The Mechanism Research Of Rho Kinase Inhibitor Fasudil In Anti-leukemia Therapy

Experimental objectiveIn vitro fasudil can influence proliferation, apoptosis, cell adhesion molecules oflekemia cell line K562cells,it clarifies the possible mechanism of action in anti-leukemia,to provide a theoretical basis for clinical admiral fasudil as new drug for the treatment ofleukemia.Experimental materials and experimental methodsThe concentration of0、50、100、150umol/L;monitoring point in time the main0-24hour（0、6、12、24H）;K562cells are reserved regular batches by the lab of hematologydepartment of Shanxi Provincial People’s Hospital;the experiment confirmed that caninhibit the growth of K562cells by the MTT method; with optical microscope to observethe influence of fasudil on K562cell morphology;using fluorescent microscope observenuclear shape change（DAPI stained）;using the flow cytometry to analysis of early andlate apoptosis rate,the expression percentage of Fas/FASL and adhesion molecules CD54in K562cells;Western bloting to detect Casepase-3,PARP protein expression;RT-PCR todetect the expession level of VEGF165mRNA.The experimental results1.Detection of fasudil by the MTT method on K562cells growthinhibition:Different concentrations of fasudil(0、50、100、150umol/L) can inhibit the proliferation of K562cells at6,12,24h,and its with time and concentrationdependence;2.Optical microsope and fluorescence microscope(after DAPI staining) observe thechanges of cell morphology;The concentration of fasudil (0、50、100、150umol/L)effects on K562cells to observe the changes in cell morphology after6H,a small part ofthe experimental group (50umol/L) cells were irregular changes in cell morphology;thenumber of the experimental group (100umol/L) cells decreased significantly and morecells were irregular changes in cell morphology，more of the cells with chromatincondensation,nuclear pyknosis,nuclear margination, especially more apoptotic bodies;thenumber of150umol/L cells in experimental group showed reduced moresignifiantly,appeared nuclear pyknosis,cytoplasm showed a larged amount of apoptoticbodies,cell debris and formation of cavity;however the control group cells were competeuniformity,good refraction;3.Detection of apoptosis rate by flow cytometry:The use of flow cytometry to detecteach monitoring point in time,normal cells group of early and late apoptosis percentagewere（1.62±0.34）%（6h）,（1.98±0.04）%（12h），（2.24±0.11）%（24h）;percentageof early and late apoptosis cells in experimental group were（6.12±0.43）%(6h、50umol/L)，（8.78±0.49）%（6h、100umol/L），（10.92±0.62）%（6h、150umol/L），（7.9±0.58）%（12h、50umol/L），（19.73±1.29）%（12h、100umol/L），（29.4±0.81）%（12h、150umol/L），（20.97±0.96）%（24h、50umol/L），（36.03±6.28）%（24h、100umol/L），（51.18±1.45）%（24h、150umol/L）;different concentrationsof fasudil treated cells at the same time,through the comparison between each other canfind a statically significant difference(P＜0.05),fasudil effect of the same concentrationfor different time，through the comparison between each other can find a statisticallysignificant difference（P＜0.05）;4.Detection of FASL and Fas expression rate by flow cytometry:Differentconcentrations of fasudil treat K562cells in6H,Fas expression rates were(6.8±0.61)%(50umol/L)、(11.25±1.06)%(100umol/L)、(12.4±1.25)%(150umol/L),FASL expression rates were(67.4±6.79)%(50umol/L)、(70.5±1.95)%(100umol/L)、(72.5±1.83)%(150umol/L), nomal cells group FASL、Fas expression rates were (0.23±0.15)%、(0.4±0.1)%;these results show that as the dose increased FASL、Fas expression ratesincreased at the same time；there was statistically significant difference between differentconcentrations of experimental group and control group(P＜0.05),the comparison of100umol/L experimental group and150umol/L experimental group,the expression rate ofFas was no significant difference (P＞0.05);the comparison of the experimental groupand the control group,the expression rate of FASL was statistically significant(P＜0.05),comparison of each experimental group,no significant difference in the expressionrate of FASL（P＜0.05）;5.Detection of adhesion molecules CD54expression rate by flow cytometry:Normalcells group CD54expression rates was (97.34±0.70)%,50umol/L experimental groupCD54expression rates was (91.21±1.94)%,100umol/L experimental group CD54expression rates was (77.84±1.03)%,150umol/L experimental group CD54expressionrates was (53.36±1.80)%;different concentrations of fasudil treat different groups ofcells,the CD54expression of each group was statistically significant difference（P＜0.05）;6.Detection of VEGF165mRNA expression by RT-PCR method:With differentconcentrations (0、50、100、150umol/L) of fasudil treatment of K562cells with the sametime（6H）compared with the control group decreased the expression of VEGF165mRNA;with the increase of fasudil dose,the expression VEGF165mRNA gradullyreduced,in a dose dependment manner;7.Detection of apoptosis protein Western blot method:The activation level of normalcells group Casepase-3appeared in100umol/L,in the150umol/L up to peak;activationfragment of PARP protein in50umol/L is beginning to appear,as the dose increases itsexpression increased;Experimental conclsion:Fasudil could inhibit the proliferation of K562cells,on the basis of the related mechanism of its anti-leukemia possible,these results provide a good experimental basisand theoretical foundation，so we have reason to believe that fasudil is hoped to become anew anti-leukemia drug, whether it is widely accepted in the clinical needs further study.