胰岛素生长因子1在颅缝闭合中调节作用的实验研究

目的：研究胰岛素生长因子1对大鼠颅缝细胞的骨诱导作用和体外调节小鼠矢状缝闭合的作用。方法：获取新出生的SD大鼠的矢状缝细胞进行培养和出生8d的CD1小鼠矢状缝进行体外无血清器官培养基培养，加入胰岛素生长因子1（insulin lilce growth factor 1 IGF1），浓度分别为10ng/ml和40ng/ml，并设立不加IGF1者为对照组，应用RT－PCR检测颅缝细胞的成骨细胞表型碱性磷酸酶、骨钙素和骨桥蛋白mRNA表达，ELISA法检测培养液I型胶原的分泌，光镜观察小鼠矢状缝闭合的情况。结果：加入IGF1的矢状缝细胞的成骨细胞表型碱性磷酸酶、骨钙素和骨桥蛋白mRNA表达以及培养液I型胶原分泌量较对照组明显增高。有IGF1干预的矢状缝移植体培养8d时，颅缝内侧面骨板开始接近，培养20d时，颅缝小部分闭合，培养30d时颅缝大部分闭合，无IGF1干预的对照组培养30d，颅缝未发生闭合。结论：胰岛素生长因子1通过增强颅缝细胞的骨诱导促进颅缝的闭合。     

碱性成纤维细胞生长因子对骨髓间充质干细胞扩增及分化潜能的影响

目的 观察碱性成纤维细胞生长因子(bFGF)对大鼠骨髓间充质干细胞(BMSCs)体外增殖及其分化潜能的影响.方法 体外培养大鼠BMSCs,分别设对照组和bFGF处理组,bFGF作用浓度分别为1、10、100 μg/L.作用72 h后,噻唑蓝(MTT)测吸光度(A)值反映细胞增殖.细胞免疫组织化学测细胞CD44的累积A值;逆转录-聚合酶链反应(RT-PCR)检测细胞CD44的mRNA相对表达量.结果 在1 μg/L bFGF作用下其MTT A值为0.334±0.036较对照组A值0.251 ±0.033明显增高(P＜0.05).在1 μg/L bFGF作用下其细胞免疫组织化学测得CD44的累积A值较对照组明显增高(P＜0.01);其RT-PCR测得CD44的mRNA相对表达量较对照组明显增高(P＜0.01).结论 作用浓度为1 μg/L的bFGF可促进BMSCs的体外扩增并有助于保持BMSCs的分化潜能.     

骨形态发生蛋白和碱性成纤维细胞生长因子激发纤维环细胞成骨的潜能

目的研究联合使用重组人骨形态发生蛋白（recombinant human bone morphogenetic protein-2，rhBMP-2）和碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）椎间盘纤维环细胞成骨潜能的激发作用。方法向体外培养的纤维环细胞中分别及联合加入rhBMP-2和bFGF，观察纤维环细胞的表型表达特点。结果联合使用rh-BMP-2和bFGF能够明显促进椎间盘细胞增殖，提高细胞内碱性磷酸酶活力，增加I型胶原分泌，提高钙盐沉积程度，提高骨钙素的表达水平。结论联用rhBMP-2和bFGF能够诱导纤维环细胞向成骨细胞方向分化，分泌钙盐并形成钙结节。     

甘露糖敏感型绿脓杆菌制剂对肝癌细胞周期的作用机制研究

目的 探讨甘露糖敏感性绿脓杆菌制剂(pseudomonasaeruginosa mannose sensitive haemagglutination vaccine,PA-MSHA)对肝细胞癌(hepatocellular carcinoma,HCC)细胞周期的作用及其机制.方法 培养人肝癌细胞株MHCC97L及HepG2,以不同剂量的PA-MSHA作用于肝癌细胞.MTT实验检测PA-MSHA对细胞增殖的影响.流式细胞技术检测PA-MSHA对细胞周期的作用.Western blot检测PA-MSHA作用前后细胞周期蛋白Cyclin D1、细胞周期蛋白依赖性激酶2(CDK2)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)以及细胞周期蛋白激酶抑制剂p21和p27的表达情况.结果 与对照组相比,PA-MSHA显著抑制MHCC97L和HepG2细胞的增殖,其作用呈剂量及时间依赖性(P＜0.05).PA-MSHA显著诱导肝癌细胞周期阻滞,PA-MSHA作用后G0-G1期和G2-M期细胞比例显著增高,而S期细胞比例则显著下降(P＜0.05).PA-MSHA显著抑制Cyclin D1、CDK2、PCNA蛋白的表达,而促进p21和p27的表达.外源性甘露糖可显著抑制PA-MSHA对肝癌细胞增殖和周期的作用(P＜0.05).结论 PA-MSHA通过调控细胞周期相关蛋白来诱导HCC细胞周期阻滞,抑制细胞增殖.这一作用是通过肝癌细胞表面的甘露糖的介导实现的.     

