TMPyP4-PDT对人卵巢癌A2780细胞的杀伤作用及其对MCM2和CA-Ⅸ mRNA表达的影响

Effects of TMPyP4-PDT on human ovarian carcinoma A2780 cells in vitro and expressions of MCM2 and CA-Ⅸ mRNA

目的:观察四-(N-甲基-4-吡啶基)卟啉(TMPyP4)对人卵巢癌A2780细胞的光动力学杀伤作用及其对微型染色体维持蛋白2(MCM2)和碳酸酐酶Ⅸ(CA-Ⅸ)mRNA表达水平的影响,阐明TMPyP4-PDT(光动力疗法)对人卵巢癌A2780细胞的杀伤作用机制。方法:体外培养人卵巢癌A2780细胞株,按不同处理因素分为空白对照组、单纯TMPyP4组、单纯PDT组和TMPyP4-PDT组。采用CCK-8法检测TMPyP4-PDT对A2780细胞株的增殖抑制作用。流式细胞术测定不同浓度TMPyP4对A2780细胞株凋亡的影响。光镜下观察各组细胞HE染色的形态学变化。实时荧光定量PCR法检测MCM2和CA-IX mRNA表达水平的变化。结果:与空白对照组比较,单纯TMPyP4组随着药物浓度的增加,细胞的增殖抑制率逐渐增加(P<0.05);单纯PDT组细胞增殖抑制率增加不明显(P>0.05);TMPyP4-PDT组细胞增殖抑制率随着药物浓度及激光能量密度的增加而增加(P<0.05)。流式细胞术检测,TMPyP4-PDT可诱导A2780细胞凋亡,在激光能量密度为4J.cm-2的条件下,3、6、15、30和60μmol.L-1 TMPyP4作用4h,A2780细胞凋亡率分别为(9.46±0.04)%、(17.7±0.3)%、(25.4±0.2)%、(30.2±0.2)%和(66.3±0.3)%。光镜下可观察到空白对照组及单纯PDT组贴壁细胞多,无核固缩及核碎裂样改变;单纯TMPyP4组及TMPyP4-PDT组贴壁细胞减少,出现核固缩、空泡及部分胞浆溶解样改变。实时荧光定量PCR法检测,在激光能量密度为4J.cm-2的条件下,3、6、15、30和60μmol.L-1TMPyP4作用4h,MCM2和CA-IX mRNA表达水平明显降低,与空白对照组比较差异有统计学意义(P<0.01)。结论:TMPyP4-PDT对人卵巢癌A2780细胞有明显杀伤作用,其机制可能与负性调节MCM2和CA-Ⅸ的表达有关。

Objective To investigate the effects of photodynamic therapy mediated by meso 5, 10, 15, 20-Tetrakis- （N-methyl-4-pyridyl） porphine（TMPyP4） on human ovarian carcinoma A2780 cells in vitro as well as the expression levels of MCM2 and CA-IX mRNA in A2780 cells and to clarify the destruction mechanism of the TMPyP4-PDT on human ovarian carcinoma A2780 cells. Metho0s The human ovarian carcinoma A2780 cells were cultured in vitro and divided into 4 groups as follows： blank control group, simple TMPyP4 group, simple PDT group and TMPyP4- PDT group. The increase of inhibitory effects of TMPyP4-PDT on proliferation of A2780 cells were detected by CCK-8 assay. The apoptosis of A2780 cells after treated with different concentrations of TMPyP4 were detected by flow cytometry（FCM）. The morphological changes of cells in various groups were observed with HE staining under light microscope. The expression levels of MCM2 and CA-IX mRNA were detected by RT-PCR. Results The inhibitory rates of cell proliferation in simple TMPyP4 group were gradually increased with the increase of the dosage（P^0.05）. The increase of inhibitory rates of cell proliferation in simple PDT group was not obvious（P~〉 0.05）. The inhibitory rates of cell proliferation in TMPyP4-PDT group were increased with the increase of the dosage and the light energy density （P % 0.05）. FCM analysis showed that TMPyP4-PDT could induce the apoptosisof A2780 cells; the apoptotic rates of A2780 cells were （9.46--＋_0.04）%, （17.7~0.3）~, （25.4--＋-- 0.2） ~, （30.2~0.2） ~ and （66.3~0.3） ~/00 when the light energy density was 4 J ~ cm-2 and the doses of TMPyP4 were 3, 6, 15, 30 and 60 /,mol ~ L 1 and the treatment time was 4 h. More adherent cells without nuclear pyknosis and nuclear fragmentation in blank control group and simple PDT group were observed under light microscope; Less adherent cells as well as nuclear pyknosis, vacuole and some cytoplasmic dissolution in simple TMPyP4 group and TMPyP4-PDT group could be observed under light microscope. RT-PCR analysis showed that compared with blank control group , the expressions of MCM2 and CA-IX mRNA were decreased significantly after treated with 3, 6, 15, 30 and 60 /,mol ~ L-1 TMPyP4 for 4 h （P ~ 0. 01） under the conditions of the light energy density of 4 J ~ cm-2. Oonclusion TMPyP4-PDT has distinctive inhibitory effects on human ovarian carcinoma A2780 cells, and the mechanism may associate with the negative regulation of the expressions of MCM2 and CA-IX .