| Renal Cell Carcinoma (RCC) was dormant and most patients were found lately. For patients with later stage, chemotherapy will be necessary to delay the patient survival. But RCC was less sensitive to chemotherapy and the conventional chemotherapy regimens effect was poor. Sorafenib as a representative of the targeted drugs bring hope to RCC chemotherapy. However, with the deepening of the research, researchers found that the long-term effect of Sorafenib treatment to advanced RCC was limited. The other also found that target therapy not only can induce tumor resistance, but also may increase the malignant degree of tumor remnants.Cancer stem cell (CSC) theory believes that the existence CSC is the root cause of a tumor occurrence, metastasis, recurrence and resistance to chemotherapy. At so we speculate that the existence of CSC in RCC may be the reason of its targeted therapy resistance. CSC most was in G0, chemotherapy only was useful for split phase cells, survived CSC awaken again and differentiated after chemotherapy, causing the failure of chemotherapy. The identification of reliable biomarkers is the key of CSC selection; studies have confirmed markers CD133, CD34, CD105 and CD44 expressed in other cancer stem cells with different extent. CSC characteristics maintain depends on the expression of certain genes, these genes involved in regulating CSC differentiation and drug resistance, studies have shown that genetic 0tc3/4, Bmil,β-catenin and Nanog was associated with the CSC in other tumors. Side group of cells (SP) in the tumor is considered to be some possible cells that are rich with CSC, can show strong characteristics of CSC. Transport protein of ABCG2 is not only related to the SP cells, also proved to be one of the markers of CSC likely. HIF can promote the expression of ABCG2 protein, and may be related to the drug resistance, tumor malignant degree and CSC.CSC studies were earlier in other tumors, but RCC cancer stem cell research was still relatively less. We selected FCM, Western Blot, immunofluorescence, RT-qPCR as technology, study the effects of sorafenib expression of some markers, gene, SP cells,ABCG2, HIF-la and HIF-2a in RCC cell lines 786-O from two levels of cell experiments and animal experiments, respectively. This topic will help the expanding of RCC study, help explore the reasons of targeted therapy tolerance, help the diagnosis, prognosis judgement and provide new clues and chemotherapy.1. The inhibitory effect and the influence of the cell cycle to RCC cell lines 786-O of SorafenibObjective:Evaluated the effects of sorafenib in inhibition, promoting apoptosis and on cell cycle change to RCC 786-0.Methods:We chose 786-0 as the study cell lines,2.5μM,5.0μM,7.5μM, 10.0μM as the different concentrations of sorafenib respectively, collecting different time points (0 h,24 h,48 h,72 h,96 h) cells, we study the cell proliferation with MTS methods in different time point and different concentration group firstly, and then detected the apoptosis and cell cycle change with FCM method respectively.Results:(1) Cell proliferation test results:analysis of Cell inhibition rate with multifactor factorial design of analysis variance, the main effect analysis of the concentration and time F value are:57.681,126.0, P< 0.01, significant was different; And the interactions was also obvious (F=4.410, P< 0.01). The overall results suggest:with the increase of time and concentration extension,the inhibition function of sorafenib to RCC cell line 786-O trends to increase gradually, and the 10.0μM concentration has the strongest inhibitory effection.