生长因子在颅缝闭合中调节作用的实验研究

最近的研究表明硬脑膜和其分泌的生长因子(TGF)决定着颅缝的闭合，颅缝早闭的模型研究中发现许多生长因子的高表达，而在开放颅缝中生长因子表达微弱^[1]。我们拟进一步探讨生长因子对大鼠颅缝间质细胞的成骨诱导作用和IGF，体外对小鼠矢状缝闭合的直接作用。     

成纤维细胞生长因子促进放烧复合伤创面愈合的实验研究

采用重组的碱性成纤维细胞生长因子(bFGF),观察其对大鼠重度放烧复合伤皮肤创面愈合的作用。结果提示:bFGF治疗组第60天创面完全愈合的百分率较对照组提高28.5％,50％烧伤面积愈合所需的平均时间较对照组提前9天。病理学检查发现,bFGF可刺激成纤维细胞增殖及胶原合成,促进毛细血管增生和肉芽组织形成。在早期,bFGF尚可增强创面炎症反应,提高机体白细胞吞噬功能,加速脾细胞NK活性的恢复。     

低频振动对人成骨细胞生物学特性影响的研究

目的:研究低频振动对人成骨细胞增殖分化及基质分泌的影响.方法:取成人的髂骨松质骨,获得人成骨细胞.并在培养48h后,施加0.1、0.2、0.5、2和5Hz的低频微振动,通过流式细胞仪检测成骨细胞增殖状况:化学比色法和放射免疫法检测碱性磷酸酶(ALP)活性和骨钙素含量.结果:加载 0.2和0.5Hz可使成骨细胞的ALP活性明显增高(P＜0.01);0.1和2Hz振动后其ALP活性与对照组无明显差异;5Hz振动则明显降低ALP活性(P＜ 0.01).加载 0.2和0.5Hz振动的成骨细胞,S期细胞从10.4%增加至12.45%和16.12%,增殖指数从20.14%增加到26.21%和 28.75%.0.1和2Hz低频振动则显示出对细胞增殖指数无明显影响(P＞0.05).而5Hz使细胞增殖指数明显降低至13.22%(P＜0.05).同时,加载0.2和0.5Hz振动可明显增加成骨细胞骨钙素分泌量至1.87和 2.47ng/ml(P＜0.05),而加载0.1Hz对骨钙素分泌量无明显影响(P＞0.05),加载2和5Hz相应降低了骨钙素分泌量(P＜0.05).结论:0.2～0.5Hz的低频振动能促进成骨细胞增殖分化和成骨活性物质分泌,对低频振动应用于骨折治疗有指导作用.     

不同浓度生长分化因子-5对内侧副韧带成纤维细胞增殖及胶原合成的影响

目的 观察生长分化因子-5(GDF-5)对内侧副韧带(MCL)成纤维细胞增殖能力及胶原合成的影响. 方法 体外培养10周龄SD大鼠MCL成纤维细胞,分别加入不同浓度GDF-5,分为4组:0 ng/mL组、10 ng/mL组、50 ng/mL组、100 ng/mL组,用CCK-8法测定细胞增殖能力,羟脯氨酸测试盒测定细胞总胶原含量,逆转录-聚合酶链反应(RT-PCR)检测I与Ⅲ型胶原mRNA的表达. 结果 浓度为10、50、100 ng/mL的GDF-5均能增强MCL成纤维细胞的增殖能力,以100 ng/mL的GDF-5作用最显著,组间比较差异均有统计学意义(P＜0.05).MCL成纤维细胞中加入GDF-5后,羟脯氨酸量增加,I型胶原mRNA表达增加,组间比较差异均有统计学意义(P＜0.05),但Ⅲ型胶原mRNA表达组间比较差异无统计学意义(P＞0.05). 结论 GDF-5可以促进MCL成纤维细胞增殖及胶原合成进而促进韧带损伤愈合.     

