| Objective:Primary liver cancers are malignant caused by lesions of hepatocytes or intrahepatic bile duct epithelial cells.Hepatocellular carcinoma?HCC?accounts for more than 90%of primary liver cancers.The incidence rates of HCC rank fifth in the world,while China has the highest incidence rates.At present,the mortality rates of HCC rank third with cancer-related deaths.Its 5-year survival rates are less than 5%even in developed countries.Therefore,it is important to find a way to eradicate HCC.Currently,there are no effective methods to cure HCC,so we have proposed a new idea here.Abnormal microenvironment is crucial to tumorigenesis.The tissue stem cells will dysregulate under the abnormal microenvironment.It has shown that most properties of cancer stem cells?CSCs?are similar to normal stem cells.Therefore,Professor Jinsheng Qi proposed that CSCs may be the normal stem cellswith stagnated development at a certain stage.Maybe tumorigenesis can be considered a phenomenon of"back ancestors"of cells.So we supposethat embryonic cells may have the ability to induce differentiation of CSCs with its tissue,temporal and spatial specificity.Our previous studies have shown that the HepG2 cells could be induced to differentiate when they were co-cultured with 13.5d embryonic hepatocytes.In this study,12.5d mouse embryonic hepatocytes were used to induce differentiation of SMMC-7721 cells.In recent years,it has shown that there were three major advantages on cancer therapy with the traditional Chinese medicine?TCM?.Many of the TCM can be used in cancer therapy,such as Buddleja officinalis and Scutellaria barbata contain Apigenin?API?.As a flavonoid anticancer drug,API at high doses can induce apoptosis,while it can induce tumor cell differentiation when used at low doses.Therefore,low-dose APIs were used to induce SMMC-7721 differentiation in this study or to promote the differentiation of SMMC-7721 induced by mouse embryonic hepatocytes.Based on the previous experiments,a gradient concentration of API was used to select appropriate concentration of API inducing SMMC-7721 to differentiate in this experiment.Additionally,this concentration of API was used to interfere with the cancer cells together with mouse embryonic hepatocytes.The cell immunofluorescent staining application was carried out to detect the changes of AFP and HNF-4?protein expression in SMMC-7721cells.Quantitative real-time polymerase chain reaction?real-time PCR?was used to detect the mRNA expression level of afp and hnf-4?.The difference was calculated by 2-??Ct??Ct method.Western Blot?WB?was used to detect the expression of AFP and HNF-4?protein levels.In this study,we explore the role of the natural plant extract API in promoting the differentiation of SMMC-7721 induced by mouse embryonic hepatocytes and provide new ideas for the eradication of HCC with low toxicity and high efficiency.Methods:1 The cell culture1.1 SMMC-7721 cells:SMMC-7721 cells were maintained at 37°C in a humidified incubator with 5%CO2 and propagated in Dulbecco's modified Eagle's medium?DMEM?containing 100mg/L D-glucose and 10%fetal bovine serum?FBS?.Cells were inoculated in 150mL flasks and passaged with0.25%trypsin.1.2 The isolation and culture of primary embryonic hepatocytes:With the collagenase IV digestion solution,at first,we cut the embryo liver tissue into the tissue blocks;next,we successfully isolated the tissue blocks into a single embryo hepatocyte following digested by collagenase IV.In order to remove fibroblasts,we used selective plating technique repeatedly to purify the embryo hepatocytes.Embryo hepatocytes cultured with high glucose DMEM.2 experimental design and grouping2.1 SMMC-7721 cells were induced to differentiate by gradient concentration API for 72 hours.The experiments were divided into 6 groups:con group,API2.5?m/L,API 5?m/L,API 7.5?m/L,API 10?m/L,API 12.5?m/L.2.2 Embryo hepatocytes were co-cultured with SMMC-7721 cells containing API 24h or 48h The co-culture system of mouse embryo hepatocytes and SMMC-7721 cells was established as follows:A non-contact co-culture system of mouse embryo hepatocytes and SMMC-7721 cells was established in six-well plates of Transwell chambers.The experiment was divided into 4 groups:con group;5?m/L API group;co-culture group?the mouse embryo hepatocytes co-cultured with SMMC-7721?;co-culture+API group?SMMC-7721 cells stimulated with 5?m/L API were co-cultured with mouse embryonic hepatocytes?.3.The optimal API concentration for API-induced differentiation of SMMC-7721During the process of API-induced SMMC-7721 re-differentiation,different concentrations of API have different ability to induce differentiation of SMMC-7721.3.1 The amount of AFP and HNF-4?secretion was observed using a fluorescence microscope.3.2 Total RNA was isolated using a one-step Trizol reagent in SMMC-7721cells.Real-time PCR analysis for the expression of mRNA for afp,hnf-4?genes and?