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© 2010 by the American Institute of Ultrasound in Medicine
J Ultrasound Med 29:61-70 • 0278-4297

Technical Advance

Antitumor Effect of Docetaxel-Loaded Lipid Microbubbles Combined With Ultrasound-Targeted Microbubble Activation on VX2 Rabbit Liver Tumors

Juan Kang, PhD, Xiaoling Wu, MD, Zhigang Wang, MD, Haitao Ran, MD, Chuanshan Xu, PhD, Jinfeng Wu, PhD, Zhaoxia Wang, PhD and Yong Zhang, MD

Department of Gastroenterology (J.K., X.W., J.W.) and Institute of Ultrasound Imaging (Zhi.W., H.R., C.X., Zha.W., Y.Z.), Second Affiliated Hospital, Chongqing Medical University, Chongqing, China.

Address correspondence to Xiaoling Wu, MD, or Zhigang Wang, MD, Institute of Ultrasound Imaging, Second Affiliated Hospital, Chongqing Medical University, 74 Linjiang Rd, Yuzhong District, 400010 Chongqing, China., E-mail: wxl-sj{at}163.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective. The purpose of the study was to explore the antitumor effect of docetaxel-loaded lipid microbubbles combined with ultrasound-targeted microbubble activation (UTMA) on VX2 rabbit liver tumors. Methods. Docetaxel-loaded lipid microbubbles were made by a mechanical vibration technique. VX2 liver tumor models were established in 90 rabbits, which were randomly divided into 6 groups, including control, docetaxal-loaded lipid microbubbles alone, docetaxal alone, docetaxal combined with ultrasound, pure lipid microbubbles combined with ultrasound, and docetaxel-loaded lipid microbubbles combined with ultrasound (DOC+MB/US). The tumor volume and inhibition rate (IR) of tumor growth were calculated and compared. Apoptosis was detected by terminal deoxyuridine nick end labeling. Proliferating cell nuclear antigen and matrix metalloproteinase 2 (MMP2) protein expression was detected by immunohistochemistry. Caspase 3 and MMP2 messenger RNA (mRNA) expression was detected by in situ hybridization histochemistry. The tumor metastasis rate and survival time of the animals were compared. Results. The IR and apoptotic index of the DOC+MB/US group were the highest among all groups, and the proliferating labeling index was the lowest. Matrix metalloproteinase 2 protein and mRNA expression in the DOC+MB/US group was the lowest among all groups, and caspase 3 mRNA expression in the DOC+MB/US group was the highest. The extensive metastasis rate in the DOC+MB/US group was the lowest, and the survival time of the animals in the DOC+MB/US group was the longest. Conclusions. Docetaxel-loaded lipid microbubbles combined with UTMA could inhibit the growth of VX2 rabbit liver tumors by deferring proliferation and promoting apoptosis, which may provide a novel targeted strategy for chemotherapy of liver carcinoma.

Key Words: docetaxel • drug-loaded microbubbles • local delivery • ultrasound contrast agents • VX2 liver tumor

Abbreviations: AI, apoptotic index • 60Co, cobalt 60 • CT, computed tomography • DOC, docetaxel alone • DOC+MB, docetaxel-loaded lipid microbubbles alone • DOC+MB/US, docetaxel-loaded lipid microbubbles combined with ultrasound • DOC+US, docetaxel combined with ultrasound • HCC, hepatocellular carcinoma • IR, inhibition rate • LI, labeling index • MB+US, pure lipid microbubbles combined with ultrasound • MMP2, matrix metalloproteinase 2 • mRNA, messenger RNA • PCNA, proliferating cell nuclear antigen • TV, tumor volume • UTMA, ultrasound-targeted microbubble activation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is the sixth most common tumor worldwide, but because of its poor prognosis, it ranks as the third most common cause of death from cancer.1 Less than 10% of patients survive 5 years after the diagnosis, and the median survival period is 4 to 6 months for patients with unresectable tumors. At present, surgical resection is the main treatment. Because only small percentages of these patients (10%–15%) are candidates for surgery, transcatheter arterial chemoembolization has been widely used for treating hepatic tumors, especially when the tumors are not surgically resectable.2,3 However, the therapeutic efficacy of transcatheter arterial chemoembolization in HCC is still limited, and the overall results of treatment remain unsatisfactory. Therefore, a new strategy for treatment of HCC is needed.

