血小板衍化生长因子对人牙髓成纤维细胞DNA和胶原蛋白合成的影响

目的:观察血小板衍化生长因子(platelet-derived growth factor,PDGF)对人牙髓成纤维细胞(human dental pulp fibroblast,HDPF )DNA合成和胶原蛋白合成的影响.方法:应用3H-TdR和3H-脯氨酸掺入方法,观察PDGF对体外培养的HDPF DNA合成和胶原蛋白合成的情况.结果:20～60 ng/mL PDGF可明显促进HDPF DNA的合成,40 ng/mL浓度使细胞DNA合成在36 h达最大值;对细胞的胶原蛋白合成无明显促进作用.结论:PDGF明显促进HDPF DNA的合成,可能在治疗牙髓病中起重要作用.     

bFGF对人牙髓、牙周膜成纤维细胞生物学效应的研究

目的：了解碱性成纤维细胞生长因子（bFGF）对人牙髓、牙周膜成纤维细胞的生物学效应,为bFGF在牙髓、牙周病中的治疗研究提供新的实验依据。方法：利用3H-TdR掺入法观察在bFGF作用下人牙髓、牙周膜成纤维细胞DNA和胶原蛋白合成的情况。结果：20ng/mL～60ng/mLbFGF可明显促进人牙髓、牙周膜成纤维细胞DNA的合成（P〈0.01）,在40ng/mL浓度时牙髓,牙周膜成纤维细胞DNA合成最高,40ng/mLbFGF作用于牙髓,牙周膜成纤维细胞,24～48h可使细胞DAN合成显著增多,牙髓成纤维细胞在36h时DNA合成达最高峰,牙周膜成纤维细胞在24h时DNA合成达最高峰;bFGF对牙髓,牙周膜成纤维细胞胶原蛋白的合成无明显促进作用（P〉0.05）。结论：人牙髓、牙周膜成纤维细胞是bFGF的靶细胞,其胞膜上可能有bFGF特异性受体的存在,也表明bFGF在牙髓、牙周组织的创伤愈合中可能起重要作用。     

碱性成纤维细胞生长因子对人牙周膜细胞生长的影响

目的 探讨在碱性成纤维细胞生长因子（bFGF)作用下,人牙周膜细胞（PDLC）的增殖、DNA合成状况的变化。方法 以常规体外组织块细胞培养法获得PDLC,采用MTTI地和^3H-TdR掺入法测定bFGF对PDLC的影响,实验结果A值和CPM值经方差分析。结果 各实验组与对照组之间均有显著性差异,bFGF能显著提高PDLC的增殖活性及DNA合成,其效应随bFGF浓度增大而增大,1000ng/ml范围内未见抑制现象,最大效应一半的浓度约为100ng/ml。结论 bFGF能促进PDLC增殖及DNA合成,可望在牙周再生治疗中起到重要作用。     

生长因子网络调节对骨形成作用的研究Ⅰ、TGF—β对人胚成骨样细胞增殖及分化的影响

在对人胚成骨样细胞分离培养的基础上，进一步探讨不同剂量转化生长因子-β（TGF-β）对人胚成骨样细胞DNA，胶原蛋白和碱性磷酸酶合成的影响。结果表明：TGF-β对体外培养的人胚成髓样细胞DNA合成有抑制作用。对胶原蛋白和碱性磷酸酶合成有促进作用。这两方面的作用在TGF-β0.01-1ng/ml间呈剂量依赖性，1ng/ml时的作用达到最强，本研究提示TGF-β在骨形成中的主要作用可能在于介导机体系统因素对骨细胞的作用。以及调控局部骨生长因子之间的相互作用。     

苯妥英钠对内毒素作用下人牙周膜干细胞在牙骨质片上附着影响研究

目的 观察苯妥英钠（PHT）对内毒素作用下人牙周膜干细胞（hPDLSCs）在牙骨质片上附着的影响。 方法 本研究于2012年6月至2013年2月在滨州医学院中心实验室和西安交通大学医学院中心实验室完成。利用扫描电镜和透射电镜观察hPDLSCs分别在20 μg/mL PHT+ 100 ng/mL脂多糖（LPS）与100 ng/mL LPS作用下在牙骨质片上的附着情况以及细胞超微结构的变化。 结果 与细胞培养液中加入100 ng/mL LPS比较,培养液中加入20μg/mL PHT + 100 ng/mL LPS培养72 h后hPDLSCs胞浆内细胞器更丰富,粗面内质网扩张,高尔基复合体发达,有大量的线粒体和核糖体;培养第7 d和第21 d,细胞生长更密集,伪足较多且粗大,与牙骨质片的附着更紧密,细胞周围钙盐沉积以及胶原蛋白合成更加活跃。结论 PHT可促进hPDLSCs在牙骨片上的附着和生长。     

