【作者】 曲娟；

【导师】 陆伟； 王凤梅； 罗雁； 张捷；

【作者基本信息】 天津医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:全世界43.7%的肝癌患者在中国,严重威胁着我国人民的健康。原发性肝癌发病比较隐匿,临床诊疗中存在早期诊断难、复发转移率高、治疗缺乏针对性、治疗手段少等问题。因此对肝癌诊治新技术、新疗法的研究尤为重要和迫切。光动力疗法(Photodynamic therapy,PDT)作为一种新的肿瘤治疗方法已应用于临床,对体表和脑内肿瘤等取得了比较理想的治疗效果,但对肝脏肿瘤等腹腔内肿瘤的相关研究较少。本实验拟在初步探讨photosan—PDT在不同实验条件下对人HepG2肝癌细胞增殖的抑制作用并初步探讨其作用机制,以期为PDT成为肝癌治疗新方法提供进一步的理论依据。内容:将体外培养的人HepG2肝癌细胞常规传代后,在无光照情况下,比较不同浓度光敏剂—photosan对肝癌细胞增殖的影响,然后以photosan为光敏剂、半导体激光器(波长630nm)为光源进行激光照射,在不同浓度光敏剂经不同剂量的光照后,用MTT法测定PDT后HepG2肝癌细胞的残存率,并用流式细胞分析检测PDT对肝癌细胞周期的影响,以Annexin V-FITC/PI检测细胞凋亡。方法:HepG2肝癌细胞复苏,常规培养并传代。实验主要分四部分:(1)无光照情况下,不同浓度光敏剂对肝癌细胞增殖的影响。将细胞按实验设计转移至96孔板,常规培养,细胞贴壁后,加入不同浓度的光敏剂,使B2至G2为空白对照,既不加细胞也不加光敏剂,只加培养基;B3至G3为实验对照,只加细胞不加光敏剂;B4至G11的矩形区域为光敏剂实验组,按列分别加入2,4,6,8,10,12,14,16mg/L的光敏剂,孵育4h后,弃去光敏剂,继续孵育24h,进行MTT比色实验。(2)在激光照射下,比较不同浓度光敏剂及不同光剂量对肝癌细胞残存率的影响,并粗略比较在光剂量一定的情况下,激光强度对肝癌细胞残存率的影响。将细胞按实验设计转移至96孔板,常规培养,细胞贴壁后,加入不同浓度的光敏剂,为照光方便,每板只接受一种浓度光敏剂(6mg/L或10mg/L)。孵育4h后,弃去光敏剂,进行激光照射。为照光方便,每板只接受一种激光强度(30mW/cm~2或50mW/cm~2),调整照光时间,使D2、D4、D6、D8、D10、F2、F4、F6、F8、F10孔分别接受2,4,6,8,10,12,14,16,18,20J/cm~2的光剂量。B2至B4为空白对照,B5至B7为实验对照,B8至B10为光敏剂实验组。照光后继续孵育24h,进行MTT比色实验。相同的实验重复六次。(3)流式细胞分析检测细胞周期。肝癌细胞行PDT处理后,继续孵育24h,常规消化处理培养细胞,收集细胞,以标准程序进行流式细胞分析,结果用细胞周期拟合软件进行分析。(4)Annexin V-FITC/PI检测细胞凋亡。肝癌细胞行PDT处理后,继续孵育24h,常规消化处理培养细胞,依照Annexin V-FITC/PI双染试剂盒染色说明检测细胞凋亡。结果:(1)MTT结果显示,无光照情况下,不同浓度光敏剂对肝癌细胞的增殖不产生明显影响(P＞0.05)。(2)MTT结果显示PDT对HepG2肝癌细胞有很强的抑制作用,在一定范围条件下,随着光敏剂浓度及激光剂量的增加,细胞残存率明显下降(P＜0.05);在光剂量大于12J/cm~2后细胞残存率下降趋于缓和甚至不再产生明显下降(P＞0.05)。此外,在光敏剂浓度及激光剂量一定的情况下,30mW/cm~2的激光强度似乎比50mW/cm~2的效果更优。(3)流式细胞分析显示PDT后肝癌细胞周期被阻滞于G1、G2期,正常的细胞复制被阻断。(4)AnnexinV-FITC/PI检测发现PDT诱导了明显的细胞凋亡。结论:体外实验显示:PDT对HepG2肝癌细胞有很强的抑制作用,在一定的范围条件下,随着光敏剂浓度及激光剂量的增加,抑制作用明显增强。这种抑制作用可能由于PDT后正常的肝癌细胞复制周期被阻断导致细胞凋亡而引起的。更多还原

