Bevacizumab对人Tenon囊成纤维细胞迁移抑制作用的研究

目的：观察bevacizumab对人Tenon囊成纤维细胞迁移能力的影响,探讨青光眼术后滤过泡瘢痕化的对策。方法：采用从陕西省人民医院中心实验室细胞库中取出冻存的人Tenon囊成纤维细胞,采用细胞复苏法,遵循无菌原则,进行常规培养。创伤划痕试验：待细胞融合度达到80%时在单层细胞表面划出一条无细胞的刮除带,空白对照组加入不含血清的DMEM培养液,bevacizumab处理组加入 bevacizumab 浓度为1 mg/mL 不含血清的DMEM培养液,分时段(0,24,48,72h)观察并测量划痕宽度。结果：人Tenon囊成纤维传代细胞显微镜下观察呈长梭形,细胞核位于细胞的中部,核较大,胞浆丰富,生长时呈漩涡状排列走形,增殖能力强,符合成纤维细胞的一般形态。冻存和复苏后细胞的形态结构和生物学特点维持不变。细胞划痕试验显示：0h时,两组细胞划痕初始宽度相等；24 h 时两组细胞迁移距离基本一致；48 h 时bevacizumab处理组细胞迁移距离明显小于空白对照组；72 h时空白对照组划痕基本愈合, bevacizumab处理组细胞迁移距离较48 h无明显变化,且细胞死亡数目较多。结论：利用细胞复苏法可成功培养形态结构和生物学特性稳定的传代人Tenon囊成纤维细胞,为实验研究奠定细胞学基础。成纤维细胞本身具有较强的迁移能力,外源性bevacizumab作用可以明显抑制细胞的迁移,作用时间过长时,会造成细胞的过多死亡。抗新生血管药物对成纤维细胞的迁徙具有一定的抑制作用,未来很有可能成为眼科临床抗击青光眼术后滤过泡瘢痕化的重要手段。

Inhibition effects of Bevacizumab on migration of human Tenon capsule fibroblasts

AIM: To investigate the inhibition effect of bevacizumab on human Tenon capsule fibroblasts(HTFs)and discuss the countermeasures of bleb scarringscarring in glaucoma surgery countermeasures.

METHODS: Adopted cell recovery method and followed the aseptic principles, we performed the culture of HTFs which came from the Central Laboratory of Shaanxi People's Hospital cell library. Wound Healing assay: We scraped a cell-free zone on the cell surface when the cells reached confluence at 80%. The control group was added to serum-free DMEM medium. The HTFs of bevacizumab group were stimulated with 1mg/mL concentrations without DEME for 0, 24, 48, and 72h. The scratch width was observed and measured.

RESULTS:HTFs were long fusiform shape under microscope, the nucleus is in the center of the cell with larger nucleus, abundant cytoplasm, were arranged in a whorled growth out of shape, strong ability to proliferate, conform to general forms of fibroblast. The morphological and biological characteristics of cells after cryopreservation and resuscitation remain unchanged. Wound healing assay: 0h, equal to the initial width of the two groups, 24h when the migration distance of the two groups of cells are basically the same, 48h when the control group cell migration distance is greater than that in the bevacizumab processing group, 72h when the control group scratches basichealing, bevacizumab treated cells migrate closer than 48h no significant change, and a lot of cells died.

CONCLUSION:Cell recovery method can successfully cultured HTFs, which was stability on morphology and biological properties, laying the cellular basis for experimental research.Fibroblast itself has a strong ability to migrate, outside-derived bevacizumab can inhibit HTFs migration evidently and it will cause excessive cell death when. Bevacizumab has certain extent inhibitory effect on HTFs migration, and it is likely to become one of the important drugs for creating bleb scarring after glaucoma surgery in the future.