| Objective:Cholangiocarcinoma is not sensitive to chemotherapy, investigate ultrasonic cavitation enhance coast gemcitabine of chemotherapy cholangiocarcinoma. Firstly, filtrate the ultrasonic irradiation time-strength parameters and chemotherapy with gemcitabine dosage; Secondly, put the simple gemcitabine, simple ultrasound irradiation and microbubble single variables into proup,by using ordinary light microscope, Hoechst33342 apoptosis staining fluorescence was observed, confocal microscopy nucleus changes and MTT inhibition rate detection method, by multiple approaches,multiple methods, to evaluate the microbubble mediated by the ultrasonic cavitation act on curative effect of gemcitabine working on cholangiocarcinoma cells.Methods:1、Preparation of lipid microbubbles The DPPC, MPEG200, DPPA, PEG400, etc were blended to a suitable proportion and process procedures lyophilized freeze-dried preparation of lipid lyophilized powder, and replaced with perfluoropropane air therein, add plasmin, according to a specific program of mechanical shock, full suspension, prepare lipid microbubbles.2、The cell culture The cultured in RPMI 1640 with cholangiocarcinoma cell culture medium (10% fetal bovine serum),37℃,5% CO2 conditions subculture, taking growth in good condition, close to the logarithmic growth phase at a concentration of 1x106 cells are grouped.3、Screening the drug concentration of Gemcitabine concentration to cholangiocarcinoma cytostatic effect1) Configure gemcitabine solution:gemcitabine lyophilized powder was dissolved in PBS, prepare a plurality of concentration gradient, filter and sterilize by 0.22μm membrane, storage in-20 ℃.2) Preparation of a solution of MTT:MTT was dissolved in PBS, magnetic stir 30min, filtered and sterilized, dubbed 5mg/ml liquid, storaged in 4 ℃ dark.3) Drug toxicity test:RBE cholangiocarcinoma cells 1x104/well were seeded in 96-well plates for 24 h at 37 ℃ 5% CO2 conditions, the cells were divided into seven groups of thirteen holes were added to different the concentration gradient of gemcitabine solution 100μL,37 ℃, under 5% CO2 condition cultured 24h, each experimental group is added to wells MTT 1OμL,5 g/L, cultured 4h after aspirating hole liquid added to each well 100μL DMSO, shaker set low shock 15min, until after the blue crystals completely dissolved, using a microplate reader at a wavelength of 490nm at the detection of the absorbance (OD), calculate the inhibition rate, each repeated three times.4、Screening the parameters of Ultrasound cavitation to cholangiocarcinoma cytostatic effectLogarithmic growth phase similar to the growth state RBE cholangiocarcinoma cells were seeded in 6-well plates in a concentration of 1x106/mL, each hole 3mL, floating in 37 ℃ water bath, water bath ultrasonic probe fixed to the bottom of the lower plate 6, from bottom plate lcm, respectively, sound intensity ultrasonic irradiation (OW/cm2 increments by 0.1W/cm2) and time (Os and increments Is) as variables, divided into different treatment groups after cavitation each group by MTT assay 24h suppression rate of the cells, select the best ultrasonic cavitation parameters for the next cavitation by gemcitabine chemotherapy foundation.5、Ultrasonic cavitation increase gemcitabine chemotherapy evaluation of RBE cholangiocarcinoma1) The human cholangiocarcinoma cells were seeded in 6-well plates, each hole concentration 1x105, the culture reached 80% confluence, the growth of state election close RBE cholangiocarcinoma cells labeled with the following six groups as a cross-reference:a. control group; b. simple ultrasound irradiation group; c. simple gemcitabine group; d. gemcitabine ultrasonic irradiation group; e. microbubble mediated ultrasound cavitation group;f. gemcitabine-mediated microbubble ultrasound-cavitation sensitizing group. And click way to treat each group:a group: control group, without treatment; b groups:simple ultrasound irradiation group, the choice 1.2.4 screened the best irradiation parameters cavitation 6-well plate cholangiocarcinoma cell; c groups:simple gemcitabine group, to the plate was added 1.2.3 screened optimum gemcitabine dosage and concentration; d group: gemcitabine-ultrasonic irradiation group, while the use of group b、c conditions cholangiocarcinoma cells in 6-well plate processing; e group:ultrasound-mediated microbubble cavitation group:plates were added to 1x109 cells/mL microbubbles 1mL, using the b parameter group ultrasound irradiation plate to make holes plate microbubble cavitation effect; f group:gemcitabine-mediated microbubble ultrasound-cavitation sensitizing group