胰岛素样生长因子-1对成纤维细胞向成骨细胞转化的影响

目的 探讨胰岛素样生长因子-1对成纤维细胞(Fb)向成骨细胞转化的影响. 方法 Fb来源于成年新西兰大白兔,通过分离、纯化、培养而得到.实验分为3组:空白对照组、成骨诱导组和实验组.空白对照组:常规培养液培养,不加入任何诱导剂及干预因子;成骨诱导组:成骨诱导液为常规培养液中加入浓度为1×10-8 mol/L地塞米松、50 mg/L维生素C、10 mmol/L β-甘油磷酸钠;实验组:成骨诱导液中加入终浓度为50 ng/mL的IGF-1.采用四甲基偶氮唑蓝(MTT)比色法检测各组细胞增殖情况,检测各组细胞碱性磷酸酶(ALP)活性,测定各组细胞培养后6d后的骨钙素含量,采用钙钴染色评价培养后2周的成骨能力.结果 实验组细胞增殖较其他两组旺盛,差异有统计学意义(P＜0.05),而空白对照组和成骨诱导组差异无统计学意义(P＞ 0.05).ALP活性和骨钙素测定:实验组ALP表达明显高于其他两组,差异有统计学意义(P＜0.05);成骨诱导组ALP表达高于空白对照组,差异有统计学意义(P＜0.05).实验组钙化结节数量明显较其他两组多,成骨诱导组次之,空白对照组未见钙化结节.结论 胰岛素样生长因子-1可以促进Fb在成骨过程中的增殖以及增加其成骨能力.     

共价结合生长因子的胶原补片修补大鼠左心室室壁瘤的实验研究

目的 比较采用碳化二亚胺法(EDC法)处理共价结合不同生长因子的胶原补片修补大鼠左心室室壁瘤后的再血管化情况,及其对大鼠左心功能的影响.方法 直径5 mm的圆形胶原补片经EDC法处理作对照组,再分别结合血管内皮细胞生长因子(VEGF)或VEGF+碱性成纤维细胞生长因子(bFGF)后作实验组.成年雄性SD大鼠行左冠状动脉前降支结扎制作透壁心梗模型.4周后,心脏彩超筛选心梗面积占左心室前壁25％～ 35％者入选.随机分成3组,以不同补片行室璧瘤修补,对照组(8只),VEGF组(10只),VEGF+ bFGF组(10只).术后1周、2周、4周分别行心脏彩超监测左心功能,至实验终点取材,免疫荧光法检测补片边缘毛细血管(vWFⅧ染色)及成熟血管(SMA染色)的生成情况.结果 全组死亡比例15％(6/40只).修补1周后,3组动物心功能均明显改善;4周后,结合生长因子的两组心功能较对照组明显改善(对照组对VEGF组,P＜0.05;对照组对VEGF+ bFGF组,P＜0.01).组织学检查显示,两组结合有生长因子的毛细血管生成情况均较对照组明显改善(P ＜0.05);VEGF+bFGF组的再血管化较其他两组明显改善(P＜0.01).相关性分析显示,大鼠心功能参数(FS)与再血管化呈正相关(P=0.0297,r2=0.998).结论 EDC法可有效改善胶原补片的机械性能,共价结合生长因子后可显著增加补片内血管生成,再血管化有助于左心功能的维持.     

Cranial deformation in craniosynostosis. A new explanation.

