| BackgroundPancreatic cancer is one kind of the deadly malignant tumors in the world.Patients bearing pancreatic cancer are difficult to be diagnosised at the early stage because the relevant symptoms are not very obvious.Most of patients have lost the opportunities for surgical resection of the tumors.In this case,chemotherapy is regarded as one of the effective therapeutic methods,it can prolong the patients’ survival time and improve their quality of life.Unfortunately,pancreatic tumors usually exhibit drug resistance which would reduce the efficiency of the chemotherapy greatly.Sonochemotherapy is a brand new treatment method which utilizes low intensity ultrasound to stimulate microbubble cavitation,by using this method,it could increase the permeation of the tumor blood vessel and the tumor cell membrane.Hence,Sonochemotherapy could augment drug concentration of the tumor region and improve therapeutic effectiveness of chemotherapy.According to the recent results of research,sonochemotherapy potentially has the ability to solve the problem of chemotherapy resistance,it has attracted interests of many researchers.However,as for choosing the optimal acoustic parameters especially for selecting the appropriate mechanical index(MI)to achieve the best tumor therapeutic outcome,the specialists of this field haven’t come to an agreement.This uncertain aspect would affect the effectiveness and safety of the sonochemotherapy vastly.In terms of this obstacle,we design three parts of the experiment to investigate the effect of ultrasound-targeted microbubble destruction(UTMD)enhancing chemotherapeutic sensitivity of pancreatic cancer.Including an in vitro experiment,the measurement of acoustic parameters and in vivo animal experiment.It is expected to select the optimal MI for UTMD to augment the tumor’s blood flow and increase the targeted region’s drug concentration,which could be helpful to control the tumor.Objective1.Investigate the effects of UTMD under the same duty cycle and different sound pressures on the apoptosis of PANC-1 cells.2.Measure the cavitation related acoustic parameters of the VINNO70 machine.3.Investigate the impact of ultrasound with different MI enhanced by microbubble on chemotherapy of nude mice bearing pancreatic cancer.Materials and methodsPart Ⅰ The impact of low intensity ultrasound with different sound pressure combined with microbubble on PANC-1 cell apoptosis1.Materials1.1 Experimental machine:Therapeutic producer(DCT-700,Shenzhen,China),it is a circle-shaped,non-focusing producer with single wafer.The diameter and frequency of the producer are 2.5cm and 1MHz,respectively.1.2 Cells: Human pancreatic carcinoma cell lines(PANC-1)1.3 Contrast agent: Sonovue2.MethodsThe cultured cells were divided into 8 groups(n=6 for each group).They were group A: control group;group B: microbubble group;group C: low sound pressure group;group D: medium sound pressure group;group E: high sound pressure group;group F: low sound pressure with microbubble group;group G: medium sound pressure with microbubble group;group H: high sound pressure with microbubble group.Low、medium、high sound pressure related peak negative pressure was 1019KPa、1785KPa、2184KPa,respectively.All of the groups frequency were set as 1MHz,duty cycle=4%,time=120s.After the cells were cultured for 24 h,the ultrasound exposure was conducted through the bottom of the wells,the cells were cultured in the incubator for 24 h after the ultrasound exposure.The cells of each group were collected and then the flow cytometer was used to detect the apoptosis rate.3.Measurement indexThe apoptosis rate of PANC-1 for each groupPart Ⅱ The measurement of cavitation related acoustic parameters of VINNO701.Materials1.1 Testing object: VINNO70(produced by Feiyinuo technology Co Ltd),this machine had a Vflash module which could adjust the cavitation related acoustic parameters including MI、frequency、pulse length(PL)、pulse repetition frequency(PRF)etc.1.2 Experimental machine: membrane-type hydrophone,oscilloscope.2.MethodsThe measuring environment was a water tank with de-aerated water,the membrane-type hydrophone and oscilloscope were used to take measurements for the frequency,PL,PRF,sound field configuration and the peak negative pressure of different MI of the X4-12 L probe.3.Measurement indexCavitation related acoustic parameters of the X4-12 L probePart Ⅲ The effect of diagnostic ultrasound(DUS)with different MI combined with microbubble on enhancing the chemotherapy of nude mice bearing pancreatic tumor.1.Materials1.1 Experimental machine: VINNO70(produced by Feiyinuo technology Co Ltd),the main experimental acoustic parameters: frequency=4MHz,PRF=50Hz,pulse/interval time=0.48s/2s,PL=18cycle,line intensity=4,acoustic power and relevant MI for each