与对照组TLR4的分泌量和表达量无显著差异(P>0.05)。结论:TP对LPS作用下PDLCs TLR4的分泌和表达有一定的抑制作用。     

流式细胞技术分析骨形成蛋白对人牙周膜细胞增殖和分化的影响

目的 :检测牛骨形成蛋白 (b BMP)对离体培养的人牙周膜细胞 (PDL C)的 DNA合成和细胞周期的影响情况。方法 :培养的第 7代 PDL C分成对照组和 BMP作用实验组 ,以含 1% FBS的 DMEM培养 72 h;消化下细胞 ,固定 ,调整细胞数在 1× 10 6/m l,加入复合 DNA荧光检测试剂后 4℃染色 3 0 m in,用流式细胞仪 (FCM)测试 ,联机专用软件分析处理。结果 :实验组 PDL C的 DNA合成量 (S% )显著高于对照组 ;G1 %较对照组降低 ,反映细胞增殖活力的增殖指数 Pr I值 (S G2 M) %增高。结论 :提示 BMP具有促进人 PDL C增殖的作用 ,可能主要是通过促使处于G1 期的细胞进入 S期来实现的。     

胰岛素和转化生长因子-β对人牙周膜细胞碱性磷酸酶活性和总蛋白含量的影响

目的：探讨胰岛素和转化生长因子－β（TGF－β）对体外生长的人牙周膜（PDL）细胞生物学特征的影响,方法：通过体外细胞培养技术,酶动力学法和考马斯亮蓝法,检测胰岛素和TGF－β单独或联合应用时对人PDL细胞碱性磷酸酶（ALPase)活性和总蛋白含量的影响。结果：单独应用时,胰岛素浓度1．0－100U／L,TGF－β浓度0．1－100ug/L,明显促进人PDL细胞的ALPase活性和总蛋白含量,最佳浓度分别为10U／L和1ug/L,以二者的最佳浓度联合应用时,作用更为显著（P＜0．01）,结论：胰岛素和TGF－β均能促进人PDL细胞的分化和总蛋白合成功能,二者联合应用有协同效应。β     

诱导成体人牙周韧带干细胞向软骨细胞分化的实验研究

目的从成体人牙周组织中分离培养牙周韧带干细胞，研究其分化为软骨细胞的可行性。方法选取10～15岁的年轻患者因正畸拔除的健康牙齿，刮取根中1／3的牙周膜，采用组织块培养法得到牙周韧带细胞，待细胞达一定量后用有限元稀释法进行单细胞克隆，筛选牙周韧带干细胞（PDLSC），体外采用离心管聚集体诱导法进行软骨化诱导培养，光镜下观察诱导细胞形态学的改变，甲苯胺蓝染色、免疫组化、RT-PCR等方法检测胶原和糖蛋白的体外表达情况。结果体外离心管聚集体诱导培养3周后，实验组呈白色半透明状，甲苯胺蓝染色显示在外周区域有大量软骨基质成分沉积，组织学显示有较为明显的软骨陷窝。免疫组化及RT-PCR检测到Ⅱ型胶原表达，Ⅱ型胶原染色呈强阳性。对照组细胞团块逐渐收缩、崩解，组织学检测无软骨陷窝结构，Ⅱ型胶原免疫组化结果阴性。结论从成体人牙周组织中可分离培养出干细胞，在体外能有效增殖且保持低分化状态，具有作为软骨组织工程种子细胞来源的可能，也为牙周组织工程的应用奠定了实验基础。     

RPMI1640,DMEM及不同生长因子对大鼠颌下腺细胞生长作用的观察

目的　观察RPMI 16 4 0、DMEM及不同生长因子对大鼠颌下腺细胞增殖能力的影响 ,探讨最有利于大鼠颌下腺细胞的培养方法。方法　在RPMI 16 4 0或DMEM培养基中分别加入一定浓度的FBS(胎牛血清 )、EGF(表皮生长因子 )、HC(氢化可地松 )、INS(胰岛素 ) ,观察纤维细胞和腺细胞在不同培养基中的增殖能力。结果　在含5 %FBS、10ng/mlEGF、5 μg/mlHC、5 μg/mlINS的DMEM培养基中 ,上皮细胞生长良好 ,在只含血清的RPMI 16 4 0培养基中 ,纤维细胞生长旺盛 ,上皮样细胞生长不良 ,在不含血清的培养基中所有细胞均生长缓慢。结论　含有 5 %血清、10ng/mlEGF、5 μg/mlHC及 5 μg/mlINS的DMEM培养基最适合鼠涎腺上皮样细胞的生长     

Effect of tensile force on expression of PTHrP and thickness of hypertrophic zone in organ-cultured mouse spheno-occipital synchondroses