Skull growth after premature fusion of a single suture was described by Virchow in 1851. He observed that growth was restricted in a plane perpendicular to a fused suture. However, he failed to predict the compensatory growth patterns that produce many of the deformities recognized as features of individual craniosynostosis syndromes. The deformities resulting from premature closure of a coronal, sagittal, metopic, or lambdoid suture can be predicted by the following observations: (1) cranial vault bones that are prematurely fused act as a single bone plate with decreased growth potential; (2) asymmetrical bone deposition occurs mainly at perimeter sutures, with increased bone deposition directed away from the bone plate; (3) sutures adjacent to the stenotic suture compensate in growth more than those sutures not contiguous with the closed suture; and (4) enhanced bone deposition occurs along both sides of a nonperimeter suture that is a continuation of the prematurely closed suture. These four rules were derived by critically examining the clinical deformities observed with each form of craniosynostosis. These rules assume that cranial sutures have the capacity to compensate by depositing bone asymmetrically along their edges. Unequal growth patterns have been demonstrated in the frontonasal suture of rabbits by Selman and Sarnat. In addition, unequal bone deposition has also been demonstrated along the parieto-interparietal suture in albino rats by Baer. Human studies to determine if asymmetrical bone deposition actively occurs along cranial vault sutures in response to a stenotic suture have not been performed, however. It is also unclear whether these four guidelines apply to cranial base abnormalities observed with craniosynostosis. As new radiologic techniques develop to define the configuration of the skull in intricate detail, a skull pattern of growth explaining the pathogenesis of all deformities created by premature fusion of a cranial vault suture may become apparent.     

Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency.

Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor beta1 [TGF-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5-bromo-2'deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone-associated ECM molecules is potently up-regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture-associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF-beta1 and fibroblast growth factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF-2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than SAG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (*p < 0.001) in FRCs cocultured with SAG suture-derived dural cells as compared with FRCs cocultured alone or with PF suture-derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.     

Effect of basic fibroblast growth factor and hyaluronic acid on proliferation of rabbit chondrocytes in vitro

Objective: To investigate the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid (HA) on the proliferation of rabbit chondrocytes in vitro. Methods: Chondrocytes from the knee joints of New Zealand white rabbits were cultured, bFGF or HA or both were added into the culture medium respectively, and the proliferation of the ehondrocytes was measured with MTT 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetra-zolium bromide. (MTT, Sigma, M2128). Results: Basic fibroblast growth factor (10ng/ml) with low concentration of fetal bovine serum in the culture medium promoted the proliferation of chondrocytes significantly, and this effect reached its maximum when concentration of bFGF reached 50ng/ml. HA itself had no effect on the proliferation of chondrocytcs. However, when bFGF was used in combination with HA, especially when the concentration of bFGF was 50-500ng/ml and that of HA was 10-50ng/ml, the effect on the proliferation of chondrocytes was much more than when bFGF or HA was used alone. Conclusions: bFGF can promote the proliferation of chondrocytes. HA, which has no effect on the proliferation of the cells, can maintain a normal growth of chondrocytcs.When bFGF is used in combination with HA, more proliferation is obtained.     

两种细胞因子联合应用对骨髓基质干细胞增殖和成骨活性的影响

目的 研究骨形态发生蛋白融合基因-4/7(BMP-4/7)和碱性成纤维细胞生长因子(bFGF)联合应用对体外培养兔骨髓基质干细胞(BMSCs)增殖和成骨活性的影响.方法 体外培养BMSCs,在第3代细胞培养液中加入不同浓度的BMP-4/7和bFGF,依据加入不同基因浓度组合的不同分为5个实验组(A组:80 ng/mL BMP-4/7,B组:80 ng/mL bFGF,C组:30 ng/mL BMP-4/7+30ng/mL bFGF,D组:50 ng/mL BMP-4/7+50 ng/mL bFGF,E组:80 ng/mL BMP-4/7+80 ng/mL bFGF)和对照组(不加任何因子),采用绘制生长曲线,甲基噻唑基四唑(MTT)比色法检测细胞增殖活力,碱性磷酸酶(ALP)和降钙素(OC)活性检测法比较各组间差异,观察不同浓度的BMP-4/7和bFGF联合应用对兔BMSCs增殖和成骨活性的影响. 结果 传代后第5天对照组个别单核细胞贴壁,呈长梭形;A组细胞增殖,呈旋涡状排列;B组细胞较为密集,部分融合成片;C组细胞呈集落式生长,生长旺盛;D组细胞生长密集,可见明显的钙结节;E组细胞密集,可见细胞性钙结节形成.各组OD值、ALP含量、OC含量随着作用时间的延长而增加,各组不同培养时间的OD值差异均有统计学意义(P<0.01);且C、D、E组均高于A、B组,差异均有统计学意义(P<0.05).C、D、E组内随着作用浓度的增加,细胞增殖及成骨活性增强,呈浓度依赖关系,差异均有统计学意义(P<0.05). 结论 合理的联合应用BMP-4/7和bFGF可促进BMSCs细胞增殖,促进成骨活性,两者对BMSCs有明显的协同增强效应.     

