bFGF调节PDGF对人成骨细胞的促增殖作用

目的 探讨碱性成纤维细胞生长因子（ｂＦＧＦ）对血小板衍生生长因子（ＰＤＧＦ）促成骨细胞增殖作用的影响及其机制。方法 分离培养人成骨细胞;以ＰＤＧＦ－ＡＢ（１００ｎｇ／ｍｌ）单独培养、ｂＦＧＦ（１０ｎｇ／ｍｌ）与ＰＤＧＦ－ＡＢ（１００ｎｇ／ｍｌ）混合培养、不加生长因子等３种方式培养细胞,绘制细胞生长曲线;ｂＦＧＦ（１０ｎｇ／ｍｌ）与ＰＤＧＦ－ＡＡ或ＰＤＧＦ－ＢＢ（浓度均为４０ｎｇ／ｍｌ）以不同方式联     

bFGF对体外关节软骨细胞创伤愈合与增殖的影响

目的：探讨碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）对关节软骨细胞创伤愈合与增殖行为的影响。方法;采用体外创伤愈合模型及ＭＴＴ比色方法,对软骨细胞创伤愈合与增殖行为进行分析。结果：ｂＦＧＦ浓度为５ｎｇ／ｍｌ时,即可显著促进软骨细胞创伤愈合,浓度为５０ｎｇ／ｍｌ时,其促进作用达到最大值。     

TGFβ1，bFGF对前列腺基质细胞增殖和分化的影响

目的　探讨前列腺基质增生的机制。　方法　系用ＭＴＴ和ＴＵＮＥＬ法检测体外原代无血清培养的前列腺基质细胞的增殖,用免疫组化、ＲＴＰＣＲ 方法检测平滑肌细胞特异性标记蛋白的表达。　结果　ＴＧＦβ有多重效应：浓度为０－０１ ～１０．００ｎｇ／ｍｌ 的ＴＧＦβ可抑制指数增生期前列腺基质细胞增殖;浓度低于０－１ｎｇ／ｍｌ 的ＴＧＦβ刺激平顶期前列腺基质细胞增殖;浓度高于１ｎｇ／ｍｌ 的ＴＧＦβ促增殖平顶期的成纤维细胞向平滑肌细胞分化。ｂＦＧＦ对于指数增生期和平顶期细胞均有刺激增殖的作用,并抑制增殖平顶期的成纤维细胞向平滑肌细胞分化。　结论　ＴＧＦβ１／ｂＦＧＦ比例的改变有调节前列腺基质细胞的稳态平衡的作用。     

碱性成纤维细胞生长因子对成骨细胞粘附特性影响的实验研究

目的 针对骨组织工程中成骨细胞与基质材料间促粘附问题,研究碱性成纤维细胞生长因子（ｂＦＧＦ）对成骨细胞粘附特性的影响。方法 取兔骨髓基质干细胞来源的成骨细胞体外培养,分别用５、１０、５０、１００及２００ｎｇ／ｍｌ浓度的ｂＦＧＦ诱导培养２４小时作为实验组;不含ｂＦＧＦ的培养基作为对照组。观察接种后０．５、１、２、４、及８小时各时间点成骨细胞粘附情况,体现学计量粘附细胞数量。结果 １０ｎｇ／ｍｌｂＦＧ     

bFGF对跟腱成纤维细胞创伤愈合影响的体外研究

目的：研究ｂＦＧＦ在跟腱损伤修复中的作用及机制。方法：采用体外创伤愈合和细胞运动模型及ＭＴＴ方法,分别对ｂＦＧＦ作用下,跟腱成纤维细胞创伤愈合,运动与增殖行为进行分析。结果：ｂＦＧＦ浓度为５ｎｇ／ｍｌ时,即可显著地促进跟腱成纤维细胞的创伤愈合与增殖,当ｂＦＧＦ浓度提高至１００ｎｇ／ｍｌ,这种促进作用达到最大值。     

