氯化镧对子宫颈癌HeLa细胞增殖和迁移能力的影响

Effects of lanthanum chloride on proliferation and migration of human cervical cancer cell line HeLa cells

目的探讨氯化镧对宫颈癌HeLa细胞增殖和迁移能力的影响，为寻找新的有效治疗宫颈癌的药物提供实验依据。方法宫颈癌细胞株HeLa细胞经培养、传代后分为两组，即实验组（加入5、50、100μmol／L的氯化镧）和对照组（未加入氯化镧）。倒置显微镜下观察两组细胞的生长情况，激光共聚焦显微镜观察两组细胞核的形态变化。采用四甲基偶氮唑蓝（MTT）比色法检测两组细胞的增殖情况，双染色流式细胞仪检测两组细胞的凋亡率，体外迁移实验检测两组细胞迁移能力的变化，逆转录（RT）-PCR技术检测两组细胞中增殖、抗凋亡和迁移相关基因细胞周期蛋白（cyclinD1）、锌指蛋白（A20）及基质金属蛋白酶9（MMP-9）mRNA的表达。结果倒置显微镜下观察：实验组细胞随着氯化镧浓度的升高，细胞密度逐渐降低，细胞质内颗粒逐渐增多，颜色加深，细胞间隙增大，少量细胞变圆漂浮，脱落细胞也逐渐增多；而对照组细胞贴壁生长密集，形态清晰，胞质饱满，相邻细胞生长融合成片。激光共聚焦显微镜观察：实验组细胞核染色质浓聚边集，核体积变小，随着氯化镧浓度的升高，核染色质崩解，核膜破裂、核碎裂，直至细胞核完全碎裂；而对照组细胞核饱满，核膜完整。实验组细胞经不同浓度（5、50、100μmol／L）的氯化镧作用后，细胞生长抑制率分别为24％、51％、78％，高于对照组（0），差异有统计学意义（P〈0．05）；细胞凋亡率分别为（4．91±0．39）％、（7．30±0．71）％、（13．48±0．92）％，高于对照组的（0．89±0．11）％，差异有统计学意义（P〈0．01）；穿膜细胞数分别为（22．2±4．3）、（12．0±3．2）、（7．8±2．6）个，低于对照组的（41．2±5．4）个，差异有统计学意义（P〈0．01）。实验组细胞中cyclinDl、A20及MMP-9mRNA的表达强度随氯化镧浓度（5、50、100μmol／L）的升高逐渐减弱。结论氯化镧通过下调宫颈癌细胞中增殖、抗凋亡和迁移相关基因cyclinD1、A20及MMP-9mRNA的表达，抑制宫颈癌细胞的增殖和迁移并诱导其凋亡。

Objective To investigate the effects of lanthanum chloride on proliferation and migration activity of human cervical cancer cells in vitro which may be a new anti-cervical cancer drug and provide experimental data for cervical cancer treatment. Methods HeLa cells cultured in vitro were divided into two groups： experimental group and control group. In experimental group, the cells were respectively treated with lanthanum chloride at different concentrations, 5, 50 and 100 μmol/L, while the cells in the control group were not treated with lanthanum chloride. The cell growth was observed by inverted microscope and the morphology changes of the cells were observed by the laser scanning confocal microscope （LSCM）. Proliferation of HeLa cells in the two groups was detected by methyl thiazolyl tetrazolium （MTT） test; apoptosis rate was analyzed by flow cytometry （FCM）. Cell migration test was applied to observe the effect of lanthanum chloride on migration. Reverse transcription （RT）-PCR was employed to evaluate the effects of lanthanum chloride on proliferation gene （ cyclinD1 ） , anti-apoptosis gene （ zinc finger protein A20 ） and migration-related gene （matrix metalloproteinase 9, MMP-9 ） . Results The status of cell growth was observed under the inverted microscope： with the increased of the lanthanum chloride concentrations, the cell density of reduced, the granule in cytoplasm increased, color intensifying and intercellular space enlarged; some cells became rounding and dead, floating in the culture media; the exfoliated cells increased gradually in the experimental groups. While In the control group, the cells grew adherently, with clear morphology and plump cytoplasm, and adjacent cell grew in lamellar. Observed with LSCM： the nuclear chromatin condensated and marginated with the volume of nuclear decreased in experimental groups. With the increase of the lanthanum chloride concentrations, nuclei in the experimental groups became pyknotic and then underwent karyorrhexis. However, the nuclear of the cells in control group were inact. The growth inhibition rates of lanthanum chloride groups （5, 50, 100 μmol/L） were 24%, 51% and 78%, respectively, in which each was significantly higher than that of the control group （ P 〈 0. 05 ） ; the apoptosis rates of lanthanum chloride group were （ 4. 91 ± 0. 39 ） % , （ 7.30± 0. 71 ） % and （ 13.48± 0. 92 ） % , respectively, which were all significantly higher than that of the control group [ （ 0. 89±0. 11 ） %, P 〈 0.01 ]. The migration ability of the cells was also decreased by the treatment of lanthanum chloride, the number of migrated ceils in lanthanum chloride groups were 22. 2 ±4. 3, 12.0 ±3. 2 and 7.8 ±2.6 respectively, which were all significantly lower than that of the control group （41.2 ±5.4, P 〈0.01 ）. The expression of genes of cyclinD1, A20 and MMP-9, were all decreased by the treatment of lanthanum chloride in a dose-dependent manner. Conclusion Lanthanum chloride can inhibit the proliferation and migration of cervical cancer cells, and induce apoptosis by down-regulating cyclinD1, A20, and MMP-9 expressions in vitro.