高迁移率族蛋白B1对人牙髓细胞迁移能力和增殖效应的影响术

目的：观察高迁移率族蛋白B1（HMGBl）在人牙髓细胞（Human dental pulp cells,hDPCs）中的表达,以及对hDPCs增殖和迁移能力的影响。方法：采用组织块培养法,培养原代hDPCs,取第3-6代细胞用于实验。免疫荧光检测HMGBl在hDPCs中的表达及定位;分别用含不同质量浓度（0．1ng／mL、1ng／mL、10ng／mL、50ng／mL、100ng／mL、500ng／mL、1000ng／mL）HMGBl的培养液培养hDPCs,5天后采用CCK一8法检测细胞增殖能力;细胞划痕实验法观察1ng／mL质量浓度HMGBl对hDPCs迁移能力的影响。结果：HMGBl表达在hDPCs胞核：低浓度HMGBl（O．1ng／mL、1ng／mL、10ng／mL、50ng／mL、100ng／mL）明显促进细胞增殖;1ng／mLHMGBl明显促进hDPCs迁移能力。结论：HMGBl表达在正常hDPCs胞核中,细胞外低浓度HMGBl可以促进细胞增殖和迁移。     

牛血小板衍化生长因子对人牙周膜成纤维细胞的生物学效应

目的：观察在牛血小板衍化生长因子作用下人牙周膜成纤维细胞ＤＮＡ和胶原蛋白合成的情况。方法：采用体外细胞培养技术和核素掺入法。结果：２０ｎｇ／ｍｌ～６０ｎｇ／ｍｌ牛血小板衍化生长因子可明显促进人牙周膜成纤维细胞ＤＮＡ合成，４０ｎｇ／ｍｌ浓度使细胞ＤＮＡ合成在２４ｈ达最高峰；对细胞的胶原蛋白合成无明显促进作用。结论：牛血小板衍化生长因子对人牙周膜成纤维细胞ＤＮＡ合成有促进作用，在牙周组织的创伤修复中可能起重要作用。     

BMP-2对体外培养人牙髓细胞TIMP-1、TIMP-2表达的影响

目的：观察BMP－2对HDPF表达TIMP－1、TIMP－2的影响。方法：用4ng/ml、40ng/ml、400ng/ml BMP-2分别作用于HDPF 2d，利用免疫组化和图像分析法法半定量观察BMP－2对HDPF表达TIMP－1、TIMP－1、TIMP－2表达显著增强，结论：BMP－2通过改变HDPF表达TIMP－1、－2的量来影响胶原的代谢。     

卵黄高磷蛋白对人牙髓细胞增殖的影响

目的研究卵黄高磷蛋白对体外培养的人牙髓细胞增殖的影响。方法 4个实验组分别采用1、10、100、1000ng/mL的卵黄高磷蛋白,对照组采用10%胎牛血清,分别作用于体外培养的第6代人牙髓细胞,每组8个标本,培养3、6day后分别对每组作MTT法检测,OD值进行t检验。实验组用100ng/mL卵黄高磷蛋白,对照组用10%胎牛血清作用上述人牙髓细胞,培养48h,常规消化,固定,经DNA荧光染色后,用流式细胞仪测定DNA含量。结果 1～100ng/mL卵黄高磷蛋白作用3、6day均促进人牙髓细胞增殖。10、100ng/mL卵黄高磷蛋白作用6day,对人牙髓细胞增殖的促进作用与对照组相比有显著差异(P<0.05)。1000ng/mL卵黄高磷蛋白作用3、6day均抑制人牙髓细胞增殖。100ng/mL卵黄高磷蛋白作用人牙髓细胞,与对照组相比DNA合成前期细胞比例明显降低,而DNA合成期细胞比例和细胞增殖指数均显著增高。结论卵黄高磷蛋白具有促进人牙髓细胞增殖和DNA合成的作用,可能主要通过促进处于合成前期的细胞进入合成期来实现的。     

