颞下颌关节髁突软骨细胞体外培养中去分化现象研究

目的　研究去分化髁状突软骨细胞的细胞生物学特性。方法　连续在体外贴壁条件下培养传代正常乳兔髁突软骨细胞至第 2 0代 ,观察培养细胞的形态变化 ,免疫组化法和RT -PCR法对传代过程中的数代细胞对II型胶原与蛋白多糖聚糖体的表达情况进行连续检测 ,观察细胞软骨基质蛋白表达分泌的变化以及发生明显去分化软骨细胞的超微结构特点。结果　在贴壁培养条件下 ,髁状突软骨细胞去分化现象于细胞传代第 3代后逐渐显著 ,第 6代后培养的软骨细胞几乎完全去分化。去分化细胞胞体拉长 ,呈“成纤维细胞”样外观。去分化过程中 ,细胞对软骨基质蛋白的表达水平逐渐降低 ,细胞增殖速度增强。透射电镜观察显示 ,去分化软骨细胞细胞核明显呈分叶状 ,表现出异型核。结论　髁突软骨细胞在体外贴壁培养传代过程中会发生去分化现象 ,丧失软骨细胞特征性细胞表型。     

生长期兔髁突软骨细胞体外培养方法及其生物学特性研究

目的 建立髁突软骨细胞体外培养模型,研究其生物学特性。 方法 分离4周龄健康新西兰大白兔双侧髁突软骨,采用胰酶和胶原酶联合消化方法收集4 h和12 h 2个时间点的髁突软骨细胞,连续体外培养并传代至P10。使用倒置显微镜观察细胞形态;采用甲苯胺蓝、阿利新蓝和免疫细胞化学染色对髁突软骨细胞进行鉴定;利用细胞计数绘制细胞生长曲线;通过Western 免疫印迹分析细胞Ⅱ型胶原、Ⅹ型胶原和SOX9在蛋白水平的表达情况。采用SPSS13.0软件包对Western 印迹条带灰度值进行统计学分析。 结果 兔髁突软骨细胞体积较大,呈纺锤形或多角形,铺路石样排列,细胞爬片染色结果为阳性。随着细胞传代“成纤维样”细胞比例逐渐加重,P2代之前的细胞形态差异较小。细胞生长曲线显示,兔髁突软骨细胞的体外培养符合经典的S形生长曲线。Western 印迹结果表明,4h和12 h所收髁突软骨细胞Ⅱ型胶原、Ⅹ型胶原和SOX9表达无显著差异。 结论 使用本方法培养兔髁突软骨细胞简单有效。髁突软骨细胞体外培养存在去分化现象,但P2代以内的髁突软骨细胞生物学特性相对稳定,适合用于后期实验研究,P3代以后的髁突软骨细胞逐渐丧失原有细胞的特性。     

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。     

藻酸盐凝胶三维培养体系在TMJ骨关节病髁突软骨细胞体外培养中的应用

目的 将藻酸盐凝胶三维培养体系应用于颞下颌关节骨关节病（osteoarthritis of temporomandibular joint,TMJOA)髁突软骨细胞的体外培养。方法 利用机械与酶消化的方法从TMJOA患者手术切除患疾髁突软骨中获得髁突软骨细胞，部分细胞在二维贴壁培养条件下体外培养1周；部分细胞在高密度条件下转入藻酸盐凝胶培养介质，进行三维培养4周；分别对贴壁条件下培养细胞和藻酸盐细胞凝胶微球右蜡包埋切片进行Ⅱ型胶原和软骨特异性蛋白多糖的免疫组化鉴定。结果 从骨关节病髁突软骨组织中收获并行体外培养成活的细菌，鉴定为软骨细胞；体外培养的藻酸盐凝胶三维培养体系中的髁突软骨细胞生长状态良好；培养4周后细胞保持了良好的分化表型。结论 成功地体外培养成活了人TMJOA髁突软骨细胞；成功地将藻酸盐凝胶三维培养体系应用于人TMJOA髁突软骨细胞体外培养，在该体系中，软骨细胞生长状态与功能蛋白分泌能力良好。     

颞下颌关节盘前移位后髁突软骨细胞基质基因表达的变化

目的　探讨颞下颌关节盘前移位后髁突软骨细胞基质基因表达的变化。方法　建立40只大白兔颞下颌关节盘前移位动物模型,用地高辛标记cRNA探针原位杂交技术,检测术后不同病变时期髁突软骨细胞Ⅱ型胶原和蛋白多糖聚合体基因表达的改变。结果　正常髁突的增殖层深层、肥大层和钙化层细胞胞浆内可见大量Ⅱ型胶原和蛋白多糖聚合体mRNA表达。术后1周蛋白多糖聚合体开始下调,至2周达低谷,6周后恢复至正常水平。Ⅱ型胶原4周后开始持续下降,其表达下降程度较蛋白多糖聚合体显著,8周后逐渐恢复正常。结论　关节盘前移位后髁突软骨的基质基因表达发生有序、协调的变化,这种变化意味着适应性改建的启动。     