-actin as a control reference were carried out.4 SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hours.4.1 Use fluorescence microscopy to observe the secretion of AFP and HNF-4?.4.2 Total RNA was isolated using a one-step Trizol reagent in SMMC-7721cells.Real-time PCR analysis for the expression of mRNA for afp,hnf-4?genes and?-actin as a control reference were carried out.4.3 Whole proteins from SMMC-7721 cells were extracted by RIPA and quantified by Lowry method.Western-blotting analysis of AFP and HNF-4?protein levels was performed.5 SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 48 hours.5.1 Observe the difference of SMMC-7721 cell morphology between untreated SMMC-7721 and co-culture+API groups under light microscope.5.2 Using fluorescence microscopy to observe the secretion of AFP and HNF-4?.5.3 Total RNA was isolated using a one-step Trizol reagent in SMMC-7721cells.Real-time PCR analysis for the expression of mRNA for afp,hnf-4?genes and?-actin as a control reference were carried out.5.4 Whole proteins from SMMC-7721 cells were extracted by RIPA and quantified by Lowry method.Western-blotting analysis of AFP and HNF-4?protein levels was performed.Results:1 The purification and culture of primary embryonic hepatocytesWith the collagenase IV digestion solution,at first,we cut the embryo liver tissue into the tissue blocks;next,we successfully isolated the tissue blocks into a single embryo hepatocyte following digested by collagenase IV.In order to remove fibroblasts,we used selective plating technique repeatedly to purify the embryo hepatocytes.Embryo hepatocytes cultured with high glucoseDMEM.Embryohepatocyteswererounded,brightand three-dimensional under the phase contrast microscope.In addition,they have high survival rate and long survival time.2 After 72-hour induction of SMMC-7721 cells by gradient concentration API,the levels of AFP protein and HNF-4?protein in SMMC-7721 cells were detected by immunofluorescence.Compared with the con group,it was observed that 5?m/L of API had the strongest ability to induce differentiation of SMMC-7721.The level of AFP protein of SMMC-7721 was significantly decreased,and its HNF-4?protein level was significantly increased.3 SMMC-7721 cells were induced with gradient concentration API for 72hours.The expression of afp and hnf-4?mRNA in SMMC-7721 cells was detected by real-time PCR.After SMMC-7721 cells were treated with gradient API,the relative expression levels of afp and hnf-4?mRNA in SMMC-7721 cells were detected.Compared with con group,afp mRNA was down-regulated in 2.5?m/L group,5?m/L group,7.5?m/L group,10?m/L group,and 12.5?m/L group,among which 5?m/L group had significant effect.The hnf-4?mRNA was up-regulated in all groups,while the 5?m/L group had a significant effect,which was consistent with the results of immunofluorescence.4 After SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hours,the levels of AFP protein and HNF-4?protein were detected by immunofluorescence in SMMC-7721 cells.Compared with the con group,there was no significant change in the AFP protein level in the API group,a slight decrease in the AFP protein level in the co-culture group,and a slight decrease in the AFP protein level in the co-culture+API group,and its level was the lowest.The level of HNF-4?protein in the API group increased slightly,and the HNF-4?protein level in the co-culture group also increased slightly,but was lower than that in the API group.The level of HNF-4?protein in the co-culture+API group increased slightly,and the level was the highest among the groups.5 The expression of afp and hnf-4?mRNA in SMMC-7721 cells was detected by real-time PCR after SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hours.The relative expression of afp mRNA in SMMC-7721 cells was detected after SMMC-7721 cells were co-cultured with 12.5 