Experiment Study On Photodynamic Therapy For Hepatoma Cell

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.