Hedgehog信号传导通路相关基因在肿瘤组织中的表达及其特异性抑制剂Cyclopamine对增殖、凋亡的影响

Expression of Hedgehog signaling pathway genes in human oral squamous cell carcinoma and the effects of specific Hedgehog pathway inhibitor （Cyclopamine） on the proliferation and apoptosis of human oral epidermoid HSQ-89 cells

目的观察Hedgehog信号传导通路相关基因在肿瘤组织中的表达，及其特异性抑制剂Cyclopamine对肿瘤细胞增殖、凋亡的影响。方法免疫组织化学法检测80例人口腔鳞癌组织中Hedgehog信号传导通路成员Shh、Smo、Ptch、Gli-1表达；CCK8法检测对HSQ．89细胞增殖的影响；AnnexinV-PI双染流式细胞仪检测Cyclopamine对HSQ-89细胞凋亡的影响；逆转录-聚合酶链反应（RT-PCR）检测Cyelopamine处理后HSQ-89细胞凋亡相关蛋白表达变化。结果Hh家族成员Shh、Smo、Ptch、Gli-1在80例口腔鳞癌中的表达率分别为：61．25％、56．25％、47．50％、53．75％，明显高于正常人；在低分化或发生淋巴结转移的病例中表达更高。Cyelopamine可抑制HSQ-89细胞增殖及诱导凋亡，且抑制效应呈浓度依赖性。Cyclopamine（0、5、10、20、40、80μmol／L）作用下，细胞增殖抑制率分别为0、3．0％、4．5％、28．2％、51．2％、62．7％，凋亡率分别为3．01％、3．36％、5．70％、9．42％。经Cyclopamine处理后HSQ-89细胞中bcl-2表达下降，bcl-xL、Bid等表达不变。结论口腔鳞癌组织中Hedgehog信号转导通路有多种基因异常表达增高，Cyclopamine可通过抑制bcl-2表达而发挥抑制癌细胞增殖、诱导凋亡效应。

Objective To investigate the expression of Hedgehog signaling pathway genes in hu- man oral squamous cell carcinoma （ OSCC）, and the effects of specific Hedgehog pathway inhibitor （ Cyclopamine） on the proliferation and apoptosis of human oral epidermoid HSQ-89 cells. Methods The expression of Hedgehog signaling pathway components （ Shh, Stun, Ptch, Gli-I ） was detected in 80 cases of human OSCC by immunochemistry. CCK8 assay was used to investigate the inhibition of Cyclopamine on HSQ-89 cells proliferation in vitro. Flow eytometry （FCM） was used to examine the effects of Cyclopamine on apoptosis of HSQ-89 cells. Reverse transcription polymerase chain reaction （RT-PCR） was applied to detect the mRNA expression of bcl-2, bcl-xL, and Bid in HSQ-89 cells. Results The positive expression rate of Shh, Smo, Ptch and Gli-1 was 61.25%, 56. 25%, 47.50%, 53.75% in 80 cases of human OSCC tissue slices, respectively. After the cells were treated with different concentrations of Cyclopamine （0, 5, 10, 20, 40, 80 txmol/L, respectively）, the growth inhibition rate was 0, 3.0%, 4. 5%, 28.2%, 51.2%, 62. 7% respectively ; the apoptosis rate 3. 01%, 3. 36 % ,5.70% ,9.42% respectly. Cyclopamine significantly inhibited cell proliferation （P 〈 0. 05 ）. FCM revealed that the apoptosis rate of HSQ-89 cells was significantly higher after exposure to Cyclopamine than in control groups （ P 〈 0. 05 ）. The mRNA levels of bcl-2 were significantly decreased, but Cyclopamine had no significant effects on the mRNA level and protein secretion of bcl-xL, Bid. Conclusion The expression of main hedgehog signaling pathway components was detected in human OSCC. Cyclopamine specifically inhibited the proliferation and the mRNA expression of bcl-2 in HSQ-89 cells.