A comprehensive screen of genes implicated in craniosynostosis

Recent advances in human molecular genetics have identified mutations in the TWIST, FGFR-1, FGFR-2 and FGFR-3 genes to be important causes of craniosynostosis. Despite this, however, mutations cannot be identified in the majority of patients. This study reports the first comprehensive screen of mutations in TWIST, FGFR-1, FGFR-2 and FGFR-3 genes in a cohort of patients with craniosynostosis. This has led to the identification of Saethre-Chotzen syndrome to be a new microdeletion disorder and reports the first example of a gene-environment interaction leading to craniosynostosis. In addition, investigation of the expression patterns of the Fgfr and Twist genes in the normal developing mouse coronal suture has identified the TWIST protein to be important in cranial suture initiation and biogenesis. These findings have significant clinical implications and will form the basis of future attempts to develop novel therapies aimed at inhibiting cranial suture fusion.     

Feasibility of using early mesencephalic neural plate for intracerebral grafting

The purpose of this study was to elucidate the biological significance and the possibility of intracerebral grafting of neuroepithelial stem cells derived from the mesencephalic neural plate. Immunohistological studies of embryonic day 10.5 (E10.5) Wister rats revealed strong nestin expression in the mesencephalic part of the neural plate. Mesencephalic neural plates removed from E10.5 rats were processed to either tissue or cell dissociation culture. They were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, cell proliferation and differentiation into neurons extending long neurites were obvious in a serum-free medium, in a medium containing 3% serum, and in a medium containing 20 ng/ml epidermal growth factor. On the other hand, in a medium containing 10 ng/ml basic fibroblast growth factor (bFGF), both vigorous cell proliferation and sphere formation were recognized. Furthermore, marked neurite growth was rarely seen in this culture. When they were plated in a dissociation culture, cell proliferation and neurosphere generation were also recognized only in a medium containing bFGF, depending on the initial cell concentration. The spheres, generated 7 days after the primary cell culture, were positively stained by nestin. These data suggested that bFGF was able to amplify the stem cell population present in the mesencephalic neural plate derived from early embryos. This might make it possible to obtain a large number of stem cells as donor material for neural transplantation on demand.     

大鼠颅骨体外培养矢状缝牵引成骨模型的建立

目的探讨建直幼年SD大鼠顿骨器官体外培养矢状缝牵引成骨模型。方法取19日龄的SD大鼠颅顶骨矢状缝组织块．随机分为对照组和实验组进行体外器官培养。实验组加力约3．92×10^3N（0．4g）力Hr照组不加力。培养24h结束时,进行大体观察及倒置显微镜下观察;并经苏木精-伊红染色后进行组织学观察。结果大体观察／受倒置显微镜下观察发现,实验组骨缝逐渐明显加宽。至加力24h,所有标本欠状缝未见断裂。组织学观察发现,缝两侧区域为成骨活跃部位,两侧骨化前沿交错排列。缝内可见纤维结缔纠织、成骨细胞、成纤维细胞及毛细血管,缝内组织与硬脑膜相连。对照组骨缝保持正常发育,未见明显变化,以成骨细胞为主。结论大鼠颅骨骨缝器官可以在体外培养中成功存活并继续生长。     

生长因子在颅缝早闭症中作用的研究进展

颅缝早闭是一种较常见的先天性颅面畸形,表现为一条或多条颅缝过早闭合。多种因素可以在胚胎期及出生后影响头骨的发育,从而导致不同类型的颅缝早闭。目前研究表明,生长因子与颅缝闭合过程有着密切的联系,本文就生长因子在颅缝早闭症中作用的研究进展进行综述。