血小板衍生生长因子促进成骨细胞DNA合成的实验研究

目的探讨血小板衍生生长因子（ＰＤＧＦ）对成骨细胞ＤＮＡ合成和细胞周期变化的影响及其在骨折修复中的作用。方法从胎鼠颅骨分离、培养成骨细胞,应用流式细胞仪观察ＰＤＧＦ对成骨细胞ＤＮＡ合成及细胞周期的影响;通过透射电镜观察细胞的超微结构改变,了解ＰＤＧＦ在鼠成骨细胞分泌功能方面的作用。结果（１）ＰＤＧＦ对培养２４小时的成骨细胞Ｓ期的ＤＮＡ含量明显增高（１７７％）;（２）ＰＤＧＦ促使细胞内线粒体更丰富,粗面内质网增多。结论ＰＤＧＦ可通过刺激成骨细胞的ＤＮＡ合成,促进成骨细胞的分裂、增殖及细胞的合成分泌。     

生长抑素对人大肠癌细胞株SW480作用的体外实验研究

用ＭＴＴ比色分析法和流式细胞术研究了８肽生长抑素（ＳＭＳ２０１·９９５），ＳＭＳ）对人大肠癌细胞株ＳＷ４８０的作用。结果显示：ＳＭＳ在１．５６３～２００ｎｇ／ｍｌ浓度范围内对ＳＷ４８０具有抑制作用，并以浓度５０ｎｇ／ｍｌ时的抑制作用为最强。浓度在５０ｎｇ／ｍｌ以上时抑制作用并不继续增加，提示生长抑素（ｓｏｍａｔｏｓｔａｔｉｎ，ＳＳ）的作用是通过受体发挥的；ＳＭＳ明显抑制了大肠癌细胞ＤＮＡ及蛋白质合成，抑制ＳＷ４８０细胞从Ｇ０／Ｇ１期过渡到Ｓ期和Ｇ２Ｍ期，提示生长抑素在受体后通过抑制ＤＮＡ及蛋白质合成，抑制细胞周期循环，从而抑制大肠癌细胞的生长。     

转化生长因子β与椎间盘细胞Ⅰ型胶原基因调控的关系

目的：探讨ＴＧＦ－β在椎间盘Ⅰ型胶原代谢中的作用及作用机制。方法：应用斑点杂交和原位杂交技术检测了ＴＧＦ－β对椎间盘原代培养及传代培养的纤维环细胞、髓核细胞中Ⅰ型胶原ｍＲＮＡ,ＴＧＦ－β１浓度为１ｎｇ／ｍｌ、１０ｎｍ／ｍｌ,其作用分别是０ｎｇ／ｍｌ组的１．１８倍及１．３７倍;对于传代培养４ 纤维环细胞,其调节作用分别是０ｎｇ／ｍｌ组的１．４８倍及２．０３倍。结论：ＴＧＦ－β可以按照剂量依赖方式正向     

类胰岛素生长因子—1作用下肌腱细胞的周期改变

为了明确类胰岛素生长因子－１（ＩＧＦ－１）促进肌腱细胞生长的作用机制，以便进一步探索调控肌腱细胞生长的手段，采用体外培养的第６代肌腱细胞，加入ＩＧＦ－１共同培养后，与对照组一起，用流式细胞技术进行细胞周期亚时相的定量分析。结果表明，实验组的ＤＮＡ合成前期（Ｇ１期）时间、ＤＮＡ合成期（Ｓ期）时间和分裂前期及分裂期（Ｇ２Ｍ期）时间分别为１１．８，２１．４和６．８小时；而对照组的时间分别为２５．６，２２．６和２１．８小时。ＩＧＦ－１使肌腱细胞的Ｇ１期和Ｇ２Ｍ期所需要的时间大大缩短。说明ＩＧＦ－１对肌腱细胞生长的促进作用是通过加快Ｇ１期和Ｇ２Ｍ期的进程实现的     