Docetaxel is an anticancer agent of the taxane class, clinical trials of which have been evaluated in a variety of cancers, including advanced unresectable metastatic gastric carcinoma and HCC; these studies have shown that docetaxel is more effective than paclitaxel.4–7 However, the water solubility of docetaxel is so poor that it is solubilized in polysorbate 80 (Tween 80) for clinical use, which usually causes adverse events such as hemolysis, anaphylaxis, and peripheral neuropathies8; therefore, clinical application of docetaxel is limited. Current studies show that paclitaxel liposomes are more effective and induce fewer toxic effects than paclitaxel used alone9–12; these studies suggest that loading docetaxel into lipid materials could avoid using a solvent in vivo and compensate for the poor water solubility and inconvenient clinical application of docetaxel.

Microbubbles, which are currently used as ultrasound contrast agents, sometimes possess shells composed of lipid materials and can be known as "lipid microbubbles" or "lipid ultrasound microbubbles."13 These are now being used as carriers of drugs and genes in treating ischemic heart diseases, skeletal muscle diseases, and ovarian carcinoma.14–17 This use led us to the idea that lipid ultrasound microbubbles could be used as drug delivery vehicles, and loading docetaxel into lipid ultrasound microbubbles may provide a new and safer formulation of docetaxel used in vivo.

Recent studies have shown that ultrasound-targeted microbubble activation (UTMA) is a non-invasive drug and gene transfer technology that provides a new method of therapy for diseases. Microbubbles might be activated, thus exerting mechanical stresses, which transiently perforate the cell membrane or disrupt the capillary wall to allow delivery of bioactive agents into cells or the interstitial space.18 To explore a new, safe, convenient, and targeted method for carcinoma chemotherapy, we investigated the possibility of docetaxel-loaded lipid microbubbles combined with UTMA to inhibit the growth of VX2 rabbit liver tumors by releasing the drug locally and enhancing its delivery to carcinoma tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Docetaxel-Loaded and Pure Lipid Microbubbles
Five milligrams of 1,2-distearoyl-sn-glycero-phosphatidylcholine (Sigma-Aldrich Corp, St Louis, MO), 2 mg of 1,2-dipalmi-toyl-sn-glycero-3-phosphoethanolamine (Sigma-Aldrich Corp), 1 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (Sigma-Aldrich Corp), 2 mg of docetaxel (United Pharmaceutical Co, Ltd, Chongqing, China), and 50 µL of glycerin were dissolved in phosphate-buffered saline to a final volume of 0.5 mL in 1.5-mL vials. The vials were incubated at 40°C for 30 minutes; after cooling, gas in the vials was replaced with perfluoropropane gas; and then the vials were mechanically shaken for 45 seconds in a dental amalgamator (YJT Medical Apparatuses and Instruments, Shanghai, China). This solution was diluted by phosphate-buffered saline and sterilized by cobalt 60 (60Co) irradiation. Pure lipid microbubbles were made by the same method as docetaxel-loaded lipid micro-bubbles without adding docetaxel and were also sterilized by 60Co irradiation.

Docetaxel-Loaded Lipid Microbubble Property Determination
The concentration of the docetaxel-loaded lipid microbubbles was calculated with a blood cell count plate; the size and zeta potential were measured with a laser instrument (3000 SSA; Malvern Instruments Inc, Westborough, MA); and the drug entrapment efficiency and drug-loading capacity were detected with a high-performance liquid chromatograph (LC-2010A; Shimadzu Corporation, Kyoto, Japan) and spectrophotometer (Spectrum Instruments Co, Ltd, Shanghai, China). These custom-made micro -bubbles had a concentration of 2.2 x 109 to 3.2 x 109/mL, a mean size of 623.1 nm, and a mean zeta potential ± SD of –3.1 ± 0.9 mV; the size distribution of 100% of the microbubbles was 473.4 to 706.6 nm; the drug entrapment efficiency was greater than 50%; and the drug-loading capacity was 17.5% ± 0.8%.19 The drug-loading capacity was calculated by the following equation: drug-loading capacity = amount of docetaxel loaded into the microbubbles/total amount of lipid material x 100%.20

