| Background: Learn along with the CT, MRI etc. image to progress unremittingly technically, the diagnosis rate of the liver tumor raises continuously, the CT and MRIs can check small cancer of the liver of 1~ of 2 cms or so , but to the smaller focus then the inability is a dint. the laboratory check such as AFP etc.contributes to the earlier period diagnosis of the cancer of the liver, but can't accurate fixed position focus. So the clinic need to be a kind of urgently up since can examine a patient in early days, again can accurate fixed position focus of simple, the diagnosis method of the easy line.It is the most in common use and convenient diagnosis technique of clinic that ultrasound developing, along with such as second harmony and intermittent imaging of ultrasound etc., and the fluorine carbon microbubble agents further mature, contrast ultrasound imaging to enter the clinical application gradually from the experiment, providing to meet the new diagnosis method of above-mentioned request for the clinic, may cause new revolution of the diagnosis technique last time. Objective: The angiginosis is the tumor development, gradually, transfer of key, this research was performed firstly from integrin that is one of the most important intercellular adhesion molecule,and use targeted microbubbles imaging to raise the liver tumor check rate ,and for the clinic in early days cured the tumor provide the basis, to raise treatment effect of the liver malignant tumor .At the same time applied commercial production 'sonovue'to patients and to lean its characteristics for diagnosis and tumor monitor and evaluating to cure the tumor. After diagnosis , it may be combine cure and cure monitor right away and raise to cure effects. Methords: Physiochemical properties: Press certain comparison to mix distearoylphosphatidylcholine(DSPC) and dipalmitoylphosphatidylglycero(DPPG)and Poly ethylene glycol(PEG) to hang in the aqueous solution,.After the frozen aridity, join the live of the sugar aqua and surfaces, shake even, re-make in to mix to hang the liquid, take out in addition to the air inside the container after, join the eighth fluorine propane air( C3F8), gain the outcome to calm down to place the layering to leave pure liquid, then make into the tiny bubble needed. The microbubbles size and distribution were analyzed by Olympus BX50 optical microscope using the magnitude of ×100 and ×400. The microbubbles concentration was detected by a Sysmex KX-21 blood cell multisizer. The PH value and Zeta potential were assessed by Zeta SIZER 3000 and the viscosity was analyzed by a viscosimeter. Conjugation of antibody to microbubbles: The microbubble manufacture complete before, join carbodiimide 1.5mg, mix after assemble the material make into mix to hang the liquid together with the microbubble evenly, join the sum to settle antibodies of the anti-αvβthat the density takes to have the red fluorescence in microbubble immediately after, draw out the air in the container as far as possible, join the eighth fluorine propane air( C3F8). Flapping 45s , the products are the targeted microbubbles ,4 degrees refrigerator stay overnight.Observe fluorescence that adhere to the microbubble surface with the fluorescence microscope, observe binding microbubble with antibody. Vitro Conjugation Testing of Targeted Microbubbles: K1735 M2 cell and K1735 C23 cell with melanin tumor cells strain stably expressing αvβ3-integrins were selected to be cultured for passing on generation. When culture of anabiosis cells became stabilized, a slide was put into orifice plate to conduct cell culturing. In bloom period of cell growth, cells were washed for twice to thrice using PBS, thus to allow culture solution to be washed sufficiently. Then 0.4% paraformaldehyde was added to enable cells to be immerged completely into paraformaldehyde solution at room temperature for 2 h. After removal of paraformaldehyde solution, PBS solution was added to orifice plate 96 to the extent that it was almost full. Then targeted antibody 0.1 ml was added, with slide grown with cells being covered reversely, making targeted microbubbles and cell react thoroughly. Later it was stored at 4C°overnight, and observed using optical microscope next day. Targeted microbubbles were washed for two times using PBSto elute free microbubbles and antibody. With PBS as wash liquid, they were washed utilizing infusion pump at speed of 0.1ml/min, 0.2ml/min, 0.5ml/min, 1ml/min, 2ml/min and 5ml/min till 10ml/min, to be observed shortly to obtain number of microbubbles conjoined to surface of tumor cells, thereby to get aware of conjugation number and degree of combining power of targeted microbubbles and drone. Vivo Use-Testing of Targeted Microbubbles First sodium alginate slow-released microcapsules carrying bFGF were prepared, to observe rebirth endovascular characteristics of targeted microbubbles stimulated by bFGF so as to get preliminary information on probability of targeted microbubbles use in vivo. During test, homotypic control and targeted microbubbles were administered into peripheral vein of rat respectively, with 3 mice in microbubbles group and 2 in control group. 