| ObjectiveIn order to reduce expression of ferrochelatase in Hela and HEC-1-A cell, Through detecting validity of FECH-siRNA in applying RT-qPCR and Western-blotting; and conducting quailtative and quantitative analysis of increasing fluorescence due to PpIX conglomeration by DFOuniting with siRNA by fluorescence microscope and flow cytometry separately. Lowest induction dose of ALA is obtained. ALA dose is reduced in Tumor fluorescence diagnosis. The result ofthis research establishes a foundation of early cervical cancer fluorescence diagnosis aided bycolp oscope and early endometrial cancer fluorescence diagnosis aided by hysteroscope.Method1.To foster Hela and HEC-1-A cell, bundle FECH-siRNA using LipofectamineTM.2000, andinterfere composition of FECH.2.To detect validity of FECH-siRNA in applying RT-qPCR and Western-blotting.3.To separate Hela and HEC-1-Acell into five groups:①control；②0.1mMALA；&0.5mM ALA③0.1mMALA+15mMDFO；&0.5mMALA+15mMDFO④0.1mMALA+FECH-siRNA；&0.5mM ALA+FECH-siRNA⑤0.1mMALA+15mMDFO+FECH-siRNA；&0.5mMALA+15mMDFO+FECH-siRNAto observe the intensity of fluorescence by fluorescence microscope, to conduct quantitiveanalysis with helps of flow cytometry, and to analyze statistic of the result.Results1. Detecting by RT-qPCR, the expression level of FECH-mRNA in FECH-siRNA interferedHela cell group is lower than that in non-interfered group.2. Detecting by Western-blotting, the expression level of FECH in FECH-siRNA interferedgroup is obviously lower than that in non-interfered group after FECH-siRNA interfering Hela.Result shows the validity of FECH-siRNA is58.13%3. Observing Hela cell through the fluorescence microscope, red fluorescence is observedwhen inducing by0.5mM ALA alone. Faint red fluorescence is observed when0.1mM ALAunited with15mM DFO or FECH-siRNA. Distinct red fluorescence is observed when ALA induct concentration is0.1mM and15mM DFO is united with FECH-siRNA.4. Conducting quantitative analysis of each group of Hela cell by flow cytometry, intensityof fluorescence and ALA induct concentration is directly proportional. Comparing0.1mM ALAinduction alone with15mM DFO united with0.1mM induction, otherness of intensity offluorescence has no significance in statistics (t=1.38，P=0.24). Comparing0.1mM ALAinduction,15mM DFO,15mM DFO united with FECH-siRNA, FECH-siRNA interferenceamong15mM DFO united with FECH-siRNA induction, otherness of intensity of fluorescencehas significance in statistics (t=139.12，P=0.00；t=70.16，P=0.00).5. Observing HEC-1-A cell, through the fluorescence microscope, red fluorescence isobserved when inducing by0.5mM ALA alone. Red fluorescence is observed when0.1mM ALAunited with15mM DFO or FECH-siRNA. Distinct red fluorescence is observed when ALAinduct concentration is0.1mM and15mM DFO is united with FECH-siRNA..6. Conducting quantitative analysis of each group of HEC-1-A by flow cytometry, intensityof fluorescence and ALA induct concentration is directly proportional. With same ALA inductionconcentration, comparing ALA induction alone, ALA united with15mM DFO, with ALA unitedwith FECH-siRNA, otherness of intensity of fluorescence has significance in statistics(P=0.00).Comparing15mM DFO united with FECH-siRNA and ALA, ALA united with15mM DFO, withALA united with FECH-siRNA, otherness of intensity of fluorescence has significance instatistics(P=0.00).Conclusion1. Selected target FECH siRNA is successful to reduce the expression of FECH in Hela.2. DFO united with siRNA can increase the intensity of fluorescence of PpIX in Hela.Simply add DFO and simply add FECH-siRNA can increase the intensity of fluorescence ofPpIX as well. But it is lower than them are united application. When15mM DFO is united withFECH-siRNA, the optimum induction concentration of ALA is0.1mM.3. DFO united with siRNA can increase the intensity of fluorescence of PpIX in HEC-1-Acell. Simply applying siRNA can increase the intensity of fluorescence of PpIX as well. But it islower than them are united application. When15mM DFO is united with FECH-siRNA cell, theoptimum induction concentration of ALA is0.1mM. |