Animal Preparation and Model Establishment
The experiment was approved ethically and scientifically by our university and complied with the Practice Guidelines for Laboratory Animals of China. Ninety rabbits (2–2.5 kg ) of both sexes were anesthetized with an intramuscular injection of sodium pentobarbital (30 mg/kg), and the abdomens were depilated with 10% sodium sulfide. Active tumor tissues were taken from rabbits inoculated with VX2 tumors (VX2 squamous carcinoma cell line; Funabashi Farm Co, Kyoto, Japan), washed with normal saline, and then subdivided into small tissue pieces of about 1 mm3. The rabbits were fixed and routinely disinfected; a median incision was made below the xiphoid to expose the left lobe of the liver, where a hole about 1 to 2 mm deep was made with ophthalmologic forceps; and then 1 prepared tissue piece was implanted into each hole. Bleeding points were stopped with a gelatin sponge; the abdominal wall was sutured; and the skin incision was disinfected. After tumor inoculation, the rabbits were prevented from infection by an intramuscular injection of penicillin, fed for 2 weeks, and then used in the study.

Experimental Protocols
The 90 rabbits were divided into 6 groups: control, docetaxel-loaded lipid microbubbles alone (DOC+MB), docetaxel alone (DOC), docetaxel combined with ultrasound (DOC+US), pure lipid microbubbles combined with ultrasound (MB+US), and docetaxel-loaded lipid microbubbles combined with ultrasound (DOC+MB/US). Tumors were detected by color Doppler imaging (iU22; Philips Healthcare, Best, the Netherlands) and computed tomography (CT; LightSpeed 16-layer spiral; GE Healthcare, Milwaukee, WI) to prove that the model was established successfully. An ultrasound treatment meter (Institute of Ultrasound Imaging, Chongqing Medical University) was used to help the microbubbles rupture and promote release of docetaxel locally in tumor tissues. Ultrasound irradiation was performed at a frequency of 300 KHz and an intensity of 2 W/cm2 with 10 seconds on followed by 10 seconds off, lasting a total of 6 minutes.21 In the control group, 6 mL of sodium chloride was injected via marginal ear veins; in the DOC+MB group, 2 mL of lipid microbubbles carrying about 2 mg of docetaxel was diluted to 6 mL and injected slowly; in the DOC group, 2 mg of docetaxel was injected; in the DOC+US group, 2 mg of docetaxel was injected, and ultrasound irradiation was applied; in the MB+US group, 3 mL of pure lipid microbubbles was diluted to 6 mL and injected, and ultrasound irradiation was applied in the same mode; and in the DOC+MB/US group, docetaxel-loaded microbubbles were injected in the same dose as for the DOC+MB group, and ultrasound irradiation was applied in the same mode. The treatments were performed 3 times separately on days 1, 4, and 7.

Tumor Observation Before Treatment
Two weeks after the inoculation, ultrasound imaging and CT were used to detect tumors in the rabbit livers and measure the longest and shortest axes of each tumor.

Specimen Handling and Detection After Treatment
Twenty-four hours after the last treatment, the longest axis (L) and shortest axis (S) of each tumor were measured by ultrasound imaging and CT. The tumor volume (TV) was calculated by the formula TV = (L x S2)/2, and the inhibition rate (IR) of tumor growth was calculated by the formula IR = (Tc – Tt)/Tc x 100%, where Tc represented the TV of the control group, and Tt represented the TV of the treatment group.20,22 Then, 10 rabbits in each group were killed, and liver carcinoma tissues were retrieved rapidly. The carcinoma tissues were washed in normal saline and put into 4% polyoxymethylene stationary liquid for detection of proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase 2 (MMP2) protein expression by immunohistochemistry, MMP2 and caspase 3 messenger RNA (mRNA) expression by in situ hybridization, and apoptosis by terminal deoxyuridine nick end labeling. The 5 rabbits left in each group were fed until they died spontaneously to compare the tumor metastasis rates and survival times of the animals.