group: group A:MI=0.3,group B:MI=0.7,group C:MI=1.1,high performance liquid chromatograph(HPLC);Cri Maestro In-vivo imaging Systems.1.2 Experimental animals: 48 healthy nude mice,4-week age,the animals were supplied by the Second Affiliated Hospital of Army Medical University.1.3 Experimental agents: doxorubicin(DOX),Zhifuxian micrbubble(produced by department of ultrasound,the Second Affiliated Hospital of Army Medical University.The out layer and core of the microbubble were phospholipid and C3F8 gas respectively,the mean diameter was 2.0um,concentration was 2-9×109/ml,fluorochrome DIR.2.MethodsPancreatic tumor models were eatablished through injecting PANC-1 tumor cell suspension into bilateral hind limbs of 48 nude mice.48 nude mice bearing tumors were divided into group A、B、C evenly and randomly.The animal model was anesthetized and then was fixed on the experimental platform with supine position,and established tail vein access.For each mouse,contrast enhanced ultrasound(CEUS)was conducted before and after ultrasonic exposure/sham exposure to gain the time-intensity curve(TIC),area under curve(AUC)and peak intensity(PI).1 hour later after the ultrasound exposure/sham exposure,10 animal models were chosen randomly from 3 different group respectively.DOX concentration in vitro tumor tissue was detected by using HPLC.5 animal models were chosen randomly from 3 different group respectively.The operations were similar to before,except that the injected agent was substituted by the Di R solution.After the ultrasound exposure/sham exposure,the animals were performed fluorescence imaging through Cri Maestro In-vivo imaging Systems.1 animal model was chosen randomly from 3 different group respectively.After the therapeutic operations,the animal models were perfused with physiological saline to obtain the tumor tissues.And then all the tumor tissues were sent to make slices with HE staining.3.Measurement index3.1 The TIC、AUC、PI were obtained by using an analytical software of the VINNO70 machine.3.2 The DOX concentration of the tumor region for each group was detected by the HPLC.3.3 The fluorescence imaging for each group was gained by the Cri Maestro In-vivo imaging Systems.3.4 The hematoxylin-eosin staining of the tumor tissue.ResultsPart Ⅰ The impact of low intensity ultrasound with different sound pressure combined with microbubble on PANC-1 cells apoptosisThe apoptosis rate of group A、B、H was 3.31%±0.91%、 3.08%±1.00%、9.61%±2.92% respectively.Compared with group A and B,the apotosis rate of group H was 3 times approximately,and the outcome had a statistically significant difference(P<0.05).Part Ⅱ The measurement of cavitation related acoustic parameters of VINNO701.The measured acoustic parameters(including PL、frequency)were equal to the demonstrated acoustic parameters of the machine.2.With the MI increased from 0.09 to 1.29,the MI relative peak negative pressure also increased from 0.14 MPa to 1.62 MPa.MI and peak negative pressure had a positive correlation.3.The sound field of probe X4-12 L belonged to normal distribution at the Y axis direction,The sound field of probe X4-12 L also belonged to normal distribution at the X axis direction,and the peak negative pressure of the region of interest(ROI)was obviously higher than the out area.Part Ⅲ The effect of diagnostic ultrasound(DUS)with different MI combined with microbubble on enhancing the chemotherapy of nude mice bearing pancreatic tumor.1.The AUC and PI of therapeutic side of the nude mice bearing pancreatic tumors in group A、B、C were higher than the control side(P<0.05).2.Concentration of Doxorubicin of therapeutic side in group A was obviously higher than that of control side([1.45±0.53]ug/g vs [1.07±0.46]ug/g;t=-5.163,P<0.05).In group B and C,there was no statistically significant difference of Doxorubicin concentration between therapeutic side and control side in drug concentration(Z=-0.297,-0.357,P=0.766,0.721).3.The outcome of fluorescence imaging showed that the DIR concentration in therapeutic side of nude mice bearing pancreatic tumor was higher than the control side in group A,this phenomenon wasn’t observed in group B and C.4.Neither hemorrhage nor swelling was found in tumor tissue of both sides in the three groups.Conclusions1.When peak negative pressure=2184KPa,UTMD could increase the apoptosis rate of PANC-1 cells significantly(P<0.05).2.The measured cavitation related acoustic parameters of probe X4-12 L were approximately equal to the acoustic parameters set of the machine,and the sound field of the probe X4-12 L belongs to normal distribution.3.When MI=0.3,DUS combined with microbubbles could increase the blood flow of the xenografts and enhance the drug concentration of the tumor region. |
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