added chemotherapy with gemcitabine based on group e on capecitabine.increase curative effect of chemotherapy in gemcitabine.2) According to the following steps for each group of cholangiocarcinoma cell inhibitory effect to evaluate:1、Observe the morphological changes of the cancer cells under the light microscope; 2、Cultured 24h after detect the inhibition rate in each group treated cholangiocarcinoma cells,48h,72h, plus MTT reagent (each well 20μL), and then cultured for 4 hours, after aspirating hole culture supernatant liquid plus DMSO (each well 150μL), set the low-speed shock shaker for 15 minutes, using a microplate reader measured OD value (wavelength 490nm), and the inhibition rate was calculated, each repeated three times; 3、To detect the cell apoptosis:after treatment, each group continued to train 24,48,72h, termination of culture and digested, centrifuged and washed, Join lOul Hoechst33342 staining solution and 10ul PI staining solution, gently mix 4 ℃ dark incubated 15min, placed observe the effect of fluorescence under a fluorescence microscope.4、 Onfocal observation groups cholangiocarcinoma cell nucleus changes:The cells in each group terminate culture and trypsinized, centrifuged and washed 3 times with 500μL staining buffer resuspended by adding 5μL Hoechst 33342, fully suspended at room temperature in the dark incubated for 20min, PBS wash off excess fluorescent dye, placed in the laser confocal microscope to observe changes in the nucleus of each group.6、Statistical analysisa) Data processing using SPSS 19 statistical software, measurement data as mean± standard deviation (mea±SD) said that differences between groups of data between the significance analysis using independent sample t test, P<0.05 was considered statistically significant.b) A plurality of sample proportions were compared using x2 test analysis RxC table data; taking P<0.05 was considered statistically significant.c) All data were expressed as mean±standard deviation, using statistical software SPSS 19.0 statistical software. Among groups were compared using ANOVA or factorial design analysis of variance between the two groups were compared using LSD method, the variance of missing persons using Tamhane’s T2 method, taking P <0.05 was considered statistically significant.Results:1、Screening the best dosage of gemcitabine cholangiocarcinoma cell inhibition:a cholangiocarcinoma cell per 1x106, use 200μL, the concentration of 0.02mg/mL.2、Selecting the best Ultrasound cavitation parameters:LSD method detected by ultrasonic sound radiation intensity is between 0～0.6W/cm2 increases apoptosis rate increases, when the sound intensity≥0.6Wcm2 when apoptosis rate did not increase.Results analysis of variance was statistically significant (F= 124.30, P <0.001). At the same time, we have selected the best cavitation ultrasonic irradiation parameters:When the sound pressure 2000KPa, PRF 80Hz, center frequency 1.2MHz,1.6% duty cycle, sound intensity 0.6W/cm2, mechanical index 0.9 cholangiocarcinoma cell apoptosis rate reached a peak 40.5% best cavitation parameters.3、Each group under light microscope morphological changes. group a:the control group, most of the cells were round or fusiform adherent growth, cell connections are tight, stretch growth, the projections more refraction good; group b:simple ultrasonic irradiation group, the majority of the cells were polygonal or spindle, was "paving stone" like growth, most of the normal cell morphology, only a few cells were spindle; group c:gemcitabine alone group, adherent cells decreased, cells partial rupture occurs, the uneven distribution of nuclear chromatin, exhibited the dense fluorescence shaped visible part of nuclear condensation, fragmentation; group d:gemcitabine plus ultrasonic irradiation group, the cells were similar to c; group e:simple ultrasound microbubbles irradiation cavitation and cell morphology was similar to the group b; group f:gemcitabine-mediated microbubble ultrasound - cavitation sensitization group, cell shrinkage, membrane shrinkage, loss, decreased adherent cell number, cell majority round, most of the aggregation and fusion, and some were broken cell debris, cytoplasm decreased transparency, nuclear color deepened, and the emergence of the phenomenon of many floating cells, the cytoplasm decreased transparency, refraction enhanced.4, The inhibition rate changing by each group and the results over time:each group were detected by MTT assay inhibition was observed 24h,48h,72h discovery:a blank control group, the inhibition rate remained at around 10%, almost unchanged over time; b