神经生长因子对体外培养的人牙乳头间充质细胞增殖和细胞周期的影响

目的：观察神经生长因子对人牙乳头间充质细胞增殖和细胞周期的影响。探讨其在牙胚生长发育过程中的可能作用。方法：采用^3H-TdR掺入和流式细胞仪分析法。结果：NGF（1－100U/ml)可明显促进培养的人牙乳头间充质细胞的^3H-TdR掺入率,NGF（100U／ml)刺激细胞增殖作用最强。NGF（1000U／ml)抑制细胞的增殖。在NGF（100U／ml)作用下,细胞S％明显升高,细胞增殖指数（S G2M)%也明显增高。结论：神经生长因子可促进人牙乳头间充质细胞DNA合成,刺激G1期细胞向S期转变。     

Platelet-derived growth factor enhances proliferation and matrix synthesis of temporomandibular joint disc-derived cells

Platelet-derived growth factor (PDGF) is an essential signaling molecule for wound healing and tissue repair. This study was aimed at evaluating the effect of PDGF on the proliferation of temporomandibular joint (TMJ) disc-derived cells and extracellular matrix synthesis. The number of cultured cells were counted by COULTER Z1. The assay for collagen synthesis was performed using a sircol soluble collagen assay. Hyaluronic acid (HA) synthesis was analyzed by a high performance liquid chromatography. The expression of collagens, matrix metalloproteinases (MMPs), and the tissue inhibitors of metalloproteinases (TIMPs) were examined using SYBR Green in terms of the RNA levels. PDGF treatment significantly (P < .01) increased the proliferation rate of the disc-derived cells as compared with the controls when the dose was 5 ng/ mL or greater. Treatment with more than 5 ng/mL PDGF resulted in an amount of collagen synthesis significantly (P < .01) higher than the controls. HA synthesis was maximal with 5 ng/mL PDGF treatment. Quantitative real-time polymerase chain reaction analyses showed that treatment with 5 ng/mL of PDGF-BB upregulated the mitochondrial RNA levels of type I and II collagens, MMPs, and TIMPs within 6 hours. It is concluded that PDGF, if its concentration is optimal, enhanced proliferation and matrix synthesis of TMJ disc-derived cells, indicating that PDGF may be effective for use in tissue engineering of the TMJ disc.     

Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro

Transforming growth factor-beta (TGF-beta) is important in regulating the repair and regeneration of damaged dental pulp. For further elucidating the roles of different isoforms of TGF-beta in the healing and inflammatory processes of human dental pulp, we found that TGF-beta1, TGF-beta2 and TGF-beta3 inhibited the growth of two human dental pulp cell strains in vitro by 19-29, 18-25 and 23-26%, respectively, at a concentration of 0.5 ng/ml. TGF-beta also differentially stimulated the collagen synthesis of pulp cells. Collagen synthesis increased by 1 ng/ml of TGF-beta1 and TGF-beta2 by 42 and 51%, respectively. TGF-beta3 (0.1-1 ng/ml) lacked of stimulatory effect on collagen synthesis of pulp cells. Pulp cells have the intrinsic capacity to contract collagen lattice, leading to decreasing of lattice diameter. An 8 h exposure to TGF-beta1 and TGF-beta2 enhanced the pulp cell-populated collagen lattice contraction at concentrations ranging from 0.2 to 3 ng/ml. At similar concentrations, TGF-beta3 lacked of this stimulatory effect. When collagen lattice were detached after 24 h of exposure, TGF-beta1 and TGF-beta2 (0.6-3 ng/ml) induced the pulp cells-populated collagen lattice contraction within 4-8h of gel detachment. These results indicate that TGF-beta-induced collagen lattice contraction is a late cellular event. These in vitro results indicate that effects of TGF-beta isoforms on the growth, collagen synthesis and collagen lattice contraction of pulp cells may play crucial roles in the pathobiological processes of dental pulp.     

