多剂量光动力联合姜黄素治疗宫颈癌体外移植瘤及肿瘤组织中肿瘤坏死因子-α、半胱氨酸蛋白酶-8的表达研究

Multi-dosephotodynamic combines with curcumin in the treatment of xenograft tumor of cervical cancer and the expression of TNF-αand Caspase-8

目的探讨多剂量光动力联合姜黄素治疗宫颈癌体外移植瘤及肿瘤组织中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及半胱氨酸蛋白酶-8(Caspase-8)的表达情况。方法将宫颈癌Hela细胞接种于裸鼠背部皮下,一周后,接种部位出现黄豆大小的肿物,提示造模成功,随即分为10组,每组6只。1~9组给予50 mmol/L浓度的姜黄素100μL,于肿瘤内部多点均匀注射,1~3组光照强度为40 J/cm^(2)(435 nm波长,0.60 A。0.54 W,距离肿瘤表面3~5 cm,照射14秒),治疗次数分别为1次、2次、3次;4~6组光照强度为60 J/cm^(2)(照射21秒),治疗次数分别为1次、2次、3次;7~9组光照强度为80 J/cm^(2)(照射28秒),治疗次数分别为1次、2次、3次,组10为模型组,不给予姜黄素及光照治疗。治疗1次组在完成治疗1天后取材,治疗2次及3次组,为每天治疗1次,并在完成最后一次治疗1天后取材,模型组成模1天后取材。观察肿瘤表面情况,苏木精—伊红染色法评价组织形态学改变,免疫组织化学染色及蛋白免疫印迹法检测各组TNF-α及Caspase-8的蛋白表达情况。结果各组肿瘤表面皮肤均无发黑、干枯及坏死情况,苏木精—伊红染色观察发现各组肿瘤组织具有不同程度的细胞凋亡和坏死,其中以组3(小剂量3次治疗组)坏死程度最显著。TNF-α免疫组化及蛋白免疫印迹表达:在相同光照剂量,不同治疗次数的情况下,多次治疗TNF-α表达最显著,而在相同治疗次数,不同光照剂量的情况下,TNF-α以小剂量光照表达最显著。Caspase-8免疫组化的表达:相同光照剂量,不同治疗次数的情况下,多次治疗时Caspase-8表达最显著,而不同光照剂量,相同治疗次数的情况下,小剂量光照时Caspase-8的表达最显著。Caspase-8的蛋白免疫印迹表达:在光照剂量为40 J/cm^(2)和60 J/cm^(2)时,Caspase-8的表达随着治疗次数的增加而增加。结论在用中药单体姜黄素联合光动力治疗宫颈癌体外移植瘤时,治疗参数以小剂量多频次效果最佳,能够有效的杀灭肿瘤细胞,其机制与TNF-α/Caspase-8通路有关。

Objective To investigate the efficacy of curcumin combined with photodynamic therapy for cervical cancer and the expression of tumor necrosis factor-α(TNF-α)and Caspase-8.Methods Cervical cancer HeLa cells were inoculated subcutaneously into the back of nude mice,one week later,a soy-sized mass appeared at the inoculation site,indicating successful modeling,andimmediately divided into 10 groups.6 mice in each group.The 1~9th group were given 100μL of curcumin at a concentration of 50 mmol/L and uniformly injected into the rumor at multiple points.The light intensity of 1~3th group was 40 J/cm^(2),(435 nm wavelength,0.60 A,0.54 W,3~5 cm from the tumor surface,irradiation for 14 seconds).The treatment processes needed 1,2 and 3 times respectively.In the 4~6th group,the light intensity was 60 J/cm^(2)(21 seconds),and the treatment times were 1,2 and 3 times respectively.The 7~9th group received 80 J/cm^(2)(28 seconds)of light intensity,and the treatment times were 1,2 and 3 respectively.The 10th group was the model group,crucumin and light were not given.The samples were collected after completion of treatment in the once treatment group,Two and three times of treatment groups were treated once a day,and samples were collected 1 day after the completion of treatment.Histological changes were observed by HE,and the expressions of Caspase-8 and TNF-αin each group were detected by immunohisto-chemistry and Western-Blot.Results No blackness,dryness and necrosis were observed on the tumor surface skin in each group.Different degrees of apoptosis were observed in each group by HE,and the 3th group(low-dose multiple treatment group)had the best degree of apoptosis.Immunohistochemistry and Western-Blot expression of TNF-α:Under the same light dose and different times of treatment,The expression of TNF-αwas the highest in the multiple treatment times,while under the same treatment times and different of light dose,the expression of TNF-αwas the highest in the lower dose of light.The expression of Caspase-8 in immunohistochemistry:in the case of the same light dose and different treatment times,the expression of Caspase-8 was the most significant in the case of multiple treatments,while in the case of different light dose and the same treatment times,the expression of Caspase-8 was the most significant in the case of small dose of light.Western-Blot expression of Caspase-8:when the light dose was 40 J/cm^(2)and 60 J/cm^(2),the expression of Caspase-8 increased with the treatment times.Conclusion In the treatment of cervical cancer with curcumin combined with photodynamic therapy,small dose and multiple treatment times have the best effect,which could effectively kill tumor cells,and the mechanism is related to TNF-α/Caspase-8 pathway.