Estrogen And Progesterone Effects On The Growth Of Cervical Adenocarcinoma Cell Hela In Vitro And In Vivo

ObjectiveInterfering the cervical adenocarcinoma cell Hela cultured in vitro and tumor-burdened nudes with sex hormone E2,P or E2 + P,figure out the changes of the proliferation and the migration ability of Hela cell;Evaluating the effect of Hela cell caused by hormone therapy preliminary,provide experimental evidence for clinical hormone replacement therapy of cervical adenocarcinoma.Methods1.Detecting the expression of Er and Pr in Hela cells cultured in vitro with immunohistochemical method.2.Culturing the Hela cells with different concentrations of E2,P and E2 + P respectively to observe proliferation of Hela cells;Detecting the OD value of each group with CCK8 after Hela were cultured for 24 hs,48hs and 72 hs.3.Figuring out the migration and invasive ability of Hela cells cultured with E2,P and E2 + P for 48 hours in vitro with Transwell board.4.Measuring the cell cycle and apoptosis of Hela cells cultured with E2,P and E2 + P for 48 hours in vitro with Flow Cytometer.5.Injecting subcutaneously Hela cell suspension liquid into bilateral back of nude mice's dorsal legs,24 hours later injecting intraperitoneally E2,P and E2+ P and repeat every day to establish tumor-burdened nude mice;Observing differences in successful tumor inoculation rate,tumor sizes and survival time of nude mices.Results1.Er,Pr expressions of Hela cells cultured in vitro detected by immunohistochemical method were strong positive;2.It was revealed that hormones(E2,P and E2 + P)inhibited the proliferations of Hela in vitro when it's concentrations recached 10?mol/L or 100?mol/L,especially when Helas were cultivated for 72 hs,and the difference was statistically significant compared with the control group(p < 0.05).The 100?mol/L groups showed stronger inhibitory effect on Hela than 10?mol/L groups,while the other three groups(0.01?mol/L,0.1?mol/L,1?mol/L)made no difference on the growth of Hela,and compared with the control group,there was no statistical significance(p > 0.05).So choose the strongest inhibitory group(drugs concentration 100?mol/L)for follow-up experiments and no acceleration effect on the growth of Hela was observed for all experimental groups.3.After Hela cultured with hormone(E2,P,E2+P)for 48 hours,apoptosis rates of groups(blank control group,E2 group,P group and E2+ P group)tested by Flow Cytometry were: 5.40±2.38%,20.1±2.20%,20.03±1.91%,24.40±2.40%,apoptosis rate of the four groups were increased and the differences were statistically significant compared with control group(P < 0.05).No apoptosis reduction was observed in all experimental groups;Each group was characterized by an increasing in G0 / G1 phase,decreasing in S + G2 / M phase,and the difference was statistically significant compared with control group(P < 0.05).4.After Hela cultured with hormone(E2,P,E2+P,drug concentration:100?mol/L)for 48 hours,the migration and invasion ability of Hela had no obvious changes in the single E2 or P group,there was no statistical difference compared with control group(P > 0.05),while the migration and invasion ability of Hela was abated in E2+P group,and compared with the control group,the difference was statistically significant(p< 0.05);No migration and invasive promotion of Hela cells was observed in all our experimental drug concentrations.5.Tumor-burdened nude mice model was successfully established one week later.The successful tumor inoculation rate of the four groups(blank control group,E2,P and E2+P groups)was:87.5%(14/16),100%(15/15),81.25%(13/16),100%(16/16),respectively,and there was no obvious difference between groups(P > 0.05).After given hormones for 82 days,the tumor size of the blank control group,E2,P and E2+ P group 105.89 mm3,450.17mm3,107.96mm3 and 168.94mm3.The tumor size of E2 group was bigger than the control group,and the difference was statistically significant(p< 0.05);while there was no obvious difference between other groups(P > 0.05).Tumor-burdened nude mice with the biggest tumor size was found in the E2 group.Abdominal metastasis was detected in the No.1 nude of E2 group(weight 19.5g).Head transfer and abdominal metastases were found in NO.2 and NO.7 nudes of E2 + P group(weight 15 g and 9.9g,respectively),and both nudes were dead in 81 th day.Metastasis rate of the two groups were 12.5%(1/8)and 25%(2/8),and the metastatic tumors volume was 171.5mm3 in E2 group,while 1070mm3 and 600mm3 respectively in E2 + P group,the metastasis tumors of E2 + P was obvious bigger.ConclusionsProliferation of Hela cells cultivated in vitro with E2,P and E2 + P were inhibited in a certain concentration had inhibition effect on the proliferation of Hela.A certain concentration E2 + P can decrease the invasion and migration ability of Hela cultured in vitro.In the nude mice in vivo,a certain concentration of E2 promoted tumor growth and E2 + P may promote the early metastasis of tumor cells.