摘要
目的
1研究ALA-PDT在体外对宫颈癌细胞的生长抑制作用特点,以寻找对ALA-PDT最敏感的宫颈癌细胞株和影响细胞增殖的最佳作用参数。
2探讨ALA-PDT对宫颈癌细胞的生长抑制机制,为深入探讨光动力作用杀伤宫颈癌细胞的生物学机制提供理论依据。
3分析ALA-PDT对宫颈癌裸鼠移植瘤的体内疗效,对不同给药途径的PDT治疗效果比较,以寻找光毒反应最小的给药方式。
4结合体外和体内实验,探讨ALA-PDT诱导宫颈癌细胞凋亡的作用途径。
方法
1 MTT法检测ALA-PDT对宫颈癌细胞增殖的影响,并筛选ALA-PDT对宫颈癌细胞增殖影响的最佳作用参数。
2通过直接观察细胞形态、May-Grunwald Giemsa染色、Annexin V-FITC/PI双染结合流式细胞仪检测细胞凋亡、Hoechst 33342荧光染色结合荧光显微镜观察ALA-PDT对宫颈癌细胞凋亡的影响、流式细胞仪分析ALA-PDT后细胞周期变化、Western blot检测ALA-PDT对宫颈癌细胞Survivin蛋白表达的影响、相对实时荧光定量PCR法检测ALA-PDT对宫颈癌细胞RNA的影响,以明确ALA-PDT对宫颈癌细胞的生长抑制机制。
3选择对ALA-PDT最敏感人宫颈癌Me180细胞系建立裸鼠宫颈癌移植瘤模型,通过人宫颈癌裸鼠移植瘤体内抑瘤实验、苏木精-伊红(HE)常规染色、免疫组化法检测ALA-PDT对宫颈癌肿瘤组织Survivin和VEGF蛋白表达的影响等方法,对局部给药和尾静脉给药两种给药方法进行比较研究。
4相对实时荧光定量法检测ALA-PDT对宫颈癌肿瘤组织mRNA的影响,结合体外实验结果探讨ALA-PDT对宫颈癌细胞的生长抑制机制和作用途径。
结果
1 MTT结果表明,ALA-PDT在体外对宫颈癌细胞均有生长抑制作用,其中宫颈癌Me180细胞株是8种肿瘤细胞株中对ALA-PDT最敏感的细胞株,其IC_(50)为6-7×10~(-4)M。单独使用10 mM以下的ALA对宫颈癌细胞没有明显的毒性反应,10—30 J/cm~2的激光剂量对宫颈癌细胞也没有明显的毒性。照射后4 h为ALA-PDT作用后的最佳时间。
2 Annexin V-FITC/PI双染结合流式细胞仪检测结果表明:ALA-PDT可以显著的诱导Me180细胞凋亡,作用后3h的凋亡主要表现为早期凋亡,到24h的时候,早期凋亡率降低,晚期凋亡率增高,总凋亡率降低,考虑为凋亡的时相问题。ALA-PDT作用后3h,1mM和2mM的ALA-PDT组的总凋亡率增高非常显著,高达52.45%和77.92%。
3直接观察细胞形态、May-Grunwald和Giemsa染色、Hoechst 33342荧光染色结合荧光显微镜均观察到细胞的凋亡现象。
4周期分析结果表明,ALA-PDT作用后4h,Me180细胞的周期分布无明显改变;ALA-PDT作用后24h,宫颈癌细胞Me180的周期分布出现了明显的改变,1 nM~2 nM的ALA-PDT均有显著的G_0-G_1期阻滞的作用,S和G_2/M期明显减少。
5相对实时荧光定量法检测不同组别的凋亡相关基因Survivin、P53、Bax、Bcl-2、Bad mRNA表达水平。证明PDT治疗后凋亡抑制基因Survivin和Bcl-2的mRNA的表达明显降低(P<0.001),而促凋亡基因P53、Bax、Bcl-2、Bad mRNA表达水平稍有增高(P>0.05)。
6 Western blot检测结果表明,ALA-PDT可显著的抑制Me180细胞的Survivin蛋白表达,0.1mM、1mM、2mM的抑制率分别为48.42%、71.22%、73.84%。
7 ALA-PDT对宫颈癌Me180细胞移植瘤生长的影响:治疗后瘤体早期出现发红、出血,24小时后肿瘤表面和切面出现局部坏死和变黑。静脉给药PDT组和局部给药PDT组的肿瘤体积与对照组相比明显减小,差异有显著意义(P<0.001),其中局部给药PDT组在7-14天时体积最小,且体积减小最明显。
8 HE常规染色可观察到:对照组、单纯静脉给药组、单纯局部给药组、单纯激光照射组无明显变化,而静脉给药PDT组和局部给药PDT组肿瘤受激光照射的皮下细胞区域坏死明显。
9免疫组化蛋白检测结果表明,ALA-PDT可显著的抑制宫颈移植瘤组织的Survivin和VEGF的蛋白表达。
10移植瘤组织mRNA的相对实时荧光定量结果证明PDT治疗后凋亡抑制基因Survivin和Bcl-2的mRNA的表达明显降低(P<0.001),而促凋亡基因P53、Bax、Bcl-2、Bad mRNA表达水平稍有增高,但差异无统计学意义。与体外实验结果基本一致。
结论
1 ALA-PDT在体外对宫颈癌细胞有明显的生长抑制作用,在,一定浓度范围内均呈现剂量-效应关系。宫颈癌Me180细胞株是8种肿瘤细胞株中对ALA-PDT最敏感的细胞株。
2早期诱导宫颈癌细胞株Me180细胞发生凋亡是ALA-光动力抑制作用的重要机制之一。
3线粒体依赖性途径在ALA-PDT诱导宫颈癌细胞株Me180细胞发生凋亡机制中起重要作用。
4结合体内和体外实验,ALA-PDT对宫颈癌治疗是有效的。
5临床用光动力治疗宫颈癌或癌前病变患者应尽量使用局部给药方式,以减少PDT光毒作用的危害和不便。
Objective
1 To study the effects of ALA-PDT on the growth inhibition in cervical cancer cell lines, so as to search the most sensitive cervical cancer cell lines and the optimal parameters of PDT.