days of embryonic liver cells with 5?m/L API.Compared with con group,afp mRNA was down-regulated in API group,co-culture group and co-culture+API group,among which co-culture+API group had significant effect.Except that the expression level of hnf-4?mRNA in the API group did not change significantly,the expression level of hnf-4?mRNA in the other groups was up-regulated,and the effect of the co-culture+API group was significant.6 SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hours,and then the changes of AFP protein and HNF-4?protein levels in SMMC-7721 cells were detected by WB method.The relative expression of AFP protein in SMMC-7721 cells was detected after SMMC-7721 cells were co-cultured with 12.5 d embryonic liver cells with the 5?m/L API.Compared with the con group,the relative expression levels of AFP protein in the API group,the co-culture group,and the co-culture+API group were all down-regulated,and the effect of the co-culture+API group was significant.The relative expression levels of HNF-4?protein in each group were up-regulated,and the effect of co-culture+API group was significant.The results were consistent with the results of immunofluorescence and real-time PCR.7 The morphology of SMMC-7721 cells in the co-culture+API group was observed under light microscope.After 48 hours of culture,the growth of SMMC-7721 cells was significantly inhibited,and some of the cells were retracted,rounded and finally disintegrated;another part of the cells did not change.Continued cultivation will not make it proliferate.8 After SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hours,the levels of AFP protein and HNF-4?protein in SMMC-7721 cells were detected by immunofluorescence.Compared with the con group,the AFP protein level in the SMMC-7721cells was slightly decreased in the API group,and the AFP protein level in the co-culture group was significantly decreased.The level of AFP protein in the co-culture+API group decreased significantly,and its level was the lowest among the groups.The HNF-4?protein level in SMMC-7721 cells increased significantly in the API group,while the HNF-4?protein level in the co-culture group increased significantly,but was slightly lower than that in the API group.The level of HNF-4?protein in the co-culture+API group increased significantly,and its level was the highest among the groups.9 The expression of afp and hnf-4?mRNA in SMMC-7721 cells was detected by real-time PCR after SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hoursAfter SMMC-7721 cells were co-cultured with 12.5 days of embryonic liver cells with 5?m/L API,the relative expression of afp mRNA in SMMC-7721 cells was detected.Compared with the con group,afp mRNA was down-regulated in the API group,co-culture group,and co-culture+API group,among which the co-culture+API group had significant effects.The hnf-4?mRNA was up-regulated in all groups,and the effect of co-culture+API group was significant.10 After SMMC-7721 cells induced by the 5?m/L API were co-cultured with mouse embryonic hepatocytes for 24 hours,the changes of AFP protein and HNF-4?protein levels in SMMC-7721 cells were detected by WB method.The relative expression of AFP protein in SMMC-7721 cells was detected after SMMC-7721 cells were co-cultured with 12.5 d embryonic liver cells with the 5?m/L API.Compared with the con group,the relative expression levels of AFP protein in the API group,the co-culture group,and the co-culture+API group were all down-regulated,and the effect of the co-culture+API group was significant.The relative expression levels of HNF-4?protein in the API group,the co-culture group,and the co-culture+API group were all up-regulated,among which the co-culture+API group had significant effects and was consistent with the results of immunofluorescence and real-time PCR.For the co-culture+API group,the results obtained after 48 hours of culture were more pronounced than those of 24 hours.Conclusion:1.Low doses of API?5?m/L?can induce differentiation of hepatoma cells SMMC-7721.2.Embryonic hepatocytes of mouse can induce differentiation of SMMC-7721cells at a specific time?12.5 days?.3.API can promote the induction and differentiation of SMMC-7721cells when they were co-cultured with mouse embryonic hepatocytes,and the synergistic effect reached the peak after 48 hours. |
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