| Objective: To further investigate the tumour inhibiting mechanism ofGenistein by studying the effects of genistein on the proliferation, apoptosis,distribution of cell cycle and detecting relevant protein apoptosis in vitro, so asto guide and support the esophageal carcinoma treatment from both medicaland theory level.Methods:1.cultivate Esophageal Carcinoma Cell Line TE-13in vitro, usetetrzolium-based colorimetric assay method to investigate the inhabitationeffect of negative control treatment and genistein treatment with differentconcentration (10mg/L,20mg/L,40mg/L,80mg/L) on TE-13cells after24h,48h and72h respectively.2. Choose the right concentration, and use inverted microscope andtransmission electron microscope to observe the morphology andultrastructure changes of the cells in the presence of genistein.3. Choose reasonable dose and time based on the above MTT observationresult, analyze the effect of genistein on the TE-13cell cycle distribution andapoptosis with flow cytometry method.4. Observe the expression of Survivin and Caspase-3protein in TE-13ofthe negative control treatment and genistein treatment with differentconcentration48h after.Results:1.MTT colorimetric method showed that: compared with the negativecontrol treatment, the genistein treatment (s) with the concentration lower than10ml/L can promote the TE-13growth when the treatment period is shorterthan24h, however with higher concentration (with10ml/L is the threshold)and prolonged treatment, which have the negative effect on OD index, the genistein is turned out to inhibit the TE-13growth. The results showed thateither of the higher treatment dose or the prolonged treatment has positiveeffect on the TE-13inhabitation. According to the observation results, therewas statistically significant difference between control treatment and everygenistein treatment group (P<0.05).2. Observe the morphology of the untreated TE-13cells with invertedmicroscope, most of them in regular round and elliptic form. As time passed,the cell number increased gradually and came into contact each other tightly inflake form and stick to the wall of the culture flask. While for the genisteintreatment, the cells morphology became irregular, and more and more cellshrinked and broken as the concentration increasing.3. Observe with the transmission electron microscope, theuntreatedTE-13cells were in elliptic form with a great quantity of bloomecphyma on its complete cell membrane. In the cytoplasm, there were affluentcell organs, such as mitochondria, rough endoplasmic reticulum,freeribosomes and euchromatin. While for the treated group, the Microvilli onthe cell membranes were decreased even disappeared, andmany irregular bulging appeared (the signal of early apoptosis). There weremany vacuoles in the cytoplasm, nuclear chromatin shrinked, and becamecrescent form; the bulk of organelles became smaller and number of the cellsdecreased.4. According to flow cytometry detection: after40h treatment bygenistein of different concentration (20mg/L,40mg/L,80mg/L), comparedwith the control group, as the concentration increasing, the cells number inG0/G1phase increased gradually, no significant changes in S phase no anddecreased in G2/M phase. The genistein can inhibit the cell cycle in G0/G1phase in a dose-dependent manner. The apoptosis rare of control groups anddifferent concentration genistein groups were(3.92±0.56)%,(4.17±0.71)%,(11.54±0.31)%,(22.23±0.92)%. respectively,showing that the apoptosis rate increased along with theincreasing concentration. There was statistically significant difference between the control group and different concentration genistein groups (P<0.05).5. The immunohistochemistry results showed: Survivin and Caspase-3were mainly expressed in the cytoplasm, with part nucleus expressed inclaybank color. With different concentration treatment, the Survivin value ofthe genistein decreased gradually as the concentration increasing based onImmunohistochemical score value method, while the Caspase-3valueincreased gradually as concentration increasing. With respect to the HIS Valueof Survivin and Caspase-3, there were statistically significant differencebetween control group and Genistein treatments with different concentration(P <0.05).Conclusions:1. Genistein could inhibit the proliferation of TE-13cells in certaindose-dependent and time-dependent manner.2. Genistein could inhibit the growth of Carcinoma of esophagus TE-13Cell and the cell cycle in G0/G1phase in a dose-dependent manner3. Genistein could induce apoptosis of Carcinoma of esophagus TE-13Cell, its mechanism might have something to do with down-regulating andup-regulating the expression levels of Survivin and Caspase-3.4.Genistein could inhibit proliferation and induce apoptosis of Carcinomaof esophagus TE-13Cell, which may serve as the new theoretical foundationfor developing and researching low side effect and cost saving medicine. |
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