髁突增殖层细胞在颞下颌关节适应性改建中的作用

目的 探讨颞下颌关节盘前移位后髁突软骨增殖层细胞在关节适应性改建中的作用。方法 32只日本大白兔，在全麻下建立关节盘前移位动物模型，术后1周、2周、4周、6周、8周处死，HE染色、Ⅱ型胶原和蛋白多糖聚合体mRNA原位杂交染色观察。结果 正常髁突的增殖层浅层有蛋白多糖聚合体mRNA的少许表达。但无Ⅱ型胶原表达。增殖层深层Ⅱ型胶原和蛋白多糖聚合体mRNA均有大量表达。术后1周，受力区软骨组织变薄，尤以增殖层明显。蛋白多糖聚合体mRNA表达开始下调。2周后Ⅱ型胶原mRNA表达也开始下降。4周后蛋白多糖聚合体mRNA表达开始回升，增殖层浅层也可见表达，但Ⅱ型胶原mRNA表达继续下降。术后8周两基因表达水平恢复至正常。结论 增殖层细胞具有向软骨细胞分化的潜能，成年兔髁突软骨增殖层具有再生能力，但损伤过度，可导致骨关节病样改变。     

人胚髁状突软骨细胞的分离、培养和生物学性状

目的:研究体外培养的正常人胚髁状突软骨细胞的生物学特性.方法:体外培养人胚髁状突软骨细胞,观察其从原代至第6代的形态变化;测定细胞数量的变化及其生长曲线;测定冷冻复苏后的细胞存活;观察细胞的超微结构.结果:从原代至第3代,髁状突软骨细胞为圆形、多角形,从第4代开始逐渐转变为梭形的成纤维细胞.体外培养至第6天髁状突软骨细胞的数量是接种时的3倍.冷冻复苏后经苔盼蓝拒染试验检查,发现细胞存活率为92%.体外培养髁状突软骨细胞的超微结构正常.结论:单层培养髁状突软骨细胞的表型至少能够保持3代稳定,且形态结构正常,具有正常的生长增殖能力,并可经深低温冷冻长期保存.     

不同静压力对大鼠髁突软骨细胞生物学特性的影响

目的探讨不同静压力对髁突软骨细胞生物学特性的影响。方法对培养到第3代的新生SD大鼠髁突软骨细胞加载12、24、36 kPa的静压力，1 h后立即收集样本，同时设不加载力的对照组，用免疫组织化学技术对样本细胞进行染色，用病理图像分析仪分析细胞胞浆中胶原表达强度的变化。结果对于对照组、12、24 kPa组，随着压力的增加，髁突软骨细胞表达Ⅰ、Ⅱ、Ⅲ型胶原及蛋白多糖（ PG）增加。与24 kPa组相比，36 kPa组髁突软骨细胞表达Ⅰ型胶原及PG减弱，而Ⅱ、Ⅲ型胶原表达则增加。结论适当的压力刺激可能会使髁突软骨细胞分化更加成熟，但是过大的应力则可能导致髁突软骨细胞生物学特性减弱，从而表现出去分化的某些特征。     

兔髁突软骨组织学分层的实验研究

目的：探讨髁突软骨的组织分层及其功能。方法：成年日本大耳白兔6只，处死后通过HE染色、免疫组化、原位杂交和透射电镜等方法进行软骨细胞PCNA、FGFR3、Ⅱ型胶原和蛋白多糖聚合体基因表达及超微结构等研究。结果：FGFR3在增殖层的浅层细胞中阴性表达，增殖层深层细胞开始为阴、阳性染色相间，逐渐变为全阳性染色细胞。PCNA在增殖层浅层细胞为阴、阳性染色相间，深层细胞均为阳性表达。增殖层的浅层细胞有蛋白多糖聚合体mRNA表达，但不表达Ⅱ型胶原mRNA。增殖层的深层细胞均强表达Ⅱ型胶原和蛋白多糖聚合体mRNA。电镜下见增殖层的深层细胞具有幼稚细胞和软骨细胞的双重表型。结论：髁突关节软骨应分为纤维层、增殖层、过渡层、软骨层和钙化软骨层5层，过渡层细胞可能属于前软骨干细胞。     

