Focused Ultrasound On Human Cervical Carcinoma Hela Cells In Vitro Study

Objictive: The purpose of our laboratory study was to investigate the cellular effect of Puled Focusd Ultrasound(PFU)on human cervical carcinoma HELA cell in vitro and layed laboratorial foundation for the treatment of human cervical carcinoma. Method: It can be divided into six staged:First, digesting HELA cells in logarithmic growth time with trypsin ,then irradiating them with PFU (the frequency of ultrasound is 1MHz,and its sound intensity is 83.6W/cm2)in different time,examining mortality of HELA cells with TPL staining method;Second,adjusting the density of HELA cells after irradiating them, and observing the growth inhibitory effect of HELA cells after PFU sonication with MTT colormetric assay; Third,collecting HELA cells after six hours, twelve hours, twenty-four hours and forty-eight hours and examining their cell cycle repartition by Flow Cytometry(FCM); Forth,adjusting the density of HELA cells density after PFU sonication ten minutes,and cultivating them for six hours, then examining apoptosis of HELA cells with tenminal deoxynucleotity transferase mediated dUTP nick end labeling (TUNEL). Fifth,collecting HELA cells after six hours , observing the morphologic change and apoptosis of HELA cells under the Electron Microscopy(EM); The last,adjusting the density of HELA cells after ten minutes, examining the expression of Bcl-2,Bax,p53 in HELA cells after PFU sonication with immunohistochemical staining assessment. Results: TPL results are that mortality of HELA cells is 19.8%,24.3%,46.25,65.36%,70.51% corresponding to PFU sonication durations of 10s,20s,30s,40s,50s.The results show that the mortality of HELA cells increase along with the entension of time, they are direct proportion impact. MTT showed that the absorbance value(A570) of HELA cells significantly decreased ,compared with the control group and the function of PFU and the inhibition effect of cells proliferation increase along with the time extention. FCM showed that there are more HELA cells during phase of G0/G1 than the control group(P<0.05), and there are less HELA cells during phase of S,M than the control group(P>0.05).The cycle of cells were stagnated in G0/G1 phase after PFU sonication. No statistic significance had gotten from the results between twelve houes and twenty-four houes in FCM. TUNEL methods showed that the apoptotic cells nucleus are brown-colored and they became round and small(P<0.05)while no deep color was found in cells of control group . The Electron Microscopy showed that there were no change in control group cells while there were some liqid drops and myeloid –like structure appearing in the cytoplasn,chromatin condensed,quite a few apoptosis bodies formed in a budding way,the mitochondrion,endoplasmic reticulum and cytoplasm swelling. In immunohistochemical staining we can see that P53 protein were weaker expression in cell nucleuses in the treated group than that of control group(P<0.05),Bcl-2 protein were also weaker expression inendoplasm than the control group(P<0.05), and Bax protein were stronger expression in endoplasm than the control group(P>0.05). Conclusion: PFU has an effect on human cervical carcinoma HELA cells and could inhibit proliferation of human cervical carcinoma HELA cell in vitro and induce apoptosis by letting HELA cells stagnate static phase. During the apoptotic processs ,P53, Bcl-2 ,Bax proteins joined them, and PFU play a role in inhibiting P53,Bcl-2 proteins expression ,increasing Bax proteins expression , resulting in the increase of Bax/ Bcl-2 ratio rising.