Effects of growth factors on temporomandibular joint disc cells

The effects of growth factors on cartilaginous tissues are well documented. An exception is the temporomandibular joint (TMJ) disc, where data for growth factor effects on proliferation and biosynthesis are very limited. The purpose of this study was to quantify proliferation of and synthesis by TMJ disc cells cultured in monolayer with either platelet derived growth factor-AB (PDGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-I (IGF), at either a low (10 ng/ml) or high (100 ng/ml) concentration. Proliferation was assessed with a DNA quantitation technique, collagen synthesis was measured via a hydroxyproline assay, and GAG synthesis was determined with a dimethylmethylene blue dye binding assay at 14 days. Overall, the most beneficial growth factor was bFGF, which was most potent in increasing proliferation and GAG synthesis, and also effective in promoting collagen synthesis. At the high concentration, bFGF resulted in 96% more cells than the control and 30 to 45% more cells than PDGF and IGF. PDGF and bFGF were the most potent upregulators of GAG synthesis, producing 2-3 times more GAG than the control. IGF had no significant effect on GAG production, although at its higher concentration it increased collagen production by 4.5 times over the control. Collagen synthesis was promoted by bFGF at its lower concentration, with levels 4.2 times higher than the control, whereas PDGF had no significant effect on collagen production. In general, higher concentrations increased proliferation, whereas lower concentrations favoured biosynthesis.     

IGF-1通过PI3K/AKT信号通路促进牙髓细胞增殖及碱性磷酸酶活性的研究

目的:研究IGF-1对牙髓细胞增殖和碱性磷酸酶(ALP)活性及PI3K/AKT信号通路的影响。方法:采用酶消化法分离培养原代人牙髓细胞。Western blot检测牙髓组织中IGF-1蛋白表达,不同浓度的IGF-1处理牙髓细胞7d,CCK8法检测细胞增殖。用IGF-1(100 μg/L)及LY294002(10 μmol/L)分别单独或同时处理牙髓细胞,培养7 d后MTT实验检测细胞增殖;在培养第3、5、7、14天时检测细胞ALP 活性;在培养第7天时用Western blot检测AKT和p-AKT蛋白表达情况。结果:IGF-1在牙髓炎组织中低表达, IGF-1在20~100 μg/L浓度范围内从作用第3天开始能够显著促进牙髓细胞增殖(P<0.05或P<0.01),且具有剂量和时间依赖效应。LY294002能够抑制牙髓细胞的增殖和碱性磷酸酶活性,具有时间依赖性。IGF-1能促进p-AKT蛋白表达,而 LY294002能减低IGF-1对p-AKT蛋白表达的促进作用。结论:IGF-1可以促进牙髓细胞增殖和碱性磷酸酶活性,且具有浓度和时间依赖性,作用机制可能与PI3K/AKT信号通路相关。[关键词] 牙髓细胞 细胞增殖 碱性磷酸酶     

脂多糖对大鼠牙髓细胞ALP、BSP、DSPP表达的影响

目的:研究脂多糖(lipopolysaccharide,LPS)对大鼠牙髓细胞牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein,BSP)及碱性磷酸酶(alkaline phosphatase,ALP)表达的影响。方法:采用组织块法获得大鼠牙髓细胞,体外常规培养并进行鉴定,以含0.1、1、10、100和10000 ng/mL牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)的LPS作用牙髓细胞1、3、5 d,用实时定量PCR检测DSPP、ALP、BSP mRNA表达的变化,采用SPSS17.0软件包对数据进行统计学分析。结果:镜下贴壁后的细胞形态多样,多呈成纤维样细胞形态,还有部分多角形细胞,胞质突起。实时定量PCR结果显示,与对照组相比,1、10 ng/mL LPS组大鼠牙髓细胞DSPP、ALP、BSP的mRNA表达增高,100、10000 ng/mL LPS组DSPP、ALP、BSP的mRNA表达均降低;在1、3、5 d时,1、10、100和10000 ng/mL LPS组mRNA表达逐渐减少。0.1 ng/mL LPS对mRNA的表达无显著影响。3种因子呈现相似的表达变化趋势。结论:低剂量P.g LPS能促进牙髓细胞ALP、BSP、DSPP的表达,高剂量时则抑制ALP、BSP、DSPP的表达;随着培养时间的延长,促进作用逐渐减弱,抑制作用逐渐增强。     

碱性成纤维细胞生长因子对人牙髓细胞增殖和分化的作用

目的 探讨碱笥成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）对培养人牙髓细胞增殖和分化的效应。方法 采用四唑盐法、^3Ｈ－ＴｄＲ掺入法、图象分析,检测重组人ｂＦＧＦ（ｈｂＦＧＦ）对细胞ＤＮＡ、胶原蛋白、纤维为连蛋白、碱笥磷酸酶、骨形成蛋白合成和凝集素表达的影响。结果 ｈｂＦＧＦ浓度在１～１０μｇ／Ｌ时显著促进细胞增殖,浓度为１～１００μｇ／Ｌ）,Ⅰ型胶原