甲状旁腺相关蛋白在鼠胚髁突外植体软骨内成骨中的作用

目的探讨甲状旁腺相关蛋白(PTHrP)对体外培养的鼠胚髁突软骨内成骨的影响。方法体外解剖分离鼠胚髁突,行外植体培养,通过组织学、免疫组化等方法观察PTHrP对体外培养的髁突外植体软骨内成骨的影响。结果髁突外植体在无血清半固态培养系统中能正常发育。加入人PTHrP(1-34)后,实验组髁突长度的增加较对照组明显,两组间差异有显著性(P<0.05);组织学及免疫组化染色显示:加入人PTHrP(1-34)后培养的髁突外植体增殖层和肥大软骨细胞层明显增厚,同时Ⅱ及Ⅹ型胶原在增殖层和肥大软骨细胞层中表达增强。结论PTHrP可刺激髁突外植体软骨增殖层和肥大层软骨细胞的增殖,促进髁突软骨内成骨的形成。?     

生长因子对人髁突软骨细胞Ⅰ、Ⅱ、Ⅲ型前胶原基因表达的调控研究

目的：研究低血清培养条件下转化生长因子－β（ＴＧＦ－β）,胰岛素生长因子－Ⅰ（ＩＧＦ－Ⅰ）,碱性成纤维生长因子（ｂＦＧＦ）对人髁突软骨细胞Ⅰ,Ⅱ,Ⅲ型前胶原基因表达的调控作用。方法：采用体外细胞培养及ｍＲＮＡ狭缝杂交的方法。结果：ＩＧＦ－Ⅰ对３种前胶原的ｍＲＮＡ水平无显著影响。ｂＧＦＧ,ＴＧＦ－β均呈不同程度地抑制Ⅱ型胶原的基因表达,分别为对照组的０．３５２及０．６５８倍同时ＴＧＦ－β显著增加了Ⅰ     

Fine structure of cultured human gingival fibroblasts and demonstration of simultaneous synthesis of types I and III collagen

A strain of human gingival fibroblasts, obtained from clinically normal gingiva, was examined by electron and immunofluorescent microscopy to determine: (i) their fine structure; (ii) whether the cultures also contained smooth muscle or endothelial cells; and (iii) the collagen synthesis activities of single cells. Cultured gingival fibroblasts were homogeneous in their fine structure with features similar to those reported for cultured human skin fibroblasts. They differed from fibroblasts in vivo in that the cultured cells had less rough endoplasmic reticulum, no collagen-containing vacuoles and a more prominent microfilament network. Smooth muscle or endothelial cells were not seen in the cultures. By electron microscopy, none of the cells had cell-to-cell junctions typical of cultured smooth muscle cells, or Weibel-Palade bodies, which are unique to endothelial cells. By immunofluorescent microscopy, none of the cells contained type IV collagen, a phenotypic marker for endothelial cells. Furthermore, type III procollagen, a major product of smooth muscle cells, was present in only small amounts. Simultaneously staining for type I collagen and type III procollagen demonstrated that most cells contained both types of collagen, although some cells contained only type I. The latter finding suggests that functionally distinct subpopulations of human gingival fibroblasts may exist.     

Effect of methyl mercaptan on synthesis and degradation of collagen

Measurements of the conversion of [¹⁴C]-proline to [¹⁴C]-hydroxyproline were employed to assess the effect of methyl mercaptan on intra- and extracellular metabolism of collagenous proteins in human gingival fibroblast cultures. Following a 30-min pulse, 10 ng of methyl mercaptan per ml of 95% air/5% CO₂, headspace suppressed collagen synthesis by 39% and increased the intracellular degradation of newly synthesized collagen from 26% to 42%. Parallel cultures assayed for proline transport demonstrated a 29% inhibition of [¹⁴C]-proline uptake. A similar analysis of cultures exposed to methyl mercaptan for 12 h revealed an increase in intracellular degradation (20% control vs. 30% test) and a marked increase in extracellular collagenolysis (4% control vs. 55% test). While pulsing, collagen synthesis was decreased by 39%. Slab gel electrophoresis also demonstrated that treatment with methyl mercaptan caused reductions both in mature a, and α₂ chains of type I collagen and in type III procollagen. Identities of the procollagen species were confirmed by pepsin digestion. Reverse transcribed polymerase chain reaction was utilized to compare expression of a1 chains of type I procollagen with type III procollagen and indicated suppression of mRNA synthesis for type III procollagen in cultures exposed to methyl mercaptan.     

