方法：2周龄幼年健康豚鼠10只，体外培养RPE细胞，传代、鉴定后，将细胞分为Ver处理组和未处理组，Ver处理组加入80mg/L Ver作用12h。两组均进一步分为聚焦光组、离焦光组、平行光组和空白对照组，前三组分别接受聚焦光、离焦光(均为将平行光经透镜转化)和平行光照射，空白对照组不接受照射。于照射后立即采用激光扫描共焦显微镜(laser scanning confocal microscopy，LSCM)测定细胞内Ca²⁺荧光强度，分析不同光照形式与效应的关系。统计学方法采用单因素方差分析。

结果：Ver作用于RPE细胞12h后，20，40，80mg/L组的细胞凋亡情况与空白对照组相比均无统计学意义(P>0.05)，Ver可降低RPE细胞内Ca²⁺荧光强度，浓度为20，40，80mg/L时分别降低了10.36%，24.54%，58.05%，仅80mg/L组与无光照组相比差异有统计学意义(P<0.05)。未加Ver对RPE细胞进行光照，聚焦光组的Ca²⁺荧光强度较其他各组明显升高，离焦光组的荧光强度也较高，组间比较有统计学差异(P<0.05)。加入80mg/L Ver对RPE细胞作用12h后再进行光照，光照各组Ca²⁺荧光强度没有明显提高，组间比较没有统计学差异(P>0.05)。

结论：超过一定浓度的Ver可诱导RPE细胞凋亡，80mg/L可在不引起RPE细胞凋亡的前提下有效降低细胞内Ca²⁺荧光强度； 不同光照形式对豚鼠RPE细胞内Ca²⁺有明显刺激作用，聚焦光影响最大； 不同光照形式对Ver处理的RPE细胞内的Ca²⁺荧光强度无明显作用。

METHODS: Ten two-week-old healthy guinea pigs were chosen and RPE cells were cultured in vitro. Cells were divided into Ver treated and untreated groups, then each group further divided into 4 groups: focused light group, defocused light group, parallel light group and control group. The first three groups were exposed to the focused light, defocused light(2 forms of light were transformed from parallel light by passing through different lens)and the parallel light respectively, and control group was removed from exposure of light. After exposed to different forms of light, the fluorescence intensity of intracellular Ca²⁺ were detected by laser scanning confocal microscope(LSCM)immediately. Cool white light was used as light source. Cells were exposed to light with same spot diameter and degree of irradiation level(at 300 LUX)for 30 minutes respectively. Horizontal temperature of cells changed between 36.5℃-37.2℃. There was no natural light interference. In order to avoid the impaction of refraction of liquid, most of the medium were siphoned off before irradiation. The data were analysed by SPSS 13.0 statistical software, completely randomized design ANOVA and Pearson linear correlation analysis were used as statistical methods. The forms-effect relations were explored.

RESULTS: Treated with Ver for 12 hours, the apoptosis rates of RPE cells in 20, 40, 80mg/L concentration group had no significant difference compared with control group(P>0.05). Ver could reduce the Ca²⁺ fluorescence intensity of RPE cell,and there was significantly statistical difference in 80mg/L group(P<0.05). The Ca²⁺ fluorescence intensity of focused group of no Ver treated part was significantly higher than other groups, had significant differences compared with the other groups(P<0.05). After added 80mg/L Ver on the RPE cells for 12 hours, exposure to light did not significantly increase the Ca²⁺ fluorescence intensity, there was no significant difference among the 4 groups(P >0.05).

CONCLUSION: Focused light can significantly stimulate the concentration of intracellular Ca²⁺ of RPE cells. Ver above a certain concentration can induce apoptosis of RPE cells; 80mg/L Ver can effectively reduce intracellular Ca²⁺ fluorescence intensity under the premise of not leading to apoptosis of RPE cells. Different forms of light have no effect on the Ca²⁺ fluorescence intensity of RPE cells after treated with Ver(80mg/L)for 12 hours.