| BackgroundAt present,maligant tumor is one of the most serious diseases threatening human life and health, the treatment of maligant tumor is important but difficult for the medical profession. At the present stage, the chemotherapy is the main treatment way for it. But because the chemotherapeutic drugs are used widely in clinical and the multidrug resistance situation is happening regularly, the difficulty of the treatment is added which reduce the success rate of the chemotherapy and is the biggest obstacle of the treatment. Now, one of the most important problems is to explore a kind of way to enhance sensibility and reduce toxicity of the chemotherapeutic drugs. In recent years, the technology of Ultrasound-targeted microbubble destruction (UTMD) develops quickly. It can promote the absorption of chemotherapeutic drugs by tumor and show a bright prospect.Therapeutic ultrasound instrument has been used in many research,but it possesses limitation because of no exact location and potential hidden dangers because of high sound intensity. Therefore, two-dimensional diagnostic ultrasound was used by researchers to avoid the limiations, the results showed that combination two-dimensional diagnostic ultrasound with microbubbles could enhance permeability of the normal hepatic vessels and the vessels of transplanted subcutaneous hepatocellular carcinoma in rats. But the acoustic beam of two-dimensional diagnostic ultrasound travels in a very narrow solid angle, it possesses disadvantsges of small irradiation area and uneven irradiation. Three-dimensional diagnostic ultrasound is a stereoscopic scanning technology of ultrasound, it scans like a pyramid in space and can cover tumor completely. The way of intratumoral injection makes the microbubbles avoiding the blood barrier and contact with tumor cells;reduces the icrobubbles attenuation by pulmonary circulation, extents the remaining time of microbubbles in tumor. The way of intratumoral injection is not only reducing distances between tumor cells and microbubble, and rasing the local microbubble concentration but also lowering drug concentration the most out of it and reducing the side effects. The normal tissue between probe and tumor can’t be destroyed because of microbubbles limited in tunor. However, there were few reports about the experimental study on the chemotherapy effect enhanced on tumor by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles. In consequence, the tumors were underwent by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles in this research. This study is to explore the feasibility of the chemotherapy effect enhanced on tumor by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles.Objective1.To explore the feasibility of improving tumor cell membrane in vivo by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles, observe the effects of mechanical index, radiation time on tumor cell membrane permeability by using lanthanum nitrate electron microscopy and achieve coustic parameters for treatment.2.To explore the chemotherapy effect enhanced on VX2 transplanted tumor by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles.Materials and methods1.Main experimental materialsExperiment instruments:ViVi E9(made in company GE) with 3V-D probe whose mechanical index is 1.3, frequency is 1.5-4.0 MHz was used for sonication radiation, the mechanical index and pulse-interval time can be changed. Philips iU22 color Doppler ultrasonography with L12-5 high frequency linear array probe containing contrast-enhanced ultrasonography mode was used. Hitachi H-7650 transmission electron microscopy.Leica EG1160 paraffin embedding mechine (made in Germany). Leica RM2235 paraffin slicing mechine (made in Germany).Ultrasound microbubbles:Microbubbles with diameter range from 2.5～4.0μm concentration of 0.8-2.2 X 109/ml are Quan fuxian made by Nanfang Hospital. Perfluoropropane is filled in Human albumin used as filming materials.3 ml saline solution is added for dilution and the mixture is oscillated and waved for contrast-enhanced ultrasonography examination and intratumoral injection.Other reagent:Doxorubicin, Sumianxin, Atropin, Pentobarbital, TUNEL apoptosis assays kit, lanthanum nitrate.2. Animal model production90 healthy New Zealand rabbits of either gender with 2.5-3.0 kg body mass weight were used as model rabbits. Another 4 healthy New Zealand rabbits were used for passaging (All animals were provided by the Animal Experimental Center of Southern Medical University Nanfang Hospital).2 ml VX2 tunor tissue suspension geted by the frozen VX2 cell line was injected into the right lateral hind linb of passaging rabbits. When the tumor diameters of passaging rabbits were about 2 cm,0.35 ml/kg Sumianxin Ⅱ and 0.25 ml/kg Atropin were injected into the right lateral hind linb