OBJECTIVE: Responses of spheno-occipital synchondroses to direct tensile stress have not been identified before. This study was, therefore designed to evaluate expression of PTHrP, and thickness of hypertrophic zone in spheno-occipital synchondroses in response to such stress, using mouse in vitro model. METHODS: Spheno-occipital synchondroses together with adjacent structures were excised from fifty-five 2-day-old mice that were randomly assigned to 6 control and 5 experimental groups for 5 experimental periods (n=5). In the experimental groups, tensile force of 0.2g was applied across the synchondroses, using helical springs. In 5 control groups, the springs were made inactive. Both groups were then cultured for 6, 24, 48, 72 h and 7 days. Another control group was cultured without any springs for 7 days to compare with natural growth of the synchondroses from a group of five 9-day-old mice. Alcian blue-PAS staining was used to study growth of the synchondroses; immunohistochemical staining to identify PTHrP and type X collagen expression. The area of PTHrP expression and thickness of hypertrophic zone, demarcated by type X collagen expression, were measured. RESULTS: Quantitative analysis showed that PTHrP expression increased significantly at hour 24 of the force application in the experimental group (p<0.05), then reduced from hour 24 to 72 with a significant drop from hour 24 to 48 (p<0.01); and the thickness of hypertrophic zone significantly increased at hour 48 (p<0.01). CONCLUSIONS: Our findings suggested that the growth of spheno-occipital synchondroses could be modified by tensile stress; and a light continuous force could enhance its growth, as evidenced by an increase in PTHrP expression and thickness of hypertrophic zone.     

In vitro cytotoxicity and in vivo biocompatibility of contemporary resin-modified glass-ionomer cements.

OBJECTIVES: To evaluate the effects of current resin-modified glass-ionomer cements (RMGICs) applied on culture of cells or implanted into subcutaneous tissue of rats. METHODS: Experiment 1 - Thirty round-shaped samples of every RMGICs: Rely X Luting Cement (RL), Vitremer (VM), and Vitrebond (VB) were placed into wells with 1.1 mL of culture medium (DMEM), and incubated for 24, 48 or 72 h. The extracts from every sample were applied on the MDPC-23 cells. Fresh DMEM was used as control group. The MTT assay was carried out for mitochondrial respiration. Experiment 2 - Fifty-four polyethylene tubes filled with the experimental materials were implanted into the dorsal subcutaneous tissue of rats. At 7, 30, and 90 days the animals were killed and the biopsies were processed for histological evaluation. RESULTS: Experiment--Both time of elution and material significantly influenced cell respiratory activity. In general, the extracts obtained at 24 h were less cytotoxic than 48 and 72 h incubation. The cytotoxic effect of VM and RL were not statistically different (p < 0.05) for the 24-hour period. VB showed the highest cytotoxic effect. Experiment 2--All RMGICs elicited at 7 days a moderate to intense inflammatory reaction which decreased over time. However, connective healing occurred for most of samples at 90-day evaluation. SIGNIFICANCE: Glass-ionomer cements may cause noticeable inflammatory response when in direct contact to connective tissue. The toxic effects of this kind of soluble material depend on the amount of components released in the aqueous environment.     

Direct contact between enamel matrix derivative (EMD) and osteoblasts is not required for EMD-induced cell proliferation

OBJECTIVE: The purpose of this study was to determine whether the effect of enamel matrix derivative (EMD) on osteoblast proliferation is dependent on direct contact between EMD and the cells. STUDY DESIGN: MC3T3-E1 cells were seeded onto 6-well culture plates at an initial density of 5000/cm(2) in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). Serum was removed from the culture medium after 24 hours with or without the addition of EMD. Four groups were evaluated: group 1, DMEM only; group 2, DMEM with 100 microg/mL EMD directly added to the culture medium; group 3, DMEM with a culture plate insert (30-mm diameter; 0.4-microm pore size) only; group 4, DMEM with 100 microg/mL EMD added onto a culture plate insert. The porous membrane of the insert prevented direct contact between EMD and the cells. After 3-day incubation, cell morphology was examined and the total cell number per well was counted and analyzed using 1-way ANOVA. RESULTS: EMD formed precipitated aggregates on the membrane of the culture insert with the same appearance as when it was added directly to the medium. The culture plate insert alone did not cause any changes in cell morphology or proliferation. The addition of EMD significantly increased cell number regardless the presence of the culture plate insert. CONCLUSION: This study suggests that direct contact between EMD and osteoblasts is not required to induce cell proliferation. Soluble peptides released from EMD may contribute to the stimulating effects of EMD on cell proliferation.     

The effect of Swedish and American smokeless tobacco extract on periodontal ligament fibroblasts in vitro

Use of moist snuff is widespread in Sweden. In 2004 approximately 8oo,ooo Swedes were daily users which corresponds to 22% of the male population and 3% of the female population. The aim of the present study was to evaluate the effect of Swedish moist snuff extract on PDLfibroblast growth and hard tissue production and compare with moist snuff extract from USA. Periodontal ligament cells (PDL-cells) were obtained from 3 healthy subjects (1 female 14 years, 2 males 14 and 17 years) from the root surface of premolars extracted for orthodontic reasons. The cells were isolated from explants and grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FBS) and cultivated in 37 degrees C with 5% CO2 in air. Snuff extract in concentrations 0.3%, 1% and 3% (in DMEM with 1% FBS) was tested. Cells from each individual were tested three times, each time in triplicate. Photographs were taken at o and 24 hours with a digital camera and analysed in terms of growth and morphology. Then the cell suspension was frozen and later thawed for examination of the production of alkaline phosphatase after exposure to different snuff concentrations. This in vitro study has shown that PDL cells from 3 different subjects demonstrated a reduced number of cells at exposure to 3% of both Swedish and American snuff extract.The production of alkaline phosphatase after 2 hours was similarly reduced from cells exposed to 3% snuff extract. Further studies have to be made to understand the effect of smokeless tobacco on periodontal tissues. However, from this study can be concluded that smokeless tobacco has biological effects in terms of reduced PDL cell growth and production of alkaline phosphatase