不同浓度碱性成纤维细胞生长因子对体外培养肌腱细胞增殖的影响

发布时间：2018-01-02 06:21

本文关键词：不同浓度碱性成纤维细胞生长因子对体外培养肌腱细胞增殖的影响　出处：《宁波大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的随着对肌腱愈合机制研究的不断深入及分子生物学等的不断发展，发现多种生长因子及其受体在肌腱愈合过程中起着关键的调控作用，其中碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)在临床中促创面愈合已得到广泛应用，且临床疗效已得到证实，使其已成为研究促肌腱愈合的外源性生长因子的热点。本实验的目的在于探讨不同浓度的bFGF对体外培养兔肌腱细胞增殖的影响，选择出促进肌腱细胞增殖的最佳浓度，并比较未冻存与冻存后复苏细胞体外培养增殖差异，从而为肌腱损伤的修复和组织工程肌腱种子细胞的培养提供重要的基础参数。方法1、无菌条件下切取新西兰乳兔的双下肢趾屈肌腱，在显微镜下剥离肌腱外膜，采用Henderson分步酶消化法分离出肌腱细胞，并用含20%胎牛血清的F-12培养液进行培养、传代并冻存第2代腱细胞；2、倒置显微镜下观察细胞形态变化及生长情况，，并对所得肌腱细胞行免疫细胞化学染色法测定细胞合成胶原类型；3、在体外培养第2代兔肌腱细胞的培养液中分别加入不同浓度（0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、20ng/ml、30ng/ml、40ng/ml和50ng/ml）的bFGF，继续培养48h，MTT法检测不同浓度bFGF组光密度(optical density,OD)值，选出促肌腱细胞增殖的最佳bFGF浓度；4、复苏冻存1月后的第2代肌腱细胞，使用最佳浓度的bFGF继续体外培养，测定OD值，比较未冻存细胞与冻存后复苏细胞增殖的差异；5、试验数据用SPSS18.0统计软件包进行统计学分析，以P0.05作为判断差异有统计学意义的标准。结果1、通过不同的酶消化分离可获得较为纯正的肌腱细胞，体外培养肌腱细胞可表现出良好的细胞增殖能力和传代能力；2、用鼠抗兔胶原I、Ⅲ抗体染色，DAB显色试剂盒显色，肌腱细胞表现为阳性反应，证明所获细胞为肌腱细胞；3、相比于对照组OD均值：5ng/ml、10ng/ml、20ng/ml、30ng/ml、40ng/ml和50ng/mlbFGF组差异有显著性(P 0.05)；随着bFGF浓度的增加，各组OD值先逐渐增大(5～20ng/mL),达到最高值（20ng/mL）后,又逐渐降低(20～50ng/mL)；4、应用最佳促肌腱细胞增殖bFGF浓度后，冻存组和冻存后复苏组之间细胞增殖无统计学差异（P0.05）。结论1、肌腱细胞能够在体外分离、扩增和传代，为研究肌腱愈合及构建组织工程化人工肌腱所需种子细胞的获取提供了可靠的试验基础；2、bFGF有明显促肌腱细胞增殖的作用，且与浓度有一定相关性，即促肌腱细胞增殖的起始bFGF浓度为5ng/ml，而20ng/ml时可达到促肌腱细胞增殖的最佳浓度；3、肌腱细胞冻存复苏后并不会影响其体外培养增殖。
[Abstract]:Objective with the development of the mechanism of tendon healing and molecular biology, found that several growth factors and their receptors in tendon healing process play a key role, including basic fibroblast growth factor (basic fibroblast, growth factor, bFGF) in the clinical wound healing promotion has been widely used, and the clinical the effect has been confirmed, it has become a study of promoting tendon healing of exogenous growth factors focus. The purpose of this experiment is to study the effect of different concentration of bFGF on proliferation of rabbit tendon cells in vitro culture influence, choose the best concentration to stimulate the proliferation of tendon cells, and compare the unfrozen and frozen thawed cells in vitro the proliferation of differences, so as to provide essential parameters for training and repair of seed cells for tendon tissue engineering tendon injury.
Methods 1 harvested neonatal New Zealand rabbit leg flexor tendon, tendon membrane stripping under the microscope, isolated tendon cells by Henderson step enzyme digestion method and cultured with F-12 containing 20% fetal bovine serum culture, subculture and cryopreservation of the second generation of tendon cells; 2, cell morphology was observed change and growth under the microscope, and the collagen synthesis was determined by cell type the tendon cells by immunocytochemical staining; 3, cultured with different concentration were cultured in second generations of rabbit tendon cells in vitro (0.5ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml and 50ng/ml) bFGF and continue to culture 48h, MTT method was used to detect the different concentration of bFGF groups (optical density, OD optical density) value, select the best concentration of bFGF to promote the proliferation of tendon cells; 4, resuscitated in January after the second generation of muscle tendon cells, with the optimal concentration of bFGF To determine OD value culture, in vitro, comparison of unfrozen between cells and frozen thawed cell proliferation; 5, the test data were analyzed using SPSS18.0 statistical software package, using P0.05 as the judgment standard. The difference was statistically significant
The 1, can obtain more pure tendon cells by enzyme digestion of different separation, tendon cells can show good cell proliferation and passage culture in vitro; 2, using mouse anti rabbit antibody staining of collagen I, III, DAB coloration kit, tendon cells showed positive reaction, that the cells were tendon cells; 3, compared to the control group mean OD 10ng/ml, 20ng/ml, 5ng/ml, 30ng/ml, there was significant difference between 40ng/ml and 50ng/mlbFGF group (P 0.05); with the increase of bFGF concentration, the group OD value gradually increased (5 ~ 20ng/mL), reached the highest value (20ng/mL), and gradually reduce (20 ~ 50ng/mL); 4, application of the best to promote proliferation of tendon cells bFGF concentration after cryopreservation group and frozen thawed group had no significant difference between cell proliferation (P0.05).
Conclusion 1, tendon cells in vitro isolation, amplification and passages, and provide the experimental basis for the study of tendon healing and reliable construction for tissue engineered tendon for seed cells; 2, bFGF can obviously promote the proliferation of tendon cells, and the concentration has certain correlation, i.e. the initial concentration of bFGF tendon cell proliferation of 5ng/ml, and 20ng/ml can reach the optimal concentration of promoting the proliferation of tendon cells; 3, tendon cells after cryopreservation does not affect its proliferation in vitro.

【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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