低剂量TGF-β1维持三维培养模型中牛角膜基质细胞生长和缓解细胞外基质纤维化的作用

Effects of low-dose of TGF-β1 on maintaining bovine corneal stromal cell growth and retarding extra cellular matrix fibrosis in a three-dimensional culture model

背景转化生长因子（TGF）-β1在角膜损伤修复过程中发挥重要作用，不同剂量的TGF-β1对角膜细胞外基质（ECM）的合成具有不同影响，从而影响瘢痕形成的程度。既往研究多集中于高质量浓度TGF-β1对二维培养的角膜基质细胞的影响，而低质量浓度TGF-β1对角膜基质细胞ECM合成的影响鲜见研究。目的研究低质量浓度TGF-β1对体外三维培养的角膜基质细胞生长状况及ECM合成的影响。方法采用两步胶原酶消化法从新鲜牛眼中分离牛角膜基质细胞并置于含体积分数10%胎牛血清（FBS）的DMEM/F12培养基，利用Pellet体外三维培养模型对牛角膜基质细胞进行培养。将细胞分为0.25 ng/ml TGF-β1＋5% FBS组和0.50 ng/ml TGF-β1＋5% FBS组，分别于培养后48 h、1周、2周、3周观察Pellet培养模型的形态变化。于培养后3周采用苏木精－伊红染色法对Pellet培养模型进行常规组织形态学检查，采用钙黄绿素-AM/碘化丙啶法（Calcein-AM/PI法）于激光扫描共焦显微镜下检查角膜基质细胞的死亡率；采用实时荧光定量PCR及免疫荧光细胞化学法分别检测细胞中α-平滑肌肌动蛋白（α-SMA）、纤维连接蛋白（FN）、Ⅰ型胶原（ColⅠ）和Col Ⅲ mRNA及其蛋白的相对表达量；采用实时荧光定量PCR法检测细胞中角膜蛋白（KERA）mRNA和基膜聚糖（LUM）mRNA的表达。结果培养后48 h、1周、2周和3周Pellet细胞均成团生长。苏木精－伊红染色可见，Pellet球内均有大量红色淡染的胶原纤维以及大部分正常的成纤维细胞和少量坏死的成纤维细胞，坏死细胞可见细胞形态破坏，呈均质红染，且0.25 ng/ml TGF-β1＋5% FBS组和0.50 ng/ml TGF-β1＋5% FBS组培养细胞形态无明显差别。0.25 ng/ml TGF-β1＋5% FBS组和0.50 ng/ml TGF-β1＋5% FBS组细胞死亡率分别为（33.60±1.65）%和（30.90±0.78）%，差异无统计学意义（t＝0.144，P＝0.887）。0.25 ng/ml TGF-β1＋5% FBS组Pellet培养模型中α-SMA、FN、Col Ⅲ蛋白表达量均低于0.50 ng/ml TGF-β1＋5% FBS组，差异均有统计学意义（tα-SMA＝4.622，P＝0.010；tFN＝2.973，P＝0.040；tCol Ⅲ＝7.845，P〈0.001），0.25 ng/ml TGF-β1＋5% FBS组ColⅠ的表达量明显高于0.50 ng/ml TGF-β1＋5% FBS组，差异有统计学意义（tColⅠ＝4.022，P＝0.016）。在转录水平和蛋白表达水平，0.25 ng/ml TGF-β1＋5% FBS组Col Ⅲ/ColⅠ比值均低于0.50 ng/ml TGF-β1＋5% FBS组，差异均有统计学意义（tmRNA＝－3.039，P＝0.038；t蛋白＝3.215，P＝0.032）。培养后48 h、1周、2周Pellet培养模型中KERA mRNA和LUM mRNA均呈阳性表达，0.25 ng/ml TGF-β1＋5% FBS组LUM mRNA相对表达量随培养时间延长而逐渐增加；0.50 ng/ml TGF-β1＋5% FBS组LUM mRNA相对表达量于培养后1周时达峰值。2个组Pellet培养模型中KERA mRNA相对表达量均在培养后1周达峰值。结论低剂量的TGF-β1既能够维持Pellet体外三维培养模型中角膜基质细胞的生长并合成ECM，也使ECM组成成分的合成倾向于正常状态，以减少瘢痕化修复的趋势。

BackgroundTransforming growth factor-β1 （TGF-β1） plays an important role in corneal wound healing.The effects of TGF-β1 on the synthesis of extra cellular matrix （ECM） vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β1 on keratocytes under the two-dimensional culture condition, and the effect of low concentration of TGF-β1 on the synthesis of ECM in keratocytes remains unclear.ObjectiveThis study was to investigate the growth of Pellet, a three-dimensional model of corneal stroma cells in vitro, and its ECM synthesis under a low concentration of TGF-β1.MethodsBovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10% fetal bovine serum （FBS）. Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β1＋ 5% FBS and 0.50 ng/ml TGF-β1＋ 5% FBS were established, respectively.The morphology of Pellets was observed under the natural light at 48 hours, 1 week, 2 weeks and 3 weeks after culture.In 3 weeks after culture, the cell structures was observed by hematoxylin-eosin staining, and Calcein-AM/propidium （Calcein-AM/PI） staining was used to assay the cell viability.Real-time fluorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin （α-SMA）, fibronectin （FN）, type Ⅰ collagen （ColⅠ） and type Ⅲ collagen （Col Ⅲ） mRNA and proteins.RT-PCR was employed to detect the expressions of lumican （LUM） mRNA and keratocan （KERA） mRNA in the cells.ResultsCells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β1＋ 5% FBS group and 0.50 ng/ml TGF-β1＋ 5% FBS group, and most cells possessed complete structures.The death rate of the cells was （33.60±1.65）% in the 0.25 ng/ml TGF-β1＋ 5% FBS group and （30.90±0.78）% in the 0.50 ng/ml TGF-β1＋ 5% FBS group, showing an insignificant difference between them （t=0.144, P=0.887）. The expressions of α-SMA, FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β1＋ 5% FBS group were lower than those in the 0.50 ng/ml TGF-β1＋ 5% FBS group （tα-SMA=4.622, P=0.010; tFN =2.973, P=0.040; t Col Ⅲ=7.845, P〈0.001）, but the expression of Col Ⅰ in 0.25 ng/ml TGF-β1＋ 5% FBS group was higher than that in 0.50 ng/ml TGF-β1＋ 5% FBS group （tColⅠ=4.022, P=0.016）. The ratio of Col Ⅲ/ColⅠ in 0.25 ng/ml TGF-β1＋ 5% FBS group was lower than that in the 0.50 ng/ml TGF-β1＋ 5% FBS group in both mRNA and protein level （tmRNA =-3.039, P=0.038; tprotein=3.215, P=0.032）. The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β1＋ 5% FBS group increased over time.While in 0.50 ng/ml TGF-β1＋ 5% FBS group, the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks.ConclusionsLow-dose TGF-β1 in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β1, implying a scarless expression.