生长因子对人髁突软骨细胞DNA及胶原合成的影响

目的:研究胰岛素样生长因子(IGF-)、碱性成纤维样生长因子(bFGF)及转化生长因子-β1(TGF-β1)对人髁突软骨细胞增殖及代谢的调节作用。方法:采用体外细胞培养技术及同位素掺入法。结果:在0.4%新生小牛血清(NCS)条件下,bFGF对人髁突软骨细胞DNA合成有显著的促进作用,浓度在1ng/ml以上具显著性(P<0.05)。IGF-在10～100ng/ml呈剂量效应地促进3H-TdR的掺入,而TGF-β1无显著作用。bFGF在0.1～100ng/ml可显著促进人髁突软骨细胞3H-Proline掺入,最大效应剂量为10ng/ml(增加60%)。IGF-在10～100ng/ml能够明显促进细胞胶原合成,最大值在100ng/ml(增加98%)。TGF-β1在1～10ng/ml明显抑制3H-Proline掺入,最大抑制浓度为1ng/ml,抑制率约为24%。结论:生长因子可有效促进软骨细胞增殖及基质合成,为关节软骨损伤性疾患的治疗提供一种有效的途径     

Antisense oligonucleotide against 47-kDa heat shock protein (Hsp47) inhibits wound-induced enhancement of collagen production

It is well known that excessive collagen synthesis during the wound-healing process causes scar formation. Our recent in-vivo study indicates that antisense treatment against 47-kDa heat shock protein (Hsp47), a collagen-specific molecular chaperone, relieves scar formation following skin wounds in rats [Wang et al., Plast. Reconstr. Surg., in press]. In order to understand the mechanism of this phenomenon, we examined the effects of antisense treatment on the expression of mRNAs and proteins of Hsp47 and collagens in fibroblasts derived from wounded rat tongues. Hsp47 and procollagen alpha1(I) and alpha1(III) mRNAs were consistently increased after wounding and were maximal at day 5 post-injury. Treatment with antisense oligonucleotide against Hsp47 efficiently blocked the production of procollagen alpha2(I) and alpha1(III) proteins, but had little effect on their mRNA levels. Therefore, we conclude that antisense oligonucleotide against Hsp47 inhibits the production of procollagen type I and III proteins in fibroblasts derived from wounded tongues, overcoming the increase in their mRNAs.     

Increased expression of collagen prolyl 4-hydroxylases in Chinese patients with hereditary gingival fibromatosis

OBJECTIVES: Hereditary gingival fibromatosis (HGF) is characterized by excess accumulation of interstitial collagen. However, until now, there has been controversy about the mechanism of collagen accumulation in HGF gingivae. The present study aimed to clarify the pathogenic mechanisms potentially involved. DESIGN: Gingival fibroblasts from three Chinese HGF patients and three healthy subjects were cultured. Cell proliferation was assessed by MTT assay. The mRNA levels of type I collagen, MMP-1, MMP-3, TIMP-1, prolyl 4-hydroxylase (P4H)alpha(I), alpha(II), alpha(III) and P4Hbeta were analyzed in gingival fibroblasts by RT-PCR. The protein production of type I collagen and P4H was examined respectively by ELISA and Western blot. RESULTS: In culture, HGF gingival fibroblasts showed similar growth characteristics to fibroblasts isolated from control gingivae. The mRNA and protein levels of type I collagen and P4Halpha in HGF fibroblasts were higher than those in controls. There were no detected differences in mRNA expression levels of MMP-1, MMP-3, TIMP-1, P4Halpha(II), alpha(III) and P4Hbeta between HGF and control fibroblasts. CONCLUSIONS: These data suggest that increased collagen post-translational modification by P4H may be one mechanism by which increased collagen accumulation occurs in some forms of HGF.     

Distribution of procollagen type III, collagen type VI and tenascin in oral submucous fibrosis (OSF)

The distribution of procollagen type III, collagen type VI and tenascin was studied in biopsy specimens from the buccal mucosa of 19 Indian women with confirmed oral submucous fibrosis (OSF) using the immunogold-silver staining technique. Immunohistochemistry revealed a loss of stainable procollagen type III and collagen type VI in the fibrotic zones of oral submucous fibrosis compared to normal oral mucosa. Tenascin was noted only very faintly at the subepithelial basement membrane. The present study showed that procollagen type III and collagen type VI in OSF were expressed in a specific pattern which allows a clear differentiation between fibrotic areas and adjacent apparently normal connective tissue stroma. Loss of procollagen type III, and therefore a probable predominance of collagen type I in collagen fiber bundles, and an almost complete loss of collagen type VI might explain the stiffness of the oral mucosa in patients with OSF. The immunohistochemical findings provided evidence that the process of fibrosis starts in the deeper subepithelial connective tissue stroma and not close to the subepithelial basement membrane. Further studies are required to determine whether OSF is due to increased or altered synthesis and deposition of extracellular matrix proteins, altered fibrolysis or both.