【摘要】 目的：观察粉防己碱（tetrandrine,Tet）对体外培养人翼状胬肉成纤维细胞(human pterygium fibroblast，HPF）增殖的影响, 研究粉防己碱对翼状胬肉成纤维细胞增殖的作用,寻找辅助治疗翼状胬肉的新方法。方法：用不同浓度（0～160μmol/L）Tet作用体外培养的HPF, 观察24～96h Tet对HPF的影响。MTT法检测细胞生长抑制率, 免疫细胞化学法检测Tet干预前后HPF增殖细胞核抗原(PCNA)的表达情况。结果：在Tet 20, 40, 80, 160μmol/L浓度作用24～72h范围内,可剂量和时间依赖性的抑制HPF增生(P<0.05)。Tet干预后PCNA蛋白表达下降，当Tet的浓度在20～160μmol/L范围内能浓度依赖性地抑制细胞表达PCNA(P<0.05) 结论：Tet可显著抑制翼状胬肉成纤维细胞增生,在一定浓度和时间范围内抑制作用呈剂量与时间依赖性 。但在高浓度时（>160μmol/L）可能存在细胞毒性作用。
【关键词】 粉防己碱 翼状胬肉 成纤维细胞 增殖
INTRODUCTION
Pterygium is local fibrovascular tissue proliferation of bulbar conjunctiva, which violates the cornea. It is one of the most common clinical eye diseases, which could not only cause eye irritation signs and appearance defects but also influence the visual acuity to different extents, and at present its pathogenesis is not clear. There are various surgical methods for pterygium, but it always recurs and the therapeutic effects are not satisfying. So the aim of current studies is to find the effective therapy with fewer side effects[1]. Through our experimental study about the inhibition of Tetrandrine (Tet) on fibroblasts of pterygium in vitro, we hope to find a new method as complementary therapy for pterygium.
MATERIALS AND METHODS
Materials Human pterygium fibroblasts (HPF) cultured in vitro, Tet was made by the pharmaceutical factory in Jinhua, Zhejiang Province, China (powder, purity> 98%); 1∶250 trypsin, propidium iodide (PI), RNA enzymes, EDTA Hanks solution were provided by the Tongji Medical University; MTT was purchased from Sigma company and DMEM medium and neonatal bovine serum was purchased from GIBCO corporation; mouse antihuman keratin monoclonal antibody and mouse antihuman PCNA monoclonal antibody were supplied by Wuhan Boshide company, Hubei Province.
Methods
Preparation of Tet storage solution A small amount of DMSO was joined to dissolve, and DMEM was added to make 10mmol/L solution of storage, and it was packed and preserved at 20 ℃ avoiding light. The period of validity lasted two weeks. When used, DMEM medium was diluted to the required final concentration, and the concentration of DMSO was less than 1‰.
Cultivation, identification and passage of HPF Pterygium tissues were obtained from ophthalmic surgical resection in our hospital. Mass culture was applied to observe block adherence, medium color, cell growth and morphological characteristics after inoculating for one day. It was observed that 48 hours later some cells climbed out from organization block (Figure 1) and every three to four days the culture solution was replaced once. After the cells grew to 80% from the bottom of bottle, 2.5g/L trypsin was used to digest cells, and passage culture was carried out in the proportion of 1∶3. Subculturing cells were inoculated in 6 preset orifices of glass coverslips. When cells overgrew on glass coverslips, cells were fixed by cold acetone and ethanol mixed 1∶1 for 15 minutes. Then after they were dried, we dyed them with HE staining (Figure 2), and with antivimentin and keratin antibody immunohistochemical appraisal was performed (Figure 3).Table 1 The effect of Tet on HPF proliferation drug concentration (略)
Effect of Tet on proliferation of HPF MTT colorimetric assay was adopted. HPF cells were cultured with 100mL/L bovine serum, 100U/mL penicillin, and 100U/mL streptomycin in DMEM medium at 37℃, 50mL/L CO2 incubator. And the cells were digested and subcultured with 2.5g/L trypsin. HPF cells of logarithmic growth phase were taken and inoculated in the 96hole boards with 1×105/mL, and each was plus 200μL solution. Blank group without inoculation, Tet group and control group were established. The fluid was changed after 24 hours, and Tet was not added to control group. The final concentrations of Tet group were 20μmol/L, 40μmol/L, 80μmol/L and 160μmol/L respectively. A sixpoint hole was set up for each concentration at each time point. 20μL of 5g/L MTT was added at 24, 48, and 72 hours respectively after Tet intervention. It was continued to culture for 4 hours and then the culture solution was aspirated. 150μL DMSO was added to each hole and the solution was vibrated for 10 minutes until the crystallization was completed dissolved. In ELX800 Reader 490nm wavelength was elected and blank group was adjusted to zero, and the absorbance (A) of each hole was determined. The inhibition rate of cell growth was calculated as follows: inhibition rate = (A value of the control groupA value the experimental group/A value of the control group)× 100%.
PCNA expression of HPF Immunocytochemistry method was employed. HPF cells of logarithmic growth phase were taken and inoculated in the 6hole boards with 1×105 /mL. The solution was changed after 24 hours. Tet was not added to control group. In Tet group the final concentrations of Tet were 20μmol/L, 40μmol/L, 80μmol/L, and 160μmol/L respectively. After it was cultured for 48 hours, it was washed three times with 0.01mmol/L PBS and fixed with cold acetone plus ethanol in the proportion of 1∶1. SP method was adopted and operation proceeded according to the manual. The dilution of mouse antiPCNA monoclonal antibody was 1∶50. PCNApositive nucleus was of brown color. The expression intensity of PCNA was determined with highresolution color image analysis system (Tongji Technology Group, Qianping Image Corporation). The optical density of five visual fields was measured and the average was adopted. 100 cells were randomly selected (magnification 20×10) and all of the positive and negative cells were counted and listed.
Statistically Analysis The experimental results and data were expressed with ±s, and analysis of variance was used to analyze the relationship between MTT inhibition rate and concentration and time. SPSS11.0 software was used in statistical analysis, and ttest was employed to analyze the effects of Tet of different concentrations on cells compared with control group. The expression of PCNApositive cells was analyzed through chisquare test. P<0.05 was regarded as statistically significant
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