(2) Apoptosis detection results:FCM detecting apoptosis in the 0 h,24 h,48 h, 72 h,96 h point. Overall apoptosis rate of each time groups was to compared to Oh group, through analysis of ANOVA,F=1893.68, P values< 0.001,24 h group vs the control group was with no significant difference (P> 0.05), and the difference of the other time group vs the control group were significant (P< 0.05)(3) Cell cycle test results:FCM to detect the cell cycle,0 h,24 h,48 h,72 h,96 h the proportion of Gl phase cell at each time point respectively was:57.37±1.03%、 58.82±1.34%、61.03±1.28%、64.56±2.61%、70.90±1.11%, showed a trend of increased gradually. Each time groups compared with 0 h control group by the analysis of ANOVA, F=34.95, P< 0.001,48 h,72 h,96 h groups was different significantly(P< 0.01)Conclusions:The inhibitory effect of Sorafenib for RCC cell line 786-0 in vitro was significantly. Sorafenib can significantly promote the apoptosis and blocked its differentiation in G1 phase, so as to achieve the purpose of inhibition to tumor; And G1 phase after cell proportion increase, may be associated with the presence of RCC cancer stem cells.2. The influence of Sorafenib on the expression of CSC related markers in RCC 786-O cell linesObjective:Explore the expression of markers related to CSC, such as CD133, CD105, CD44, CD24, and CD34 in RCC cell line 786-0, and analyze the change effect of sorafenib to these markers.Methods:In cell experiments, the stem cell marker such as CD133, CD105, CD44, CD24, and CD34 was detected by FCM..Then we choose 10.0μM as the concentration treated the 786-O RCC cells, to detect markers chang in different time points (0 h,24 h,48,72 h,96 h).later we try to built the animals tumor model on BALB/C-NU mice, the control group and experimental group mice each have 6. After the success of the model, sorafenib was used to lavage treatment to dose group mice for 14 days, put to death these mice and handle tumors, choose immunofluorescence method, detected the expressions of CSC markers in the dose and control group tumor tissue samples.Results:(1) The results of FCM in cell experiments: ① The expression of CD133 was low, control group only have 0.55±0.05%,96 hours group increased to 4.14±0.15%. Each time group compared with 0 h control group, the variance analysis result:F= 291.585, P< 0.001, the difference was significantly (P<0.05), except the 24 h group. ②The expression of CD105 was relatively higher, control group have 4.04±0.08%, 96 hours group increased to 12.36±1.23%. Each time group compared with 0 h control group, the variance analysis result:F= 4.853,P<0.001,all the difference was significantly (P<0.05). ③ The expression of CD34 was low, control group have 0.23±0.02%,96 hours group increased to 1.28±0.05%. Each time group compared with 0 h control group, the variance analysis result:F= 714.105, P< 0.001, all the difference was significantly (P<0.05).④The expression of CD44 was high. Each time group compared with 0 h control group, the variance analysis result:F= 2.019, P>0.054, all the difference was not significantly (P> 0.05).⑤The expression of CD24 was high. Each time group compared with 0 h control group, the variance analysis result:F=1.075, P=0.419, all the difference was not significantly (P> 0.05).(2) The results of immunofluorescence in animal experiment:the Animals tumor model was good. The immunofluorescence results show that the CD 133, CD 105 and CD34 expressed lowely in the control group tumors, but the expression was increased obviously in dose group. CD44 and CD24 both has high expression in the control and dose group organization, there was no significant difference between two groups.Conclusions:CD133, CD105 and CD34 has a small amount of expression in RCC cell line 786-0, the expression level could be improved by sorafenib. CD44 and CD24 expression level was high and could not be improved by sorafenib. CD133, CD105 and CD34 may be the CSC marker in RCC cell line 786-0, but CD44 and CD24 was not suitable.3. The influence of Sorafenib on the expression of CSC related Genes in RCC 786-O cell linesObjective:To detect the expression of CSC related genes Oct3/4、Bmi1、 β-catenin、Nanog in RCCcell line 786-0, study the influence and mechanism of sorafenib on the changes of these gene expression.Methods:This part research was based on the chapter 2, we study the expression of CSC related genes Oct3/4、Bmi1、β-catenin、Nanog in RCC cell line 786-O from two aspects of in vitro cell experiments and animal experiments by FCM, respectively.Results:(1) The genetic testing results in Cell experiment:① Gene Oct3/4 mRNA expression of 24h、48h、72h、96h groups was:1.21±0.10、3.28±0.35、4.38±0.41、 7.55±0.36,respectively;②Gene Bmil mRNA expression of 24h、48h、72h、96h groups was:1.12±0.11、2.96±0.27、6.12±0.44、10.68±0.78,respectively;③Gene β-catenin mRNA expression of 24h、48h、72h、96h groups was:1.12±0.12、 3.10±0.16、4.26±0.22、8.46±0.68, respectively;④Gene Nanog mRNA expression of 24h、48h、72h、96h groups was:1.21±0.15、1.48±0.13、4.35±0.29、6.82±0.36, respectively. Each time group compared with 0 h control group, the variance analysis result shown the difference was significantly, except for the 24h group.