碱性成纤维细胞生长因子对犬角膜上皮细胞增殖影响的研究

发布时间：2018-05-08 21:13

本文选题：碱性成纤维细胞生长因子 + 角膜上皮细胞　；参考：《华中农业大学》2007年硕士论文

【摘要】： 碱性成纤维细胞生长因子(bFGF)是一种广谱促有丝分裂原，对几乎所有来源于中胚层和神经外胚层的细胞具有广泛的生物学活性，它具有良好地神经保护、心肌保护、缺血保护以及促进创面愈合作用，使其临床治疗日益受到关注，同时bFGF在眼科领域的研究也日益深入。制作角膜上皮细胞，用DMEM／F-12培养液培养，将培养到第三代的角膜上皮细胞用四甲基偶氮唑盐(MTT)比色法观察碱性成纤维细胞生长因子在不同浓度(0ng／mL，1ng／mL，10ng／mL，，100ng／mL)和不同时间点(24h，48h，96h)对犬角膜上皮细胞培养增殖性能来研究碱性成纤维细胞生长因子对犬角膜上皮细胞的生长和增殖的影响，检测所的的结果表示为光密度值(OD)值，OD值的大小与细胞密度呈正比。结果表明，从1ng／mL开始，随浓度递增，OD值逐渐上升，10ng／mL时达到峰值，此后随浓度递增OD值下降。bFGF组从1ng／mL开始各浓度组OD值均大于对照组(P＜0.05)，说明bFGF对犬角膜上皮细胞有明显的促分裂作用。10ng／mL组在24h、48h、96h均高于其他剂量组。说明碱性成纤维细胞生长因子有明显促进犬角膜上皮细胞增殖的作用，在一定范围内呈剂量依赖性，10ng／mL为促进犬角膜上皮生长的最佳剂量。此外，本研究建立了犬角膜环钻损伤模型，分析碱性成纤维细胞生长因子对犬角膜损伤的治疗效果。将24只杂交犬随机分成四组：生理盐水对照组、0.1μg／mL bFGF组、0.5μg／mLbFGF组、1μg／mLbFGF组。每组使用不同浓度的bFGF点眼，每日三次，每次两滴。并在术后第1d、第3d、第5d、第7d同一时间用荧光染色素钠染液点眼，照相观察角膜上皮愈合效果。并于术后第3d、第7d、第14d取角膜各5只制作病理切片观察角膜上皮愈合情况及胶原纤维排列情况、成纤维细胞生长情况。荧光染色结果表明，环钻损伤后第1 d，0.5μg／mL剂量组愈合率为27.47％，1μg／mL剂量组愈合率为31.23％，与对照组相比差异极显著(P＜0.01)。环钻损伤后第3d、第5d不同浓度的bFGF各组愈合率均极显著高于对照组(P＜0.01)，第7d时除对照组有两例愈合不良外各组均基本愈合。显示碱性成纤维细胞生长因子能促进犬角膜损伤后角膜上皮愈合，早期应用即能发挥作用。病理切片观察发现添加碱性成纤维细胞生长因子的犬角膜上皮修复快于对照组，表现为细胞层数比对照组多，细胞排列整齐，焱性细胞浸润少，另外碱性成纤维细胞生长因子还能促进犬角膜损伤后角膜基质有序修复。未发现碱性成纤维细胞生长因子对犬角膜损伤后角膜新生血管生长有明显促进或抑制作用。
[Abstract]:Basic fibroblast growth factor (bFGF) is a broad-spectrum mitogen with extensive biological activity to almost all cells from mesoderm and neuroectoderm. The protection of ischemia and the promotion of wound healing have attracted more and more attention in clinical treatment. Meanwhile, the research of bFGF in ophthalmology has been deepened. Corneal epithelial cells were prepared and cultured in DMEM/F-12 medium. The third passage corneal epithelial cells were cultured by MTT colorimetry to study the effect of basic fibroblast growth factor (BFGF) on the proliferation of canine corneal epithelial cells at different concentrations (0 ng / mL ~ (-1) 10 ng / mL ~ (-1) ~ 100 ng / m ~ (-1) and at 24 h / m ~ (-1) for 48 h / 96 h) to study the effect of alkaloids on the proliferation of canine corneal epithelial cells in vitro. Effects of fibroblast growth factor on the growth and proliferation of canine corneal epithelial cells The results show that the OD value is proportional to the cell density. The results showed that from the beginning of 1ng/mL, the OD value gradually increased to a peak value of 10 ng / mL with increasing concentration. From then on, the OD value of each concentration group was higher than that of the control group (P < 0.05) from the beginning of 1ng/mL, which indicated that bFGF had obvious mitogenic effect on canine corneal epithelium cells. 10 ng / mL group was significantly higher than other dose groups at 24 h and 48 h to 96 h. The results showed that basic fibroblast growth factor (bFGF) could obviously promote the proliferation of canine corneal epithelial cells. In a dose-dependent manner, 10 ng / mL was the best dose to promote the growth of canine corneal epithelium. In addition, a canine corneal annular injury model was established, and the therapeutic effect of basic fibroblast growth factor (bFGF) on canine corneal injury was analyzed. 24 crossbred dogs were randomly divided into four groups: saline control group, 0. 1 渭 g/mL bFGF group, 0. 5 渭 g/mLbFGF group and 1 渭 g/mLbFGF group. Each group was given two drops of different concentrations of bFGF three times a day. The corneal epithelium healing was observed with fluorescein sodium at the same time on the 1st, 3rd, 5th and 7th day after operation. At the 3rd, 7th and 14th day, the corneal epithelium healing, collagen fiber arrangement and fibroblast growth were observed. The results of fluorescence staining showed that the healing rate of the 0.5 渭 g/mL group was 27.47 and the healing rate of the 1 渭 g/mL group was 31.233.The difference was significant compared with the control group (P < 0.01). The healing rate of different concentrations of bFGF was significantly higher than that of the control group on the 3rd and 5th day after drilling injury (P < 0.01). On the 7th day, there were two cases of poor healing in the control group. The results showed that basic fibroblast growth factor (bFGF) could promote corneal epithelial healing after corneal injury in dogs. The results of pathological section showed that the corneal epithelium repaired by adding basic fibroblast growth factor was faster than that of the control group, which showed that the number of cell layers was more than that of the control group, the cells were arranged neatly, and the infiltration of Yan cells was less than that of the control group. In addition, basic fibroblast growth factor can also promote corneal stroma repair after corneal injury in dogs. It was not found that basic fibroblast growth factor could significantly promote or inhibit corneal neovascularization after corneal injury in dogs.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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