目的探讨纤维蛋白粘合剂(FS)与全飞秒激光小切口透镜取出术(SMILE)来源的人角膜成纤维细胞(HCFs)的生物相容性。
方法人角膜组织取自2018年3—4月于广西医科大学第一附属医院行SMILE患者12例24眼术中取出的角膜基质透镜，体外分离培养HCFs，观察HCFs在FS表面的生长状态。将HCFs分为2倍浸提液组和正常对照组，分别与2倍浸提液和完全培养基共培养，采用吖啶橙(AO)/溴化乙锭(EB)双染色法观察并比较各组细胞凋亡情况。将HCFs分为3个组，2倍浸提液组和正常对照组细胞每孔分别加入2倍浸提液和完全培养基各100 μl，空白对照组无细胞，每孔加入100 μl完全培养基，采用四甲基偶氮唑蓝(MTT)法检测并比较3个组HCFs的细胞增生能力，并进行细胞毒性分级。将HCFs分为1倍浸提液组、2倍浸提液组和正常对照组，分别用1倍浸提液、2倍浸提液和完全培养基培养，采用Annexin V-FITC/PI流式细胞术检测并比较3个组细胞凋亡率。
结果HCFs在FS表面生长良好，形态正常。MTT法检测结果显示，2倍浸提液组与正常对照组的HCFs具有相似的增生趋势，2倍浸提液组HCFs的0～72 h毒性评级为0～1级。AO/EB染色结果显示，2倍浸提液组和正常对照组的HCFs状态均正常，仅可见极少量早期凋亡细胞。流式细胞术检测结果显示，正常对照组、1倍浸提液组和2倍浸提液组的细胞凋亡率分别为(4.96±1.09)%、(3.66±1.35)%和(2.88±0.66)%，差异无统计学意义( F＝2.89， P＝0.13)。
结论FS无体外细胞毒性，与HCFs有良好的生物相容性。
ObjectiveTo study the biocompatibility of fibrin sealant (FS) and human corneal fibroblasts (HCFs) obtained by small incision lenticule extraction (SMILE).
MethodsThe human corneal stromal tissues were selected from corneal stromal lens in 24 eyes of 12 patients underwent SMILE in the First Affiliated Hospital of Guangxi Medical University from March to April 2018.HCFs were isolated and cultured in vitro within 1 hour after the corneal stromal lens were extracted and the growth status of HCFs on FS surface was observed.HCFs were divided into 2-fold leaching solution group and normal control group, and the cells in the two groups were treated with 2-fold leaching solution or complete medium according to grouping, respectively.The apoptosis of HCFs in the two groups was observed by acridine orange (AO)/ethidium bromide (EB) double staining.The proliferation of HCFs in the two groups was assayed by methyl thiazolyl tetrazolium (MTT) method.HCFs in logarithmic phase were divided into 2-fold leaching solution group, normal control group, and the cells were treated with 2-fold leaching solution or complete medium according to grouping, respectively.In addition, a blank control group without HCFs was also set and treated with complete medium.The absorbance value and relative growth rate of HCFs in the three groups were compared.HCFs in logarithmic phase were divided into 1-fold leaching solution group, 2-fold leaching solution group and normal control group, and the cells were treated with 1-fold leaching solution, 2-fold leaching solution or complete medium culture according to grouping, respectively.The apoptosis of HCFs in the three groups was compared by Annexin V-FITC/PI flow cytometry, and the cytotoxicity of the three groups was graded.Written informed consent was obtained from each patient before the operation.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (No.2018[022]).
ResultsHCFs grew well on FS surface and the morphology was normal.MTT assay showed that HCFs in the 2-fold leaching solution group and the normal control group had a similar proliferation tendency, and the toxicity index of HCFs in the 2-fold leaching solution group was graded 0-1 at 0-72 hours after changing solution.After AO/EB staining, the HCFs in the 2-fold leaching solution group and the normal control group were normal, and only a small amount of early apoptotic cells were observed.Flow cytometry showed that the apoptosis rates of the normal control group, once leaching solution group and the double leaching solution group were (4.96±1.09)%, (3.66±1.35)% and (2.88±0.66)%, respectively, with no significant difference among them ( F=2.89, P=0.13).
ConclusionsFS has no cytotoxicity and has good biocompatibility with HCFs in vitro.
叶青,蒋林志,陈文琳,等. 纤维蛋白粘合剂与人角膜成纤维细胞的生物相容性[J]. 中华实验眼科杂志,2021,39(02)：113-118.
DOI:10.3760/cma.j.cn115989-20190605-00244版权归中华医学会所有。
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