of passaging rabbits for anesthesia, and established intravenous line for injecting pentobarbital. The tumor-bearing rabbits were maintained in the lateral position. After cleaning and sterilizing the tumor site and its 1.5 cm around, the tunor tissue was stripped under aseptic condition. The surrounding fascia, connective tissue and central areas of necrosis quickly. The rest was dipped into the normal saline and sheared into tissue blocks about 1 mm3.90 experimental rabbits were anesthetized and sterilized with above method, the tissue blocks were implanted on the inside of the right lateral hind linb of the rabbits.3. Experimental procedure1.Investigation of the effects of influence factors on VX2 tumor cell membrane by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles:(1) 45 VX2 xenograft tumor models were randomized into 2 groups for the impact factors including mechanical index and radiation time, each impact factors were divided into 5 levers which MI was 0、0.1、0.5、1.0、1.3 and radiation time was 0、5 min、10 min、15 min、20 min. Each lever repeated 5 times.When inspecting levers of one factor, the other factor was fixed.The intracellular lanthanum particles were acted as the standard of judgeing the change of the tumor cell membrane permeability. The effects in tumor cell membrane permeability by mechanical index, radiation time were quantitstively investigated, and the best acoustic parameters were achieved for treatment.(2) Operational process:Before irradiation, the volume of microbubbles injected in tumor directly was about lml×the volume of the tumor (cm3).3V-D probe was used for irradiation tunor based on every impact factors. After irradiation, the tumor tissue was stripped with stain fo lanthanum for observeing the ultrastructure of tunor cells and intracellular lanthanum particles under the transmission electron microscopy.(3) lanthanum particles scoring criteria:10 tumor cells were selected randomly in the same slice.Zero:there were no lanthanum particles in the tumor cell; 1:1-3 lanthanum particles were saw in the tumor cell; 2:4-9 lanthanum particles were saw in the tumor cell; 3:10-14 lanthanum particles were saw in the tumor cell; 4:15-19 lanthanum particles were saw in the tumor cell; 2:20 or more lanthanum particles were saw in the tumor cell.2.Experimental study of chemotherapeutic effect of VX2 xenograft tumor enhanced by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles:(1)Protocol of ultrasonic irradiation:35 VX2 xenograft tumor models were randomized to one of 7 groups with 5 in each group. Three-dimensional diagnostic ultrasound+intratumoral injection microbubbles+ Doxorubicin group (3D+MB (intratumoral injection)+Dox):after injection Doxorubicin intravenously, microbubbles were injected intratumorally, then tumor was irradiated by three-dimensional ultrasound;Three-dimensional diagnostic ultrasound+intravenous injection microbubbles+ Doxorubicin group (3D+MB(intravenous injection)+Dox): after injection Doxorubicin intravenously, microbubbles were injected intravenously, then tumor was irradiated by three-dimensional ultrasound; Two-dimensional diagnostic ultrasound+intratumoral injection microbubbles+ Doxorubicin group (2D+MB(intratumoral injection)+Dox):after injection Doxorubicin intravenously, microbubbles were injected intratumorally, then tumor was irradiated by two-dimensional ultrasound; Doxorubicin group (Dox):after injection Doxorubicin intravenously, sodium chloride were injected intravenously instead of microbubbles, then tumor was falsely irradiated by ultrasound; Three-dimensional diagnostic ultrasound+ Doxorubicin group (3D+ Dox):after injection Doxorubicin intravenously, sodium chloride were injected intravenously instead of microbubbles, then tumor was irradiated by three-dimensional ultrasound; Three-dimensional diagnostic ultrasound+ microbubbles group (3D+MB (intravenous injection)):after injection sodium chloride intravenously instead of Doxorubicin, microbubbles were injected intratumorally, then tumor was irradiated by three-dimensional ultrasound;Control group:after injection sodium chloride intravenously instead of Doxorubicin, sodium chloride were injected intravenously instead of microbubbles, then tumor was falsely irradiated by ultrasound; The irradiation time was 10 minutes, and the mechanical index was 1.3, the volume of microbubbles injected in tumor directly was about 1 mi×the volume of the tumor (cm3), the volume of Doxorubicin was 1.25ml/kg, the volume of sodium chloride was eqal to the reagent which it instead of. Tumor in each group was treated in the 1st,3rd,5th,8tfh day, the total was 4 times. In the 10th day after treatment, the tumor tissue was stripped for the cells apoptosis examination and HE staining. The growth rate of the tumor volume was calculated.