| Part Ⅰ The effect of shRNA on the expression of Fibulin-3 in malignant pleural mesothelioma cells.Objective:The study was to construct Fibulin-3 overexpression and the effect of silence on the Primary cell of malignant pleural mesothelioma(MPM),and to look for new ways to treat MPM.Methods:Constructed Fibulin-3 expression,short hairpin RNA carrier,transfected Control、Fibulin-3 overexpression vector、overexpression Control vector、interference vector、interference Control vector with MPM cells.used flow cytometry to detect cell cycle and apoptosis,and applied Real-time PCR and Western blotting to detect the expression of Fibulin-3 before and after interference.Results:After transfected the MPM Primary cells,the results of flow cytometry test showed that compared with the Control group,the G2 phase of Fibulin-3+Exp was increased(p<0.05)and shRNA2 was reduced(p<0.05),the difference was statistically significant.The result of apoptosis was that compared with the Control,the apoptosis of Fibulin-3+Exp cells were decreased(p<0.05),and the apoptosis of the shRNA2 cells were increased(p<0.05),the difference is statistically significant.The results of molecular level detection showed that compared with Control group,the expression level of Fibulin-3 was increased.And shRNA group was down-regulated,the difference was statistically significant.Conclusion:We built Fibulin-3 gene expression and silence successfully,confirmed the role of fibulin-3+Exp,shRNA in vitro culture of MPM Primary cell growth.Fibulin-3 as targets of RNA silencing means to provide a theoretical basis for clinical MPM treatment.Part Ⅱ The effect of shRNA’s silence fibullin-3 gene on the transplanted growth of malignant pleural mesotheliomaObjective:Discussed the effect of shRNA’s silence fibulin-3 gene on the growth of MPM nude mice,provided new treatment for further diagnosis and treatment of MPM.Methods:Constructed Fibulin-3 expression,short hairpin RNA carrier,transfected Control、Fibulin-3 overexpression vector、overexpression Control vector、interference vector、interference Control vector with MPM Primary cells.Divided the nude mice into eight groups randomly,and each group of three,selected the subcutaneous hypodermic injection point in the lower part of each of the naked mice,kept and observed the growth status of each group of transplanted tumors carefully.Used morphological observation to observe the change of the cell of eight groups of MPM nude mice.applied Real-time PCR and Western blotting to detect the expression of Fibulin-3 before and after interference.Results:The weight and volume of the Fibulin-3 overexpression vector group were significantly greater than that the negative control group and the blank control group,and the interference group were significantly smaller,the difference was statistically significant(p<0.05).There was no significant difference between the weight and volume of the tumor in the tumor of the tumor in the negative control group and the blank control group,without statistical significance(p>0.05).HE staining indicated that the irregular adenoid structure of tumor cells,interwoven into bundles,hyperchromatic nuclei of nuclei,the number of cell nucleus splitting and the number of fiber cables,had increased compared with the control group,while the interference group was improved.Immunohistochemistry showed that Compared with the control group,the number of Fibulin-3 positive in the expression group was increased,and the interference group was decreased significantly.The expression levels of mRNA in Fibulin-3 was significantly higher than that the negative control and blank control group(p<0.05).while the expression of interference group and transcription products was significantly lower than that the negative control group and the blank control group(p<0.05).The relative expression of Fibulin-3 proteins in the eight groups of MPM nude mice was consistent with mRNA expression.Conclusion:confirmed that the shRNA silent Fibulin-3 gene has been shown to reduce the expression of Fibulin-3 in the transcription and protein levels,thereby inhibiting the growth and apoptosis of the transplanted tumor in MPM.And provided a theoretical basis for the explicit shRNA silence Fibulin-3 gene in MPM. |
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