15 minutes later, same dosage of normal saline was injected into peripheral vein of rat to wash non-conjunct microbubbles in its blood vessel. At 30 minutes, rat's scrotum tissues with rebirth blood vessel were cut off, being fixed with 4% paraformaldehyde for 30 minutes, and observed under laser focusing microscope. Vivo Test of Melanin Tumor Model: Setting up Melanin Tumor Model: The research was divided into 3 groups, common microbubbles group, homotypic control group and targeted microbubbles group. During the experiment, same concentration of targeted contrast agent was used in each group, weighing 0.02ml/Kg and, before the experiment, to be checked using optical microscope and fluorescence microscope to get aware of microbubble quality and binding of antibody. Microbubble diameter of targeted Zhifuxian contrast agent ranged 3～10μm,98% of them below 8μm, and concentration 7×109/ml. Microbubbles were infused into caudal vein of rat or using intravenous intubation injection. Diagnosing and Monitoring Primary Carcinoma of Liver Using Commercialized Lipid Contrast Agent: Collection of patients who had been diagnosed as cancer after image and clinic examination and treated with RF in Chongqing Southwest Hepatic Duct Surgery Hospital in year 2004. Diagnosis standard of liver cancer involves: 1. Primary carcinoma of liverdiagnosed by histological examination; 2. Those with clinical manifestation of liver cancer, and 3 items among isotope examination, ultrasound visualization, CT check-up, hepatic arteriography, X-ray diaphragm symptom, enzymology examination (mainly ALP and Y-GT) were identified as affirmed positive and with secondary carcinoma of liver and benign tumor excluded; 3. Over one month continuance of AFP≥500ng/ml or over 2 months AFP≥200ng/ml, eliminating other factors that result in rise of AFP. Standard of Inclusion: 1. Number of tumor is smaller than 3, diameter less than 5 cm; 2. Patients of primary carcinoma of liver who are unsuitable or unwilling to resect tumor by way of operation. Standard of Exclusion: 1. Pulmonary hypertension; 2. Extremely bad result of contrast; 3. Diffuse liver cancer; 4. Allergic constitution; 5. Late patient with widespread metastasis; 6. Patients whom we failed to visit and those who were dead. Routine ultrasonic, contrast enhanced ultrasound and CT scanning were used to check candidate patients before and after RF. After RF contrast enhanced ultrasound would at least be carried out 12 hours after RF. In the meantime, CT scanning was conducted for shadiness focus to observe it in arterial phase, portal phase and balance phase, with image data kept. Routine needle biopsy of liver was carried out for all patients listed into research; Needle biopsy of liver was repeated for postoperative shadiness patients. Results: 1.Granular diameter of Zhifuxian ultrasound contrast agent microbubbles ranged 3~10 μm, with 98% of them below 8μm, mainly 4 μm; and concentration of microbubbles was around 7×109/ml (associated with amount of remaining clear solution); PH value was 6.42; after normal saline was diluted by 25 times, viscosity of contrast agent detected by way of capillary method was 0.75; surface electric potential of microbubbles in normal saline was up to -35.5±4.0 mv. 2.Targeted antibodies with different concentrations were conjugated to microbubbles, and flow-type cell counter was used to detect amount of antibody conjugated to microbubble surface, indicating there was a given relationship between conjugation of antibody concentration and microbubbles, and targeting antibody conjugation would be more sufficient when over 45ug antibody was added. 3.After targeted microbubbles with most proper concentration antibody (antibody75ug/ml microbubbles) were conjugated to high-expressed integrins and low-expressed integrins tumor cells, it was found that conjugation of high-expressed melanin tumor cells to targeted microbubbles was obviously stronger than low-expressed microbubbles, and there was no conjugation with common microbubbles. 4.When liquid flows with different scouring powers were utilized to wash vitro targeting conjugated carbodiimide microbubbles, it was found that there still existed conjugation at flow velocity of 2ml/min. Result of preliminary test indicated this conjugation was capable of withstanding a given liquid shearing force. 