PDCD5, Astragalus And DDP For Genetic Immunization Chemotherapy Of Anti-endometrial Cacinoma In Vitro

ObjectiveThe aim of this study was to determine the joint action and mechanism of apoptosis-related protein PDCD5, biological immunity Astragalus injection and conventional chemotherapeutics cisplatin (DDP) when they were used to human endometrial carcinoma cell HEC-1-B, in order to provide experimental support for clinical applications.MethodsHuman endometrial carcinoma cell HEC-1-B was conventionally cultured to exponential phase of growth. The experiment was divided into three parts.Part 1: The unification experiment of Astragalus injection and DDP (10%, 20%, 40% Astragalus injection group, 5ug/ml DDP group, 10%, 20%, 40% Astragalus with 5 ug / ml DDP group).Part 2: The unification experiment of Astragalus injection and protein PDCD5 (5ug/ml, 10ug/ml , 20ug/ml PDCD5 group, 30% Astragalus with 5 ug / ml, 10ug/ml, 20ug/ml PDCD5 group).Part 3: The unification experiment of DDP and protein PDCD5 (5ug/ml, 10ug/ml, 20ug/ml PDCD5 with 5 ug / ml DDP group).Concrete methods:1. Cell morphological change was observed with inverted phase contrast microscope at different time after each group of drug was used.for, and ultrastructure change with transmission electron microscopy.2. Cell proliferation inhibition rate of each group was detected by MTT assay.3. The cycle change and apoptosis rate of cell stainned with EGFP-PI were detected by flow cytometry. Apoptosis cell fluorescein stain was observed by inverted fluorescence microscope.4. Cell was stainned with Hoechst33258. Apoptosis cellular nucleus morphology was observed by inverted fluorescence microscope.5. Using monoclonal antibody against protein PDCD5,Nuclear translocation of PDCD5 was examined by immunofluorescence.ResultsPart 1.1.Cellular morphology change appeared in 48 hours when Astragalus was used to HEC-1-B alone. Cyto-morphology change became more obvious with drug concentration increased and time prolonged. Cellular ultramicrostructure change was observed by transmission electron microscope. The change was earlier and more obvious. when Astragalus was in combination with DDP.2.The results of MTT assay shew that the cell inhibition rate was 0.0376 when 10% Astragalus was used to HEC-1-B cells for 48 hours, while it was 0.2565 when 10% Astragalus combined with DDP. Cell inhibition rate raised with Astragalus concentration increased and time lasted. The differences among each group were statistically significant (P<0.05).3. The results of flow cytometry shew that early apoptosis appeared when Astragalus or DDP was used for 48 hours. Apoptosis rate and advanced stage increased. The results conformed with that observed by inverted fluorescence microscope.4. It was found that nucelus morphological change occured when cells with Astragalus or Astragalus and DDP stained by Hoechst33258. The change became more intensive with Astragalus concentration increased and time lasted.Part 21.There were no significant changes in cell growth , proliferation, and morphology when protein PDCD5 acted alone.But more significant morphology change could be abserved by inverted phase contrast microscope when protein PDCD5 and 30% Astragalus acted together than 30% Astragalus. Cell morphology change became obvious with increasing PDCD5 concentration and extending the action time. 2. The results of MTT assay shew that there was no significant cell inhibition when protein PDCD5 acted alone. But the cell inhibition rate was 0.2364 when 5ug/ml PDCD5 and 30% Astragalus were used together to HEC-1-B cells for 48 hours, which was 0.0405 more than 30% Astragalus. Cell inhibition rate raised with PDCD5 concentration increased and time lasted. The differences among each group were statistically significant (P<0.05).3. Nucelus of PDCD5 and negative control group showed no phenomenon of apoptosis. While apoptosis was observed when cells with 30% Astragalus and PDCD5 stained by Hoechst33258. The changes became more intensive with A PDCD5 concentration increased and time lasted.4. Iimmunofluorescence using monoclonal antibody against PDCD5 results showed that protein PDCD5 was transferred into cellular nucleus, most around the nucleus and the rest located in the perinuclear cytoplasm. Endogenous protein PDCD5 appeard in HEC-1-B cell when DDP or Astragalus were usde. And the protein most located in cellular nucleus.Part 3.1. More significant morphology change could be observed by inverted phase contrast microscope when protein PDCD5 and DDP acted together than DDP alone. Cell morphology change became obvious with increasing PDCD5 concentration and extending the action time.2. The results of MTT assay shew that cell inhibition rate was 0.3176 when 5ug/ml protein PDCD5 and DDP were used together to HEC-1-B cells for 48 hours, which was 0.0529 more than DDP group. Cell inhibition rate raised with PDCD5 concentration increased and time lasted. The differences among each group were statistically significan(P<0.05).3. Hoechst33258 fluorescent staining showed that blue fluorescence heap and nucelus morphological changes were observed when 5ug/ml DDP combined with PDCD5. The changes became more intensive with PDCD5 concentration increased and time lasted. Conclusion1. Astragalus can inhibit and promote apoptosis of human endometrial carcinoma cell HEC-1-B, which has a-time-and-concentration dependence.2. The inhibition action are more conspicuous when Astragalus in combination with DDP than two of them used alone. And they have a synergistic anti-tumor effect.3. PDCD5 dose not have anti-tumor effect alone. But it can increase Astragalus'inhibition and pro-apoptotic role on human endometrial cancer cell HEC-1-B. And which has a concentration-and-time-dependence of PDCD5 proteinum.4. PDCD5 has sensitizing effect of anti-tumor when used combine with DDP. And which also has a concentration-and-time-dependence.5. PDCD5 promotes cell apoptosis by the way of Nuclear translocation.