光动力治疗骨与软组织肉瘤的疗效及其机制

Antitumor effect and mechanism of hematoporphyrin monomethyl ether mediated photodynamictherapy on sarcoma

目的探讨血卟啉单甲醚介导光动力疗法（hematopotphyrin monomethyl ether induced photodynamic therapy，HMME-PDT）的抗肉瘤效应及其机制。方法流式细胞仪检测骨肉瘤细胞和成肌细胞的胞内HMME摄取，MTT法检测骨与软组织肉瘤细胞存活率，流式细胞术检测骨肉瘤细胞凋亡率，Hoechst33342染色法观察细胞凋亡，不可逆caspase通路抑制刹及坏死抑制剂检测PDT介导的细胞死亡形式，蛋白免疫印迹和免疫组化检测肿瘤细胞中caspase-l、-3、-6、-9、PARP蛋白相对表达量，测定瘤体体积及瘤重评估PDT的抗肿瘤效应，HE染色检测肿瘤组织形态学改变。结果光敏剂HMME选择性的聚集于骨肉瘤细胞中，而正常细胞摄取较少，且骨肉瘤细胞的摄取呈孵育时间及浓度依赖性。HMME-PDT能显著杀伤骨与软组织肉瘤细胞。HMME－PDT可明显诱导贴壁及悬浮培养骨肉瘤细胞发生凋亡。Hoechst33342染色可见典型的捌亡改变。凋亡是HMME-PDT介导的细胞死亡的主要形式，且与easpase依赖的凋亡通路密切相关。HMME－PDT可明显上调easpase-1、-3、-6、-9、PARP蛋白表达寄HMME-PDT组肿瘤体积及瘤重均较空白组明显减小，且肿瘤区域出现广泛坏死。结论HMME-PDT能有效地抑制肉瘤生长，其抗肉瘤效应与caspase依赖的凋亡通路密切相关。

Objective To investigate the effect and mechanism of hematoporphyrin monomethylether mediated photodynamic therapy （HMME-PDT） on sarcoma. Methods Tile intracellular uptake of HMME on osteosareoma cells （LM8） and myoblast ceils （C2C12） using flow cytometry method. The bone and soft sarcoma cells viability was measured by 3-（4, 5-Dimethylthiazol-2-yl）-2, 5-diphenyhetrazolium bromide （MTT） assay. The apoptosis of osteosarcoma cells were detected by flow cytometry method with Annexin V- FITC/PI staining. The apoptnsis of cells was fnrther observed by Hoechst 33342 staining. The irreversible inhibitor of caspase （Z-VAD-FMK） and necrosis （Nec-l） was performed to detect the type of PDT-mediated cell death. The relative expression levels of caspase-l, 3, 6, 9 and PARP were detected by Western blotting and immunohistochemistry （IHC）. The efficacy of antitumor was evaluated by the size and the weight and Hematoxylin eosin （HE） staining was used to observe the histological changes of tumor. Results HMME can se- lectively accumulate in osteosarcoma cells while normal cells absorbed much less. HMME-PDT can markedly killed the sarcoma cells and the killing effect increased with HMME concentration and energy intensity. The apoptosis rates in HMME-PDT groups significantly increased both adherent and suspension cells. It was observed typical changes, by Hoechst 33342 stained. The results showed that apoptosis was the major form of cells death and might be closely with caspase-dependent apoptosis after HMME-PDT. The expression levels of caspase-l, 3, 6, 9 and PARP were significantly up-regulated in the treatment grnups. Comparing with the blank group, the tumor volume and the tumor weight were markedly decreased. The widespread necrotic areas were observed by HE staining in tumor. Conclusion HMME-PDT significantly inhibited the tumor growth in vivo, which are closely correlated with caspase-dependent pathway.