5.When C3H rat aged 6 weeks was selected to carry out experiment, it was difficult to inject ultrasound contrast agent into blood vessel of the rat. For this, duct was first put into internal jugular vein of rat to conduct test after it was well fixed. After repeated improvement, good result was also achieved when caudal vein of rat was selected as main vein for injection. Therefore it was feasible that intravenous injection of rat caudal vein allowed rat tumor to be model of tumor ultrasound contrast. 6.After bFGF treatment, vascular hyperplasia and increased vascular density in rat scrotum were visible to the naked eye. After bFGF treated micrangium of rat was administered with targeting αvβ3 antibody microbubbles and homotypic control antibody microbubbles with secondary antibodies excited red fluorescence, it was found that not a few of microbubbles with fluorescence existed in targeted microbubbles group, exhibiting notably enhanced brightness of vein. But lesser microbubbles were found in vein injected with homotypic control microbubbles. 7.Around 5 seconds after ultrasound contrast agent was infused via peripheral vein into rat, the tumor began to develop. The contrast agent lasted a relatively longer time in vein, and from 15 minutes to 30 minutes development of tumor tissues was stronger than that of peripheral tissues. During this time, 3 contrast agents were all continuously enhanced developments and, after 30 minutes, control common and homotypic control contrast agents slacked up gradually in tumor tissues, thus tumor was gradually changed to lower density focus. Moreover, targeted contrast agent remained remarkably strengthened, which slacked up gradually at 60 minutes. 8.Comparison of 3 diagnosis methods(CT, BUS and CBUS)before RF melting was made based on common index of diagnosis test with pathological result as standard.Notable difference existed between pathological and preoperative contrast result χ2=5.82,P=0.012. No difference existed between pathological and preoperative CT result χ2=2.25,P=0.146; and no difference existed between pathological and BUS result χ2=0.22,P=0.815. Positive likelihood ratio of ultrasound contrast and CT examination was 1.26 and 1.31 respectively, and sensitivity and specificity being 96.88% & 90% and 21.43% & 25%, respectively. Moreover, gray scale ultrasound positive likelihood ratio was 0.99, and sensitivity and specificity being 75.76 and 23.07 respectively, slightly worse than the former two. It can be seen from this experiment that ultrasound development has relatively high value to diagnosis of primary carcinoma of liver. Although no notable difference exists between CT examination and pathological result, CT examination for the moment is one of most approbatory methods for diagnosing primary carcinoma of liver, and both positive likelihood ratios are almost identical. Accordingly it can be held that value of two methods, ultrasound contrast and CT examination, to diagnose primary carcinoma of liver in this study, is basically the same. 9.As CT at present is recognized as method to evaluate tumor growth, comparison is made between after RF therapy ultrasound contrast result and postoperative CT result which is regarded as standard, showing that sensitivity and specificity of postoperative ultrasound contrast are 60% and 90.2% respectively, positive likelihood ratio being 6.18, and negative likelihood ratio 0.443. And notable difference exists in χ2=5.82,P=0.012. Conclusion: 1.Formation and physiochemical properties of prepared lipid ultrasound contrast agent "Zhifuxian"are physical basis of its stability, whose size and stability allow it to be intravenous contrast agent. 2.Carbodiimide is used as coupling agent to prepare successful targeted lipid contrast agent. Observation shows targeted microbubbles possess relatively high stability. 3.It is found from observation of vitro targeting function of targeted microbubbles that targeted microbubbles and corresponding ligands conjugate tightly, which can withstand a given scouring action of water flow and be able to confront shearing force of internal blood flow. 4.Increased congregation of targeted microbubbles in rebirth blood vessel indicates feasibility from vivo targeting to local zone, and targeted ultrasound microbubbles withαvβ3 monoclonal antibody possess a given targeting capability. 5.The finding that αvβ3 monoclonal antibody targeted ultrasound microbubbles aiming to tumor intravascular Integrins last longer time in animal than common microbubbles plays a role of guidance to diagnosis. 6.Ultrasound contrast can be used as routine monitoring tools for diagnosis of primary carcinoma of liver and after RF melting operation.
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