(2)Analyses:apoptotic index was calculated by TUNEL. Criteria:the positive apoptosis cells were dyed dark brown.5 horizons (10×40 times) were selected randomly for cell count under the light microscope (the total number of cells in each group were no less than 500), the number of apoptotic cells and total cells of the 5 horizons were calculated accumulativly. The apoptotic index calculation (AI)(%)= the number of apoptotic cells/the total cells x100%.4.Data analyses SPSS 13.0 software package was adopted for atatistical analysis,the volume of the tumor and lanthanum particles score were described with x±s. One-Way ANOVO was used to analysis the comparisons of the volume of the tumor among different groups; K Insependent Samples Tests was used to analysis the comparisons of the lanthanum particles score among different levels in the same factor, if the total difference was statistically significant,2 Insependent Samples Tests was used to analysis the comparisons of the lanthanum particles score between any two levels; One-Way ANOVO was used to analysis the comparisons of the volume and the growth rate of the tumor in the same time and the apoptotic index among groups, Tukey tests was used for pairwise comparison. P value less than 0.05 for the difference is statistically significant.Results1.Investigation of the effects of influence factors on VX2 tumor cell membrane by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles:Lanthanum particles wasn’t observed in the tumor cells when mechanical index was o, Lanthanum particles was characterized by linear distribution in the cell junctions. Increasing with mechanical index, Lanthanum particles in the tumor cells was added. The largest number of Lanthanum particles was when mechanical index was 1.3, its score was higher than other levels, the difference of comparison with other levels is statistically significant (P<0.01). The number of Lanthanum particles and its score were increased with the irradiation time, the difference of comparison with control group is statistically significant (P<0.01). But there was no significant change of the number of Lanthanum particles and its score for more than 10 min (P>0.05). Lanthanum particles wasn’t observed in the tumor cell necleus, but the mitochondria and endoplasmic reticulum was cavitation with Lanthanum particles in.2.Experimental study of chemotherapeutic effect of VX2 xenograft tumor enhanced by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles:The volume of the tumor (0.25-0.37 cm3)was no statistical difference among each group before treatment (P>0.05). After treament the volume of the tumor in the 3D+MB (intratumoral injection)+Dox group,3D+MB (intravenous injection) +Dox group,2D+MB (intratumoral injection)+Dox group, Dox group,3D+Dox group,3D+MB (intravenous injection group, control group were 0.80+0.05 cm3, 1.60±0.24 cm3,1.83±0.32 cm3,2.30±0.20 cm3,2.46±0.10 cm3,2.46±0.19 cm 3,3.57±0.37 cm3 respectively. The volume of the tumor in the 3D+MB (intratumoral injection)+Dox group was the smallest, the difference is statistically significant (P<0.05). The growth rate of the tumor volume in the 3D+MB (injection injection) +Dox group,3D+MB (intravenous injection)+Dox group,2D+MB (intratumoral injection)+Dox group, Dox group,3D+Dox group,3D+MB (intravenous injection group, control group were 3.19,6.19,8.30,9.62,7.40,8.43,12.16. The growth rate of the tumor volume in the 3D+MB (intratumoral injection)+Dox group was lower than other groups’, the difference is statistically significant (P<0.05). Apoptotic index:After treament, the apoptotic index (%) of the tumor cell in the 3D+MB (injection injection)+Dox group,3D+MB (intravenous injection)+Dox group,2D+MB (intratumoral injection)+Dox group, Dox group,3D+ Dox group, 3D+MB (intravenous injection group, control group were 45.99±5.64、27.65±2.74、 25.68±2.64、13.78±1.81、16.87±3.09、15.17±2.76、9.44±1.93,3D+MB (injection injection)+Dox group was the highest. The difference is statistically significant (P<0.05).Conclusions1.The VX2 tumor cell membrane can be punchinged and the cell membrane permeability can be increased by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles.Though cell nucleus can’t be punchinged, the cell membrane permeability can be changed by cavitation effect caused by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles.2.The cell membrane permeability can be effected by mechanical index and radiation time. The cell membrane permeability can be increased with mechanical index and radiation time within limits.3.The chemotherapy effect of Doxorubicin can be enhanced by three-dimensional diagnostic ultrasound combined with intratumoral injection microbubbles. This technology is damage slightly, easy accessibility, repetition and be replicated. |
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