转化生长因子β对椎间盘细胞Ⅰ、Ⅲ型胶原基因表达的调节作用

目的 探讨转化生长因子（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈｆａｃｔｏｒβ,ＴＧＦ－β）对椎间盘Ⅰ、Ⅲ型胶原基因表达的调节作用。方法 应用斑点杂交和原位杂交技术检测了ＴＧＦ－β１ 对椎间盘原代培养及传代培养的纤维环细胞、髓核细胞中Ⅰ、Ⅲ型胶原ｍＲＮＡ 的调节作用,采用ＶＩＤＡＳ软件计算杂交膜斑点及杂交涂片中细胞的平均光密度值,进行胶原ｍＲＮＡ相对定量。结果 （１）对于原代培养的椎间盘纤维环细胞Ⅰ型胶原ｍＲＮＡ,ＴＧＦ－β１ 浓度为１ ｎｇ／ｍｌ 及１０ ｎｇ／ｍｌ 时,其调节作用分别是０ ｎｇ／ｍｌ 组的１．１８ 倍及１．３７倍;对于传代培养的纤维环细胞,其调节作用分别是０ ｎｇ／ｍｌ 组的１．６ 倍及１．８４ 倍;对于传代培养的髓核细胞,其调节作用分别是０ ｎｇ／ｍｌ 组的１．４８ 倍及２．０３ 倍。（２） 对于原代培养的椎间盘纤维环细胞Ⅲ型胶原ｍＲＮＡ,ＴＧＦ－β１ 浓度为１ ｎｇ／ｍｌ 及１０ ｎｇ／ｍｌ 时,其调节作用分别是０ ｎｇ／ｍｌ 组的１．０８ 倍及１．２２ 倍;对于传代培养的纤维环细胞,其调节作用分别是０ ｎｇ／ｍｌ 组的１．２２ 倍及１．４０ 倍;对于传代培养的髓核细胞,其调节作用分别是０ ｎｇ／ｍｌ 组的１．２０ 倍及１．     

胰岛素样生长因子对体外培养豚鼠肋软骨细胞的影响

目的：了解胰岛素样生长因子(insulin-1ike growthfactor-1，IGF-1)对体外培养豚鼠肋软骨细胞分裂增殖及功能代谢的影响。方法：体外单层培养豚鼠肋软骨细胞，对照组培养液为无血流清DMEM，实验组在培养液DMEM中加入IGF-1使其终浓度分别为10ng／ml、50ng／ml、100ng／ml，作用6天后，检测细胞DNA含量和培养液中糖醛酸的含量。结果：IGF-1在10～100ng／ml浓度范围能明显增加培养软骨细胞的DNA及糖醛酸的含量，且以50ng／ml作用效果最明显，与对照组相比，有统计擘意义(P＜0．01)。结论：IGF-1对体外培养肋软骨细胞有刺激，并以剂量依赖性方式影响细胞的增殖及功能代谢。     

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.     

Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts

OBJECTIVE: To determine the effects of osteoarthritic (OA) subchondral osteoblasts on the metabolism of human OA chondrocytes in alginate beads. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 days in the absence or in the presence of osteoblasts isolated from non-sclerotic (N) or sclerotic (SC) zones of human OA subchondral bone in monolayer (co-culture system). Before co-culture, osteoblasts were incubated for 72 h with or without 1.7ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M (OSM). Aggrecan (AGG) and matrix metalloproteases (MMP)-3 and -13 mRNA levels in chondrocytes were quantified by real-time polymerase chain reaction. AGG production was assayed by a specific enzyme amplified sensitivity immunoassay. RESULTS: SC, but not N, osteoblasts induced a significant inhibition of AGG production and AGG gene expression by human OA chondrocytes in alginate beads, and significantly increased MMP-3 and MMP-13 gene expression by chondrocytes. When they were pre-incubated with IL-1beta, IL-6 or OSM, N osteoblasts inhibited AGG synthesis and increased MMP-3 and -13 gene expression by chondrocytes in alginate beads in a same order of magnitude as SC osteoblasts. CONCLUSIONS: These results demonstrate that SC OA subchondral osteoblasts could contribute to cartilage degradation by stimulating chondrocytes to produce more MMP and also by inhibiting AGG synthesis.     

