严重低氧低血清蛋白对人增生性瘢痕成纤维细胞功能的影响

Effects of severe hypoxia and low concentration of serum protein on the function of human hypertrophic scar fibroblasts

目的模拟增生性瘢痕形成和演变过程中的环境因素，探讨中、重度低氧低血清蛋白对人增生性瘢痕Fb（HSF）功能的影响。方法常规培养人HSF，取第3～6代细胞按照随机数字表法分为10．0％氧气＋10．0％小牛血清（FCS）、5．0％氧气＋5．0％FCS、0．5％氧气＋0．5％FCS组，先用不含FCS的DMEM培养液培养24h后，再采用相应体积分数的氧气和FCS培养。采用噻唑蓝法检测细胞增殖活性（结果以实际细胞数表示），天狼星红染色法检测细胞总胶原含量（结果以吸光度值表示），蛋白质印迹法检测低氧诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF）、TGF—β1、B淋巴细胞瘤2（Bcl-2）、P53的蛋白表达（结果以灰度值比值表示），原位末端标记法检测细胞凋亡率。以上实验每组样本数均为3。对数据行Kruskal，Wallis检验、Dunnett检验。结果（1）与10．0％氧气＋10．0％FCS组的（11000±1306）个比较，5．0％氧气＋5．0％FCS组细胞增殖活性较高[（13290±1500）个，P〈0．05]，0．5％氧气＋0．5％FCS组细胞增殖活性较低[（6999±765）个，P〈0．05]。（2）与10．0％氧气＋10．0％FCS组（0．0396±0．0042）比较，5．0％氧气＋5．0％FCS组细胞总胶原含量较高（0．0516±0．0051，P〈0．05），0．5％氧气＋0．5％FCS组细胞总胶原含量较低（0．0156±0．0024．P〈0．05）。（3）与10．0％氧气＋10．0％FCS组比较，5．0％氧气＋5．0％FCS组细胞的HIF-1α、VEGF、TGF—β1、Bcl-2蛋白表达量增高（P值均小于0．05），P53的蛋白表达量差异不明显（P〉0．05）；0．5％氧气＋0．5％FCS组细胞的HIF-1α、VEGF、TGF—β1、Bel-2蛋白表达量下降（P值均小于0．05），P53蛋白表达量增高（P〈0．05）。（4）与10．0％氧气＋10．0％FCS组[（1．2±0．9）％]比较，5．0％氧气＋5，0％FCS组细胞凋亡率无明显差异[（2．6±0．9）％，P〉0．05]，0．5％氧气＋0．5％FCS组细胞凋产率明显升高[（13．3±4．1）％，P〈0．05]。结论重度低氧低血清蛋白可抑制人HSF的增殖活性、总胶原生成，并诱导细胞凋亡。

Objective To simulate the environmental factors during the process of formation and evolution of hypertrophic scar, so as to explore the effects of moderate and severe hypoxia and low concentration of serum protein on the function of human hypertrophic scar fibroblasts （HSFs）. Methods Human HSFs were routinely cultured. Cells of the 3rd to the 6th passage were divided into 10.0% oxygen ＋ 10.0% fetal calf serum （FCS） , 5.0% oxygen ＋ 5.0% FCS, and 0.5% oxygen ＋ 0.5% FCS groups according to the random number table. After being cultured with DMEM nutrient solution with no FCS for 24 h, the cells were cultured with the corresponding volume fraction of oxygen and FCS. Cell proliferation activity was determined with methyl-thiazole-tetrazolium assay （ denoted as actual cell number）. Content of total collagen was detected with Sirius red staining method （denoted as absorba/lce value）. Protein expression levels of hypoxia-inducible factor 1 α （HIF-1 α） , vascular endothelial growth factor （VEGF） , transforming growth factor β1 （TGF-β1） , B-cell lymphoma 2 （Bcl-2） , and P53 were determined with Western blotting （denoted as ratio of gray value）. Cell apoptosis rate was detected by in situ end labeling method. The sample numbers of each group in the above experiments were all 3. Data were processed with Kruskal-Wallis test and Dunnett test. Results （1） Compared with 11 000 ± 1 306 in 10.0% oxygen ± 10.0% FCS group, the cell proliferation activity was higher in 5.0% oxygen＋5.0% FCS group （13 290±1 500, P 〈0.05）, but lower in0.5% oxygen ＋0.5% FCSgroup （6 999 ±765, P 〈0.05）. （2） Compared with 0.039 6±0.004 2 in 10.0% oxygen ＋ 10.0% FCS group, the content of total collagen of cells was higher in 5.0% oxygen ＋ 5.0% FCS group （0.051 6±0.005 1, P 〈0.05）, but lower in 0.5% oxygen ＋0.5% FCS group （0.015 6 ±0.002 4, P 〈0.05）. （3） Compared with those in 10.0% oxygen ＋10. 0% FCSgroup, the protein expression levels of HIF-1 α, VEGF, TGF-β1 , and Bcl-2 were increased （with P values below 0. 05） , with no obvious difference in protein expression level of P53 in 5.0% oxygen ＋ 5.0% FCS group （ P 〉 0.05） , whereas the protein expression levels of HIF-1α, VEGF, TGF-β1 , and Bcl-2 were decreased （with P values below 0.05 ） , while the protein expression level of P53 was increased in 0.5% oxygen ＋ 0.5% FCS group （ P 〈 0.05）. （4） Compared with （1.2 ±0.9）% in 10.0% oxygen ＋10. 0% FCS group, the cellapoptosis rate in 5.0% oxygen ＋ 5.0% FCS group showed no significant difference [ （2.6 ± 0.9） %, P 〉 0.05 ], while it was significantly increased in 0. 5% oxygen ＋ 0. 5% FCS group [（13. 3 ±4. 1）%, P 〈 0. 05]. Conclusions Severe hypoxia and low concentration of serum protein can inhibit proliferation activity and production of total collagen of human HSFs and induce their apoptosis.