(2) The genetic testing results in animal experiment: ①Gene Oct3/4 mRNA expression in control and dose groups was 1.25±0.37 and 9.60±3.29;②Gene Bmil mRNA expression in control and dose groups was 1.30±0.36、6.58±1.49;③Gene β-catenin mRNA expression in control and dose groups was 1.06±1.36、39.27±21.89; ④ Gene Nanog mRNA expression in control and dose groups was 0.98±0.27、 77.22±24.0. Each dose group compared with control group, the data analysis shown the difference was significantly (P< 0.05),by independent sample t test.Conclusions:There are different levels expression of gene Oct3/4、β-catenin、Nanog、 Bmi in RCC cell lines 786-O, and all the four gene expression levels could be improved by sorafenib. This result shown that Oct3/4、β-catenin、Nanog、Bmi all may be related to CSC of RCC.4. The influence of Sorafenib on the proportion of SP cells, the expression of ABCG2, HIF-1α and HIF-2α in RCC cell lines 786-OObjective:To detect the expression of proportion of SP cells, the expression of ABCG2, HIF-1α and HIF-2α in RCC cell lines 786-O, study the influence and mechanism of sorafenib on the these changes.Methods:This part research was based on the chapter 3, we study the proportion of SP cells, the expression of ABCG2, HIF-1α and HIF-2α in RCC cell lines 786-O from two aspects of cell experiments and animal experiments by Western Blot and RT-qPCR methods, respectively.Results:(1) The proportion of SP cells:The proportion of SP cells in control group and 24h,48h,72h,96h groups was:1.06±0.05%、1.21±0.12%,2.66±0.28%、14.05±1.52%, 41.27±4.74%,respectively. Each time group compared with Oh control group, the variance analysis result:F= 90.643 P< 0.001, the difference was significantly (P <0.05), except the 24 h group (P>0.05).(2) The results of Western Blot in cell experiments:chose GADPH as protein quantitative internal refer. The grey value of ABCG2, HIF-1α and HIF-2α in different time was compared with the control time group, through analysis of variance, F was: 293.85,293.85, and 281.956, P< 0.001, all the difference was significantly.(3) The results of Western Blot in animal experiments:The gray value of ABCG2 and HIF-1α in dose group compared with control group, the data analysis shown the difference was significantly (P< 0.05),by independent sample t test. However, the protein expression of HIF-2a in dose group compared with the control group was not obviously (P> 0.05).(4) The results of RT-qPCR in cell experiments:① Gene ABCG2 mRNA expression of 24h、48h、72h、96h groups was:1.21±0.19、2.34±0.19、3.23±0.24、 5.12±0.16,respectively;②Gene HIF-1α mRNA expression of 24h、48、72h、96h groups was:1.14±0.16、3.50±0.28、7.53±0.27、10.67±0.75,respectively;③Gene ABCG2 mRNA expression of 24h、48h、72h、96h groups was:1.29±0.15、6.39±0.40、 21.44±1.75、42.37±3.66,respectively; Each time group compared with 0 h control group, the variance analysis result shown the difference was significantly, except for the 24h group.(5) The results of RT-qPCR in animal experiments:the mRNA expression of gene ABCG2, HIF-la and HIF-2a in control and dose groups accordingly was 0.89±0.21:6.27±6.62,0.98±0.35:9.60±3.29,1.05±0.39:6.58±1.49. Compared with Each dose group and control group, the data analysis shown the difference was significantly (P< 0.05),by independent sample t test.Conclusions:There were few proportion of SP cells in RCC cell lines 786-0 and the proportion could be improved by sorafenib. Some amount of ABCG2, HIF-1α and HIF-2α expression was also be found in RCC cell line 786-O and the expression level also could be improved by sorafenib. These results shown that the markers ABCG2, HIF-1α and HIF-2α may be related to CSC of RCC. |
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