Proliferating cell nuclear antigen protein expression, apoptosis expression, MMP2 protein expression, and MMP2 and caspase 3 mRNA expression were detected with a microscope (CKX41; Olympus America, Inc, Center Valley, PA). The positive cell count was semiquantitatively evaluated by counting the number of positive cells in 5 to 10 randomly chosen high-power (x400 magnification) fields. The observers were blinded to the identifications of the specimens. The proliferating labeling index (LI) and apoptotic index (AI) were calculated by the formula LI/AI = number of nuclei showing positive staining (brown)/total counted number of nuclei x 100%23; MMP2 protein expression and MMP2 and caspase 3 mRNA expression were mainly located in the cytoplasm. Positive cells were counted in at least 5 fields of each section, and the positive cell rate of the specimen was calculated the same way as for the LI and AI.

Statistical Analysis
Analysis of variance (SPSS version 13.0 statistical software; SPSS Inc, Chicago, IL) was used to assess the effects on proliferation, apoptosis, protein expression, mRNA expression, and survival time. Differences in tumor metastasis rates were compared by a {chi}2 test. P < .05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tumor Volume and IR
Two weeks after the inoculation, tumor models were established successfully in all rabbits. Liver imaging of rabbits could be enhanced obviously and persistently by the docetaxel-loaded lipid microbubbles. A "fast in and out" phenomenon was typical of VX2 carcinoma. The axes of the tumor measured by ultrasound imaging were coordinated with the axes measured by CT (Figures 1Go and 2Go). There was no significant difference in the TV among all groups before treatment, but after treatment, the TV of the DOC+MB/US group was significantly smaller than that of the other groups (P < .05). The IR of the DOC+MB/US group was the highest among all groups (Table 1Go).


Figure 1
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  		Figure 1. VX2 liver tumor detection and measurement with ultrasound imaging.

 

Figure 2
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  		Figure 2. VX2 liver tumor detection and measurement with CT. The same VX2 liver tumor as in Figure 1Go was detected and measured by CT. The results showed that the longest and shortest axes of this tumor measured by ultrasound imaging and CT were similar.

 

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  		Table 1. Mean TV, IR, AI, and LI in Each Group
 
Apoptosis Expression and AI
Positive staining was shown as brown staining in nuclei. The cells with positive staining were apoptotic. The results showed that apoptotic cells were observed in all groups. There was more tumor cell apoptosis in the DOC+MB/US group; the AI of the DOC+MB/US group was markedly higher than that of the other groups (P < .01; Table 1Go and Figures 3Go and 4Go).


Figure 3
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  		Figure 3. Apoptosis expression in VX2 liver carcinoma tissues from the DOC+MB/US group (original magnification x400).

 

Figure 4
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  		Figure 4. Apoptosis expression in VX2 liver carcinoma tissues from the DOC+US group (original magnification x400). Terminal deoxyuridine nick end-labeling results showed that cells stained brown in nuclei were apoptotic cells. There were more apoptotic cells in the VX2 liver carcinoma tissues of the DOC+MB/US group (Figure 3Go) than the DOC+US group.

 
Proliferating Cell Nuclear Antigen and LI Expression
Proliferating cell nuclear antigen expression usually appeared as brown granules in nuclei, and brown granules were observed in all groups. Proliferating cell nuclear antigen expression was obviously decreased in the DOC+MB/US group. The LI of the DOC+MB/US group was significantly lower than that of any other group (P < .01; Table 1Go and Figures 5Go and 6Go).


Figure 5
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  		Figure 5. Proliferating cell nuclear antigen expression in VX2 liver carcinoma tissues from the DOC+MB/US group (original magnification x400).

 

Figure 6
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  		Figure 6. Proliferating cell nuclear antigen expression in VX2 liver carcinoma tissues from the DOC+US group (original magnification x400). Immunohistochemical results showed that PCNA expression appeared as brown granules in nuclei. Proliferating cell nuclear antigen expression in the VX2 liver carcinoma tissues was obviously lower in the DOC+MB/US group (Figure 5Go) than the DOC+US group.

 
Matrix Metalloproteinase 2 Protein and mRNA Expression
Matrix metalloproteinase 2 protein and mRNA expression was shown as brown granules, and MMP2 expression was primarily detected in the cytoplasm and partial interstitial tissues. According to our findings, MMP2 expression was significantly downregulated in the DOC+MB/US group; it was lowest in that group (P < .01; Table 2Go and Figures 7Go and 8Go).