group and e group 48h before inhibition with the control group is insignificant, after 48 hours and gradually began to cholangiocarcinoma cells suppressed; there gemcitabine factor intervention group c, d group, f group 24 hours from the beginning of the cholangiocarcinoma cells inhibited at 48h, inhibition rates were 25.1%,26.7%,38.1%, c, d group is insignificant, when 72h, c, d two similar inhibition rate were 30.1%,31.1%, group f, namely gemcitabine - mediated microbubble ultrasound-cavitation sensitization group was the highest inhibition rate of cholangiocarcinoma,72 hours up to 52%. Different groups at different times paired samples t-test results showed that:when each group 48h after inhibition was significantly higher than 24h, the difference between the three periods was statistically significant (P<0.05), f inhibition group was significantly higher than the rest 5 groups, P<0.001, statistically significant difference compared with other groups.5、Each group by fluorescence detection of apoptosis imaging result of the comparison:each group were Hoechst33342 staining apoptosis developing relatively visible:(1) control group (a group):the nucleus showed low intensity fluorescent blue uniform, shape and size more consistent, more cell survival, high activity, showed a normal nuclear fluorescence intensity of the blue fluorescence was low; (2) simple ultrasound irradiation group (b group):most of the cells were blue uniform, more consistent shape and size, a few cell fluorescence intensity, compared with a group of fluorescence slightly stronger; (3) a simple gemcitabine group (c group):the visible part of cellular chromatin density increased, the nuclei begin condensation, coagulation, Aizen increased brightness, cell fluorescence intensity, showing apoptotic state, was slightly higher intensity of the blue fluorescence; (4) gemcitabine-ultrasonic irradiation group (d group):group similar to c; (5) simple ultrasound microbubbles-ultrasound irradiation group (e group):most cells were blue uniform, more consistent shape and size, compared with b group had more cell fluorescence intensity; (6) gemcitabine-mediated microbubble ultrasound-cavitation sensitization group (f group):most cellular chromatin density significantly higher, obviously condensation nuclei, condensation, marginalization, Aizen brightness significantly increased cellular fluorescence intensity was significantly enhanced, formation of apoptotic bodies, a large number of cells showed ill-defined, cell disruption, significantly reduced the number of adherent cells, a large number of cells dead floating cells most rounded, most of aggregation and fusion, was typical of apoptosis and cell death, showed a high intensity blue fluorescence.Conclusions:This study evaluated the ultrasonic cavitation increases gemcitabine for chemot-herapy of cholangiocarcinoma.Firstly, completed ultrasonic cavitation time-parameter filter strength to find the best ultrasonic cavitation irradiation parameters: the sound pressure 2000KPa, PRF 80Hz, duty sound better than 1.6%,0.6W/cm2,m-echanical index 0.9, followed by screening test useful in the present experiment the effect of chemotherapy well, to reflect the difference, easy to detect differences in the optimum dosage of gemcitabine and concentration, a cholangiocarcinoma cell per 1x106, use 200μL, the concentration of 0.02mg/mL; Second, gemcitabine alon-e, simple ultrasound irradiation microbubble and other variables are paired single packet to ensure that a single variable between the groups to ensure that scientific tests. Eventually, after ultrasonic irradiation parameters using the best, most appropriate dose gemcitabine us by ordinary light microscope, Hoechst33342 fluorescence staining apoptosis was observed by laser scanning confocal microscope nucleus changes, MTT method to detect inhibition rate and other multi-channel,multi-method,examined the treatment groups cholangiocarcinoma cell apoptosis and inhibition effect was evaluated microbubble mediated by ultrasonic cavitation increase gemcitabine chemotherapy cholangiocarcinoma cell effect. Fully proved:ultrasonic cavitation microbubble mediated triggered the sound hole effect, in favor of more chemotherapy drug gemcitabine into tumor cells, making chemotherapy systemic low concentrations, localized high concentrations, good efficacy, side effects vision to obtain a test verification, for clinical provides an efficient, safe, convenient and treatment of cholangiocarcinoma with non-invasive chemotherapy deas and methods. |
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