胰岛素样生长因子-Ⅰ对兔关节软骨细胞增殖及代谢的影响

目的　探讨胰岛素样生长因子 - (IGF- )对体外生长的关节软骨细胞分裂增殖及功能代谢的影响。方法　采用体外单层培养的方法 ,应用兔关节软骨细胞 ,对照组培养液为 10 %小牛血清 DMEM,实验组在培养液DMEM中加入 IGF- 使其终浓度分别为 3、10、30、10 0及 30 0 ng/ ml,作用细胞 2、4和 6天 ,检测细胞 DNA含量和基质中糖醛酸的含量。结果　 IGF- 在 3～ 30 0 ng/ ml浓度范围能明显增加培养软骨细胞的 DNA及糖醛酸的含量 ,且以30～ 10 0 ng/ ml作用 4天刺激效果最明显 ,与对照组相比 ,有统计学意义 (P<0 .0 1)。结论　 IGF- 对体外培养软骨细胞有刺激 ,并以剂量时间依赖性方式影响增殖及功能代谢。     

生长因子促成年兔关节软骨细胞增殖的研究

目的　研究促进成年兔关节软骨细胞迅速增殖的方法。方法　将体外培养 2 4～ 2 8月龄的新西兰大白兔的原代关节软骨细胞中加入不同浓度的胰岛素样生长因子 - (IGF- )、碱性成纤维细胞生长因子 (b FGF)及 IGF- b FGF联合应用。于 2 4、4 8及 72小时分别进行 MTT检测软骨细胞的成活数 ;检测传 3代后各组软骨细胞的总增殖倍数 ;第 14天后 ,用流式细胞仪检测各组软骨细胞的增殖倍数 ,观察 IGF- 、b FGF及 IGF- b FGF联合应用对成年兔关节软骨细胞各时间点的促增殖作用。结果　 1施加不同浓度的 IGF- 、b FGF或两者联合应用后各时间点软骨细胞活细胞数明显高于对照组 (P<0 .0 1) ;2传 3代后 ,三组总增殖倍数也都明显高于对照组 (P<0 .0 1) ,且两种因子联合应用 ,增殖倍数明 显比应用单个因子时高 ,有统计学意义 (P<0 .0 1) ;3培养 14天后 ,增殖指数明显高于对照组 (P<0 .0 1) ,两种因子联合应用时 ,增殖指数明显比应用单个因子时高 (P<0 .0 5 ) ;而施加两次因子时 ,增殖指数比施加一次因子时高 (P<0 .0 5 )。结论　 IGF- 、b FGF对成年兔关节软骨细胞有明显的促增殖作用 ,两者之间有协同效应。     

Effects of hyaluronan on proteoglycan content of osteoarthritic chondrocytes in vitro.

PURPOSE: To investigate the effect of hyaluronic acid (HA) on proteoglycan (PG) concentration in alginate cultures of human osteoarthritic chondrocytes and to analyze whether HA exhibit anti-degradative effects in the presence of the cytokine IL-1beta. METHODS: Cartilage samples from ten patients with osteoarthritis of the knee were harvested and chondrocytes were cultivated in alginate beads. Four groups were cultured: control group with and without IL-1beta (500 pg/ml) and HA group (100 microg/ml) with and without IL-1beta (500 pg/ml). PG concentration was estimated by a dimethylmethylene blue assay. To assess cell proliferation, we measured DNA content fluorometrically. RESULTS: The proliferation rate (DNA) was unchanged in all culture groups. In the control-group PG/DNA (ng/ng) concentration was 27.1 +/- 7.2. Supplementation of the medium with HA decreased PG concentration to 25.3 +/- 6.9 (p < 0.05). After administration of IL-1beta PG/DNA concentration dropped to 23.1 +/- 6.0 (p < 0.01). By contrast HA treatment of IL-1beta stimulated chondrocytes did not further decrease PG concentration (23.9 +/- 6.1). In fact the negative effect of isolated HA application was inverted if HA was given with IL-1beta (p < 0.05). CONCLUSIONS: In osteoarthritic chondrocytes cultured phenotypically stable, HA could exhibit protective effects only in the presence of the degradative cytokine IL-1beta. Thus, the reported anti-inflammatory effects of HA to cartilage matrix seem to be more indirect by blocking degradative effects of cytokines to the matrix.     

