| Background:It is generally thought that tumor grows in two different stages, ie, a slow growth stage with avascularity and a fast growth stage with vascularity. In the slow growth stage, tumor's growth relies on the diffusion ventilation, with the maximum diffusion diameter of 150μm. When the diameter of the tumor exceeds 1-2mm, the further growth needs blood supply. It is reported that most of the solid tumors short of blood vessels grew rather slowly but developed fast with blood vessels. Doubling generation time of vascular endothelial cells (VECs) of health persons is about one year, but that of tumor is only four days! Therefore, blocking the blood supply of tumor to inhibit its growth is one of the hot spots of current research.Hepatic metastasis of malignant cancers is accomplished mainly by diffusion metastasis, lymphatic metastasis, blood route metastasis and implantation metastasis caused by malignant cell shedding. The liver is the largest parenchymatous organ of the human body, and is considered the major location of many malignant cancer metastasis because of its double blood supply. Meanwhile, hepatic metastasis is commonly seen in the blood route metastasis of colon cancer. It was reported that for the patients with colorectal cancer, about 10%-25% had been found with hepatic metastasis when they underwent primary operation, another 25% were found with hepatic metastasis during follow-up and over 50% finally had hepatic metastasis, which proved that the hepatic metastasis was the leading cause for death of patients with colon cancer. Therefore, inhibition of metastatic hepatic carcinoma is the key for the improvement of survival rate.Recently, with the development of ultrasound contrast and the application of new ultrasonic contrast agent, the problems caused by the biological effects of microbubbles contrast agents have gradually aroused a great concern. Ultrasound contrast has been extensively employed in the diagnosis of liver diseases and it's playing an important role. Physical characteristic and biological effects (heat effect, cavatition erosion and mechanical effect) of ultrasound have been widely used in many medical fields including health care, disease prevention, disease treatment (e.g. vitro macadam, hemostasis and ablation of tumor) and biotechnology (e.g.gene transfection). As a kind of artificial cavitation nuclei, ultrasonic contrast agent can not only degrade cavitation threshold and accentruate cavatition but also diminish the ultrasonic energy for cavatition. Furthermore, ultrasonic cavatition itself exerts kinds of biological effect on peripheral histiocytes, for instance, damaging cellular membrane and DNA, breaking DNA chain, inhibiting cellular proliferation, accelerating cell apoptosis, and depressing metastasis and adhesiveness of cells by recombination of cystoskeletons as well as altering distribution of carcinoma cell cycle, which are helpful in many aspects of clinical treatment including suppression of growth and metastasis of tumor and facilitation of tissue repair. Accordingly, intermission irradiation on the primary foci with high mechanical index (MI) ultrasound contrast can enhance ultrasound cavatition erosion and inhibit hepatic metastasis of colon cancer.Objective:In the study, based on an animal model of micrometastasis hepatoma, we employed high MI ultrasound contrast of intermission irradiation on primary foci to cause damage to tumor tissues and peripheral microvessels via cavatition erosion with accentruated microbubbles. In the meantime, the effect of high MI ultrasound contrast on locomotory movement, proliferation, apoptosis as well as on cystoskeletons of microfilament and microtube of carcinoma were investigated to further discuss if the high MI ultrasound contrast enhancing cavatition erosion can lead to the changes of the number and size of hepatic metastasis of colon cancers so as to explore a new way for the inhibition of hepatic metastasis of colon cancers.Methods:1.In vivo study of high MI ultrasound contrast in rats(1) Establishment of rat model of metastatic hepatic carcinoma: Lovo cells of colon cancer were cultured in DMEM medium containing 10% inactivated fetal bovine serum, when the cells at exponential phase of growth were selected for experimental study, which involved 62 male SD rats weighing 200-250g, of which 10 rats were injected with colon cancer Lovo cells via the spleen and sacrificed at days 2, 4 and 6 respectively to dissect the liver for observing the number and size of metastasis so as to determine optimal bombardment time.(2) Experimental groups: all rats were divided randomly into four groups, ie, control group, microbubbles plus ultrasound irradiation group, simple ultrasound group and simple microbubbles group, 12 rats in each group. At day 4 after inoculation with colon cancer Lovo cells, the rats were brought into the experiment. was given to depilate The abdomen of rats were depilated with 8% sodium sulfide and the they were anesthetized with the injection (40mg/kg) of 1% pentobarbitone sodium before operation. The rats were fixed in dorsal position, the high-frequency probe was used to find the spleen location and shifted to phased array (at the frequency of 1.5MHz, MI1.7, imaging depth of 4cm). The probe was put onto the spleen of SD rats. Ultrasound contrast SonoVue (1ml/kg) was injected quickly via caudal vein, followed by 1ml saline injection to wash the tube. Ultrasound radiation was performed 20 times with the intermision of 6 seconds.(3) The liver and spleen were processed with HE staining, histopathologic examination and transmission electron microscope to observe their ultrastructural changes.2.In vitro study of high MI ultrasound contrast in rats(1) Colon cancer Lovo cells were cultured in DMEM medium containing 10%inactivated newborn calf serum, then the cells at exponential phase of growth were selected for observing the refraction density and divided into four groups including control group, microbubbles plus ultrasound irradiation group, simple ultrasound group and simple microbubbles group, of which microbubbles plus ultrasound irradiation group was treated with type Vivid Five color Doppler, with wave frequency 1.5MHz, mechanical index 1.7,visualization depth 4cm,irradiation 6S,intermission 6S,total 20 times.(2) Transwell method and laser confocal technique were used to detect the cell migration ability and its effect on microfilament and microtube of the cells.