【Abstract】 Objective:Primary liver cancer severely threatens the health of people,because 43.7%liver cancer in the world is in our country.The progress of liver cancer is relatively hiding, which results in many problems in clinical diagnosis and treatment that early diagnosis is hard,the rate of relapse and diversion is high,treatments lack pertinence and treatment means is few.So,it’s important and urgent to study new technology and therapeutics for liver cancer.As a new tumor treatment modality,photodynamic therapy(PDT)has been applied to clinic and gained relatively perfect treatment effect. But,PDT is mostly applied to superficial tumor and brain tumor.The correlative study on intraperitoneal tumor such as liver cancer is rare.This experiment is to observe the inhibitory effect of PDT for hepatoma cell under different experiment conditions and to primarily observe the mechanism of PDT.Content:HepG2 human hepatoma cells were routinely cultured.The cell viability was determined by MTT assay after being treated with different concentration photosan without irradiation and being treated by PDT with different concentration photosan and different light doses and the cell cycle and cell apoptosis was detected by flow cytometer(FCM)after PDT.Methods:The HepG2 cells were recovered and cultured routinely.This experiment was divided four parts:(1)comparing the survival of hepatoma cell after being treated with different concentration photosan without irradiation.The cells were added into 96 wells culture plate according to experiment design making that the well from B2 to G2 was blank control added in only culture medium,the well from B3 to G3 was experiment control added in only cells and the rectangle area from B4 to G11 was photosensitizer experiment added in photosan by column with the different concentrations of 2,4,6,8,10,12,14,16mg/L,when the cells attached to the substratum.The experiment cells were incubated with photosan for 4 hours,then the photosan was abandoned and the cells continued to be incubated for 24 hours before the cell survival was determined by MTT assay.(2)Comparing the survival of hepatoma cell after PDT under different experiment conditions.The cells were added into 96 wells culture plate according to experiment design making that the well from B2 to B4 was blank control added in only culture medium,the well from B5 to B7 was experiment control added in only cells,the well from B8 to B10 was photosensitizer experiment added in photosan and cells without irradiation and D2、D4、D6、D8、D10、F2、F4、F6、F8、F10 wells was treated by PDT with different light doses of 2,4,6,8,10,12,14,16,18,20J/cm~2,respectively.The cells in one plate were treated with one concentration(6mg/L or 10mg/L)and one light intensity(30mW/cm~2 or 50mW/cm~2).The same experiment was repeated six times. The cell survival was determined by MTT assay,too.(3)Detecting the cell cycle after PDT by FCM.The cells were incubated for 24 hours after PDT,then collected and the cell cycle was detected by FCM.(4)Detecting the cell apoptosis after PDT by Annexin V-FITC/PI method.The cells were incubated for 24 hours after PDT,and the cell apoptosis was detected by Annexin V-FITC/PI method.Results:(1)MTT test showed that the cell viability had no significant difference(P＞0.05)after being treated with different concentration photosan without irradiation.(2) MTT test showed that the inhibitory effect of PDT for hepatoma cell was strong.The survival was descending obviously(P＜0.05)along with the increasing of the light dose and the photosan concentration in some condition.When the light dose was beyond 12J/cm~2,the descending of the survival was slow even no further change(P＞0.05).Besides,the light intensity of 30mW/cm~2 was superior to 50mW/cm~2,when the photosan concentration and the light dose were same.(3)FCM test showed that the cell cycle was blocked to G1、G2 phase after PDT.(4)Annexin V-FITC /PI test showed that the cell apoptosis was induced by PDT.Conclusion:The inhibitory effect of PDT for hepatoma cell is strong.The survival is descending obviously along with the increasing of the light dose and the photosan concentration in some condition.This inhibitory effect may be due to that the cell cycle is blocked after PDT,which induces the cell apoptosis.更多还原