2 To investigate the growth inhibition mechanism of ALA-PDT effecting on cervical cancer cell lines, and to build the theoretical foundation of biological mechanism for that study.
3 To analyze the in vivo effects of ALA-PDT on human cervical cancer xenograft implanted in nude mice, to compare the effect of various routes of administration, so as to identify the best administration with lest phototoxic reaction.
4 Combining the experiments in vivo and in vitro, to analyze the contribution of ALA-PDT on inducing apoptosis of cervical cancer cell lines.
Methods
1 Detecting the effects of ALA-PDT on the proliferation of cervical cancer cell lines by MTT colorometric assay, and to select the optimal parameters.
2 Indentifying the growth inhibition mechanism of cervical cancer cell lines by observing cellular shape, May-Grunwald Giemsa staining, cell apoptosis and DNA content analysis using flow cytometry and Hoechst 33342, protein and mRNA detected by Western blot and relative quantification in real-time RT-PCR respectively.
3 Producing the animal model of human cervical cancer by implanting the most ALA-PDT sensitive human cervical cancer cells Me180 into the dorsum of nude mice. Comparing the effects of topical administration and caudal vein administration by tumor inhibition experiment, HE staining, immunohistochemical staining.
4 Detecting the effects of ALA-PDT on the expression of xenograft mRNA by relative quantification in real-time RT-PCR, and combining the results of experiments in vitro to identify the growth inhibition mechanism and major contribution to apoptosis.
Results
1 The results of MTT demonstrate that, ALA-PDT does have the effects of growth inhibition on all cervical cancer lines in vitro, in which Mel 80 is the most sensitive cell line among the eight lines, the phototoxic IC50 concentration in Me180 is 6-7×10~4 M. There is no obvious phototoxic reaction under 10 mM and 30 J/cm~2. The optimal results come at 4 hours after ALA-PDT.
2 The results of dual Annexin V-FITC/Propidium Iodide assay by flow cytometry demonstrate that, Me180 cell death is significantly induced post ALA-PDT, in which the pattern of early apoptosis shows in 3 hours post-PDT, and the total rate of apoptosis declines in 24 hours post-PDT, which could be considered as different phase issue. The total rate of apoptosis for 1 mM and 2 mM are 52.45% and 77.92% respectively, 3 hours post-PDT.
3 Cell apoptosis is all observed by monitoring cellular shape, May-Grunwald Giemsa staining, cell apoptosis and DNA content analysis using flow cytometry and Hoechst 33342.
4 The results of cell cycle analysis demonstrate that, no significant cell cycle effects were evident at 4 hours post-PDT, and the distribution of cell cycle significantly changed at 24 hours post-PDT; an appreciable accumulation of cells residing in G0/G1 phase was induced at the 1 nM~2 nM, the reverse results shows in S and G2/M phase.
5 The results of relative quantification in real-time RT-PCR demonstrate that, the experimental cell group's mRNA expression of Survivin and Bcl-2 was down-regulated versus the control cell group (P<0.001) , on the other end, the experimental cell group's mRNA expression of P53, Bax, and Bad was up-regulated (P>0.05).
6 The results of Western blot demonstrate that, ALA-PDT can significantly inhibit the Survivin protein expression of Me180 cell line, the inhibition ratio of 0.1 mM, 1 mM and 2 mM are 48.42%, 71.22% and 73.84% respectively.
7 The results of tumor inhibition experiment in vivo demonstrate that, the tumor volume was significantly reduced post-PDT by topical and caudal vein administration (P<0.001), in which the most significant effect shows at 7-14 days post-PDT.
8 The results of Hematoxylin and Eosin staining demonstrate that, there is no appreciable change in the control group, pure vein administration group, pure topical administration group and pure laser group, however, local region necrosis show up in vein administration PDT group and topical administration PDT group.
9 The results of immunohistochemical staining demonstrate that, the protein expression of Survivin and VEGF post-PDT was down-regulated significantly versus control group.
10 The results of relative quantification in real-time RT-PCR demonstrate that, the experimental xenograft group's mRNA expression of Survivin and Bcl-2 was down-regulated versus the control xenograft group (P<0.001) , on the other end, the experimental xenograft group's mRNA expression of P53, Bax, and Bad was up-regulated (P>0.05). This result is coincide with that of experiment in vitro.
Conclusions
1 ALA-PDT can inhibit the growth of cervical cancer cell line in vitro, and the effect pattern is dose-dependent within certain concentration range. Me180 is the most sensitive cell line among the eight kind of cervical cancer cell lines.
2 Early induction of apoptosis of Me180 is one of the most important mechanisms of growth inhibition post-PDT.
3 The mitochondrion-dependent pathway makes important contribution in mechanism of Me180 cell line apoptosis.
4 Combining experiments in vivo and in vitro, ALA-PDT is effective for treatment of cervical cancer.
5 Topical administration PDT is recommended in treating cervical cancer so as to minimize th side-effects and inconvenience of phototoxic reaction brought by PDT.
引文
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