(3) MTT method was used to observe the proliferative changes of carcinoma cells.(4) Flow cytometry was used to detect the cell cycle and the apoptotic change and observe the cell proportion at stages G1, S and G2/G1.Results:1.Results of high MI ultrasound contrast on hepatic metastasis of colon cancer:(1) In the 10 rats that were injected with colon cancer Lovo cells via the spleen and sacrificed and was dissected at days 2, 4 and 6 respectively, white nodosity metastasis with diameter of 1-2mm was observed at day 4 after inoculation, so it could be selected as the optimal exposure time. In the other 52 SD rats, 4 died of anesthetic accident, 41 were found with 1-5mm white nodosity metastasis shown in the dissection of the liver 14 days after inoculation, with success rate of 85.4%.(2) Ten days after treatment in different groups, the SD rats were sacrificed, with focal lyard micrometastases and leion for 2-5mm was found in the liver in control group.The tumor micrometastases for 1-2mm was found in microbubbles plus ultrasound wave irradiation group. Compared with control group, either the number or the size of carcinoma were decreased more significantly in microbubbles plus ultrasound wave irradiation group (P<0.01). There was insignificant statistical difference in aspects of number and size of carcinoma in ultrasound radiation group and simple microbubbles group in comparison with control group (P>0.05).(3) The rats in all groups were sacrificed at the 10th day and the tissues from the liver and spleen were removed for pathological examination. Tumor grew in the liver tissue, mostly round or ova in shape, and was distributed in mass. However, there could be seen remarkable decrease of tumor cells, massive hemorrhagic necrosis and thrombogenesis in the spleen of microbubbles plus ultrasound wave irradiation group. Meanwhile, the number of thrombus in the spleen tissue of microbubbles plus ultrasound was obviously more than that in the other Groups.(4) Ultrastructural changes of the spleen tissues shown by transmission electron microscope. In simple control group, tumor cells had large cellular nuclei, low ratio of kytoplasm to nucelus and more mitochondria with no swelling, while endothelial cells had regular structure with tight conjunction and normal mitochondria. In microbubbles plus ultrasound group, transmission electron microscope showed obvious damaged ultrastructure and that the tumor cells had swelled and globular mitochondria, which was different from simple control group. At the same time, endothelial cells were varied in morphous, with swelled mitochondria and disjunction. There was no significant difference among ultrasound radiation group and simple microbubbles group in comparison with simple control group.2.Effect of high mechanical index ultrasound contrast on proliferation, apoptosis and metastasis of colon carcinoma Lovo cells:(1) Millicell method showed marked metastasis of Lovo cells in control group. While after microbubbles plus ultrasound wave irradiation, Lovo cells were found with significant decrease of metastasis (P<0.01), with large quantity of float and adiaphanous dead cells. There was no statistical difference among metastasis number in simple ultrasound radiation and simple microbubbles compared with control group (P>0.05). The metastatic cells in control group, simple ultrasound and simple microbubbles group were more than those in microbubbles plus ultrasound group.(2) Laser confocal microscopic detection of expression of microfilamin F-actin in VSMC in each group showed that microtubule staining manifested compact network structure extending towards margins in control group; no statistical difference was found in simple ultrasound group and simple ultrasound microbubbles group compared with control group. Compared with control group, expression of microtubule was weaker and sparser, network structure aligning along cell macroaxis in microbubbles plus ultrasound group. In control group, the cell microfilament staining manifested as compact filament network structure protruding many trifling and short sentus ecphyma, with obvious drawing-off character and directivity. There was no statistical difference between simple ultrasound group plus simple ultrasound microbubbles group and control group. In microbubbles plus ultrasound group, there could be seen marked decrease of network-like cells with dull fluorescence and decrease of trifling and short sentus ecphyma in kytoplasm of Lovo cells.(3) The results of cell proliferation by MTT method showed that the cell proliferation in control group was more than that in microbubbles plus ultrasound group (P<0.01), but no statistical difference was found in aspect of cell proliferation between simple ultrasound group and simple microbubbles group compared with control group (P>0.05), indicating remarkable inhibitive effect on cell proliferation in microbubbles plus ultrasound group.(4) Results of flow cytometry detecting cell cycle and apoptosis of colon carcinoma Lovo cells in all groups showed that cell ratio was decreased markedly at phase G1 (P<0.01),but increased at phase S (P<0.01). Compared with control group, there was no statistical difference upon cell cycle in simple ultrasound group and simple microbubbles group (P>0.05). In comparison with control group, cell ratio at phase G1 was elevated (P<0.01), but decreased at phase S, and apoptosis peak was added in microvesicle plus ultrasound group (P<0.01). These results indicated that high mechanical index ultrasound contrast can bring about cell cycle change, induce apoptosis of colon carcinoma cells and that microbubbles agents could markedly accentruate the effect of ultrasound inducing apoptosis.Conclusions:(1) High MI ultrasound contrast can decrease the number and size of metastatic hepatic carcinoma and inhibit hepatic metastasis of cancer.(2) High MI ultrasound contrast can induce the massive apoptosis of tumor, thrombogenesis of interior and peripheral tissues and hence result in ultrastructural changes of the tumor cells.(3) High MI ultrasound contrast can inhibit the metastasis and proliferation of colon cancer Lovo cells and induce the changes of the cystoskeleton.(4) High MI ultrasound contrast can bring about the change of colon cancer Lovo cell cycle and induce the apoptosis but inhibit the proliferation of colon cancer Lovo cells.
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