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  		Table 2. Matrix Metalloproteinase 2 Protein Expression and MMP2 and Caspase 3 mRNA Expression in Each Group
 

Figure 7
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  		Figure 7. Matrix metalloproteinase 2 mRNA expression in VX2 liver carcinoma tissues from the DOC+MB/US group (original magnification x400). could be inhibited to some extent in the DOC+MB, DOC, DOC+US, and MB+US groups, the inhibition effect in the DOC+MB/US group was the most significant, from which we could infer that the antitumor effect in the DOC+MB/US group was an intricate combination of many factors, including a cavitation effect, sonoporation, and an anti-cancer effect of the drug, and UTMA could improve the antitumor efficacy of the drug.

 

Figure 8
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  		Figure 8. Matrix metalloproteinase 2 mRNA expression in VX2 liver carcinoma tissues from the DOC+US group (original magnification x400). In situ hybridization results showed that MMP2 mRNA expression appeared as brown granules in the cytoplasm and partial interstitial tissue. Matrix metalloproteinase 2 mRNA expression in the VX2 liver carcinoma tissues was lower in the DOC+MB/US group (Figure 7Go) than the DOC+US group.

 
Caspase 3 mRNA Expression
Caspase 3 mRNA expression was mainly shown as brown staining in the cytoplasm and some nuclei. The results showed that caspase 3 mRNA expression was significantly upregulated in the DOC+MB/US group; it was obviously higher in that group than in the other groups (P < .01; Table 2Go and Figures 9Go and 10Go).


Figure 9
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  		Figure 9. Caspase 3 mRNA expression in VX2 liver carcinoma tissues from the DOC+MB/US group (original magnification x400).

 

Figure 10
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  		Figure 10. Caspase 3 mRNA expression in VX2 liver carcinoma tissues from the DOC+US group (original magnification x400). In situ hybridization results showed that caspase 3 mRNA expression appeared as brown staining in the cytoplasm and some nuclei. Caspase 3 mRNA expression in the VX2 liver carcinoma tissues was markedly higher in the DOC+MB/US group (Figure 9Go) than the DOC+US group.

 
Survival Time and Tumor Metastasis Rate
After treatment, the survival time of the DOC+MB/US group was obviously prolonged and was significantly longer than that of the other groups (P < .01). Interestingly, the abdominal cavity metastasis rate was 100% in all groups; however, the extensive metastasis rate of the DOC+MB/US group was the lowest among all groups (Table 3Go).


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  		Table 3. Survival Time and Tumor Metastasis Rate in Each Group
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Docetaxel is among the second-line chemotherapeutic drugs for preoperative and postoperative chemotherapy. It has higher antitumor efficacy than paclitaxel, and because docetaxel is fat soluble, this characteristic made it easy to dissolve with other lipid materials and loaded into lipid microbubble shells, so it was chosen to prepare the drug-loaded lipid microbubbles. In this study, docetaxel-loaded lipid microbubbles were prepared successfully; properties including the concentration, size, zeta potential, drug entrapment efficiency, and drug-loading capacity were determined; and primary stability experiments were performed, including comparison of the properties of docetaxel-loaded lipid microbubbles under different storage conditions before and after sterilizing with 60Co irradiation. The results showed that these custom-made microbubbles were more stable when stored at –20°C than at 4°C, and there was no notable difference in properties before and after sterilizing with 60Co irradiation19; however, further studies should be done to enhance the stability of docetaxel-loaded lipid microbubbles to maintain their properties and the antitumor activity of docetaxel. Perhaps we will try to change the suspension of docetaxel-loaded lipid microbubbles into a powder to make storage and application more convenient. In this study, no obvious change in the heart rate and respiration of the animals was detected after intravenous injection of the microbubbles, and no animal died of complications from microbubble injection. These studies showed that docetaxel-loaded lipid microbubbles combined with UTMA had antitumor advantages, and the custom-made microbubbles were safe for experimental animals; however, in future studies, more safety experiments should be done to evaluate the safety of docetaxel-loaded lipid microbubbles in vivo.

