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The Effection of the Signal Channel of Mtor/p-4EBP1,p-p70S6K in JEG-3 Cell Line and the Tentative Exploration of Mechanism

Author: HuangLing
Tutor: LiuXueQing
School: Chongqing Medical University
Course: Genetics
Keywords: rapamycin JEG-3 cell proliferation apoptosis invasion
CLC: R965
Type: Master's thesis
Year: 2010
Downloads: 30
Quote: 0
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Abstract


Objective:The study discusses the effection of rapamycin in JEG-3 cell line. Before and after rapa treatment ,we test the difference in proliferation, apoptosis and invasion ability of JEG-3 by MTT techology, FCM technology and Tanswell invasion. The results show that rapamycin inhibits proliferation and invasion ability of JEG-3 and stimulates apoptosis of JEG-3.Lay the foundation for a clinical treatment in vitro of choriocarcinoma.With the treatment of rapamycin test the amount of transformed RNA of mTOR and the protein’s change of mTOR、p-4EBP1 and p-70S6K by RT-PCR and Western-blot. Discuss the molecular mechanism of the change in proliferation, apoptosis and invasion.Methods:1.Cell culture.2.Experimental grouping: First group:control group; Second group: 0.01% DMSO; Third group: 0.1 ng/ml rapa; Fourth group: 1 ng/ml rapa; Fifth group:10 ng/ml rapa; Sixth group:100 ng/ml rapa.3.MTT techology:To detect different proliferation in experimental groups cell .4.FCM technology:To detect different apoptosis in experimental groups cell .5.Tanswell invasion : To detect different invasion ability in experimental groups cell .6.RT-PCR: To detect different RNA’s quantity of mTOR in experimental groups cell .7.Western-blot:To detect different protein’s quantity of change of mTOR、p-4EBP1 and p-70S6K in experimental groups cell .Results:1. The result of MTT Technology indicated that the groups with rapa concentration in 0.1,1,10ng/ml which followed concentration gradient increased with proliferation rate reduce(F=8.46, P<0.05).Compare the group of concentration in 10ng/ml with the group of concentration in 100ng/ml have no significant difference. Compare the control group with the group of DMSO have no significant difference(P>0.05).2.The result of FCM technology indicated that the groups with rapa concentration in 0.1,1,10ng/ml which followed concentration gradient increased with apoptosis rate increase(F=7.29, P<0.05).Compare the group of concentration in 10ng/ml with the group of concentration in 100ng/ml have no significant difference. Compare the control group with the group of DMSO have no significant difference(P=0.083).3. The result of Tanswell invasion technology indicated that invasion ability of the group with rapa concentration in 100 ng/ml is better than invasion ability of the control group (F=4.24, P<0.05). Compare the control group with the group of DMSO have no significant difference(P>0.05).4. The result of RT-PCR indicated that compared RNA’s quantity of mTOR in group with rapa concentration in 100 ng/ml with the control group have no significant difference(P>0.05).5. The result of Western-blot indicated that protein’s quantity of change of p-4EBP1 and p-70S6K in experimental groups cell reduced follow with Rapa concentration gradient increased(P<0.05).Conclusion:1.This study found that proliferation and the invasion ability are lower and apoptosis rate is higher in treatment guoups with Rapa through MTT techology, FCM technology and Tanswell invasion.2. The result of MTT techology and FCM Technology indicated that rapa concentration in 0.1,1,10ng/ml which followed concentration gradient increased with apoptosis rate reduce or apoptosis rate increase. Compared the group of concentration in 10ng/ml with the group of concentration in 100ng/ml have no significant difference. Demonstrated the 10ng/ml of rapa have a maximum effect in experimental groups.Mean rapa act on JEG-3 cell line have a threshold dose.3.The result of RT-PCR indicated that Compared RNA’s quantity of mTOR in group with rapa with the control group have no significant difference. Demonstrated rapa didn’t play a role directly through influence RNA’s quantity of mTOR ,but by suppressing inhibited its function.4.The result of Western-blot indicated that protein’s quantity of change of p-4EBP1 and p-70S6K in experimental groups cell reduced follow with Rapa concentration gradient increased. The signal channal mTOR /p-4EBP1,p-P70s6K in JEG-3 play an important part in proliferation, apoptosis and invasion ability.

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