bFGF对人α1(I)原胶原基因启动子活性的调控

目的　观察bFGF对人皮肤成纤维细胞增殖的影响及调控α1(I)原胶原基因序列的作用。　方法　用组织块法培养人皮肤成纤维细胞并传代,采用BrdU掺入的ELISA法,测定不同浓度的bFGF对成纤维细胞增殖的影响。构建三种人α1(I)原胶原基因5'侧翼序列与报告基因氯霉素乙酰基转移酶(CAT)的重组质粒,用FuGENE转染试剂转染成纤维细胞,同时用p-sv-b-GAL表达质粒转染细胞作为阳性对照组,ELISA法测定经bFGF处理后成纤维细胞CAT的表达量。　结果　在体积分数2%或10%小牛血清培养条件下,bFGF加入浓度从0.25ng/ml增至64.00ng/ml,作用24h后,各组细胞增殖数值之间差异有显著性意义（P<0.05）。用三种重组质粒分别转染细胞,bFGF处理24h后,CAT相对表达量测定结果提示,bFGF组与对照组相比差异有显著性意义（P<0.05）。　结论　bFGF对人皮肤成纤维细胞增殖有抑制作用,对胶原基因启动序列具有负性调控作用,且存在剂量依赖关系。     

Subchondral bone osteoblasts induce phenotypic changes in human osteoarthritic chondrocytes

OBJECTIVE: To determine the influence of osteoarthritic (OA) phenotype of subchondral osteoblasts on the phenotype of human chondrocytes. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 or 10 days in the absence or in the presence of osteoblasts in monolayer. The osteoblasts were either isolated from non-sclerotic (N) or sclerotic (SC) zones of human subchondral bone. Before co-culture, osteoblasts were incubated for 72 h with or without 1.7 ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M. SOX9, type I, II and X collagen (COL1, COL2, COL10), osteoblasts-stimulating factor (OSF)-1, bone alkaline phosphatase (ALP), parathyroid hormone related peptide (PTHrP) and its receptor (PTH-R) messenger RNA (mRNA) levels in chondrocytes were quantified by real-time polymerase chain reaction. RESULTS: In comparison with chondrocytes cultured alone in alginate beads, chondrocytes after 4 days in co-culture with N or SC osteoblasts expressed significantly less SOX9 and COL2 mRNA. The decrease of SOX9 and COL2 gene expression was significantly more pronounced in the presence of SC than in the presence of N osteoblasts (P<0.001). OSF-1 mRNA level in chondrocyte was increased by both N and SC osteoblasts, but to a larger extent by SC osteoblasts (P<0.001). PTHrP expression in chondrocytes was 21-fold increased by N osteoblasts but four-fold inhibited by SC osteoblasts. PTHrP secretion was also increased by N but reduced by SC osteoblasts. SC, but not N osteoblasts, induced a significant decrease of PTH-R gene expression in chondrocyte. In our experimental conditions, chondrocytes did not express COL1, COL10 or ALP, even after 10 days of co-culture with osteoblasts. CONCLUSIONS: In co-culture, SC subchondral osteoblasts decrease SOX9, COL2, PTHrP and PTH-R gene expression by chondrocytes but increase that of OSF-1. These findings suggest that SC osteoblasts could initiate chondrocyte phenotype shift towards hypertrophic differentiation and subsequently further matrix mineralization.