Ultrasound-targeted microbubble activation plays an ever-increasing role in enhancing some therapeutic agents’ delivery into certain important tissues, such as the myocardium, tumors, and even pancreatic islets.15,24,25 The major mechanism of the phenomenon is primarily considered a physical effect called acoustic cavitation, which can promote the rupture of microbubbles. In this study, an ultrasound treatment meter was used to enforce microbubble activation locally in tumor tissues; the ultrasound beam was not focused; the frequency of the treatment meter was fixed at 300 KHz; and the intensity was set at 2 W/cm2. Intermittent irradiation was used because of the phenomenon that quite a number of microbubbles would burst under insonation, so the interval between instances of irradiation would allow reaccumulation of microbubbles to a sufficient concentration at the tumor site. In this mode, many more microbubbles could be destroyed by ultrasound irradiation.

In our study, the tumor growth state was estimated comprehensively by the TV, IR, apoptosis expression, and PCNA expression. The results showed that docetaxel-loaded lipid microbubbles combined with UTMA could inhibit proliferation and promote apoptosis of liver tumors. Although the results showed that tumor growth

As a kind of microtubule depolymerization inhibitor (tubulin stabilizer), docetaxel can inhibit proliferation of tumor cells and angiogenesis by inducing mitotic arrest and apoptosis.26,27 Current studies show that the caspase family plays an important role in mediating cell apoptosis. Caspase 3 is a key factor in this family, and MMP2 is closely associated with tumor invasion and metastasis.28,29 In this study, caspase 3 mRNA expression was highest in the DOC+MB/US group, and MMP2 protein and mRNA expression was lowest among all groups, which hinted that caspase 3 was possibly the central factor in inducing apoptosis, and MMP2 might have been the key factor in tumor metastasis in our experiment; however, a more detailed study should be done in the future to clarify the specific mechanism of apoptosis induced by docetaxel-loaded lipid microbubbles combined with UTMA.

In this study, we found that the extensive metastasis rate was lowest in the DOC+MB/US group. This result showed that docetaxel-loaded lipid microbubbles combined with UTMA might have advantages in preventing extensive metastasis of VX2 rabbit liver tumors, including lung, brain, kidney, and bone metastasis; however, abdominal cavity metastasis, including abdominal wall, mesentery lymph node, and colon metastasis, was found in all groups. We thought that the abdominal cavity metastasis rate was possibly influenced by several factors in our experiment: for instance, the VX2 tumor tissues were superficially implanted about 1 to 2 mm deep, which made the tumor cells prone to plant in other organs in the abdominal cavity.

Current tumor chemotherapy faces the dilemma of a desire to maximize the cytotoxic drug efficacy to kill most tumor cells while minimizing side effects; therefore, targeted therapy, which could improve curative effects and reduce side effects, has attracted more and more attention. Because it is difficult to perform targeted therapy in vivo, however, it has become a controversial and troublesome issue in cancer therapy studies.30 As ultrasound contrast agents, microbubbles have been used for carrying drugs and genes, and activation of ultrasound-targeted microbubbles containing drugs or genes can release the drugs or genes in target tissues. After intravenous injection, microbubbles can arrive at the target tissues and can be destroyed by a certain ultrasound intensity in vivo, and the consequent mechanical effects and cavitation could increase the membrane permeability, causing rupture of microvessels (diameter <7 µm) and widening of endothelial cell gaps31; the drugs or genes can then get into the tissue cells through the ruptured microvessels and endothelial cell gaps. Ultrasound-targeted microbubble activation can help drug-loaded microbubbles rupture and promote drug release and absorption in targeted tissues. For these reasons, drug-loaded microbubbles combined with UTMA may provide a novel approach to targeted chemotherapy.

In the study, we used an ultrasound contrast agent as a new type of chemotherapy drug delivery vector, and the antitumor efficacy of docetaxel was markedly improved. In conclusion, the combination of docetaxel-loaded lipid microbubbles and UTMA is expected to become a new drug delivery means and may provide a novel strategy for targeted chemotherapy.


   Footnotes
 
We thank Chongyan Li for assistance with model establishment and Lianju Ma for help. This work was supported by grant 30430230 from the Key Program of the Natural Science Foundation of China and grant 2006AA02Z4FO from the Hi-Tech Research and Development Program of China.

Received April 4, 2009, from the Department of Gastroenterology (J.K., X.W., J.W.) and Institute of Ultrasound Imaging (Zhi.W., H.R., C.X., Zha.W., Y.Z.), Second Affiliated Hospital, Chongqing Medical University, Chongqing, China. Revision requested May 4, 2009. Revised manuscript accepted for publication August 10, 2009.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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