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Objective:To evaluate the ability of vascular endothelial growth factor receptor 2(VEGFR2)/integrin α vβ 3 dual-targeted microubble (MBD) to target angiogenesis of renal cell carcinoma (RCC) in vivo. Methods:Non-targeted microbubble (MBN) USphere LA was employed as a template to prepare single- and dual-targeted microbubbles which could bind VEGFR2 and/or integrin α vβ 3 (MBV and MBI) by the biotin-avidin bridging method. A total of 40 RCC nude mice models were established by subcutaneously injecting 786-O cells.Twenty of the models were all injected with MBN, MBV, MBI and MBD in a random order, and the other 20 models were registered for antibody blocking assays. The results of ultrasound images were used for quantitative analyses, and the following quantitative parameters were obtained: intensity increment (a 1), peak halving speed (a 2), curve rising slope (a 3), perfusion time (t 0), time to peak (TTP), peak intensity (PI), mean transit time (MTT) and area under the curve (AUC) for the first three minutes, peak intensity at 10 s before (P 1) and after (P 2) ultrasound destruction, and the differences of tissue enhancement (dTE) between P 1 and P 2 (dTE=P 1-P 2). All the quantitative parameters of four contrast agents and the antibody blocking assays were compared.Besides, the immunohistochemical assays were performed to evaluate the expression of CD31, VEGFR2 and integrin α vβ 3 in tumor tissues. Results:The differences of parameters of a 1, a 3, t 0, TTP, PI and P 2 among four different microbubbles had no statistical significances (all P>0.05), and all parameters between the two single-targeted contrast agents were not statistically different (all P>0.05). The parameters of AUC, MTT, P 1 and dTE all showed a trend that dual-targeted bubbles > single-targeted bubbles > non-targeted bubbles (all P<0.05). On the contrary, the trend of dual-targeted bubbles < single-targeted bubbles < non-targeted bubbles (all P<0.05) was observed for a 2. In the antibody blocking experiment, a 2 was faster after the antibody injection ( P<0.001), while AUC, MTT, P 1 and dTE were all lower than those before the antibody injection ( P<0.001), and the other parameters were not statistically different before and after the antibody injection (all P>0.05). Immunohistochemical analyses confirmed the high expression of CD31, VEGFR2 and integrin β 3 in tumor tissues. Conclusions:The VEGFR2 and integrin α vβ 3 dual-targeted microbubble has a good potential to target the angiogenesis of human RCC in vivo.
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ObjectiveTo investigate the risk factors for rebleeding within 5 years and the influencing factors for 5-year survival in patients with liver cirrhosis and severe esophagogastric variceal bleeding (EVB). MethodsA retrospective analysis was performed for 129 patients with liver cirrhosis who attended Tianjin Third Central Hospital from May 2012 to May 2014 due to severe EVB for the first time, with a follow-up time of 5 years. Related clinical data were analyzed, including age, sex, cause of liver cirrhosis, presence or absence of infection at the first time of bleeding, liver stiffness measurement (LSM), splenic stiffness measurement (SSM), portal vein diameter, biochemical parameters, rebleeding time, and prognosis. Esophagogastric variceal rebleeding was defined as the primary endpoint and death was defined as the secondary endpoint. The t-test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups; a logistic regression analysis was used to investigate the independent risk factors for rebleeding, and a Cox regression analysis was used to analyze the predictive indicators for 5-year survival in EVB patients; the Kaplan-Meier curve was used to analyze the cumulative non-rebleeding rate. ResultsAmong the 129 patients, 87(67.4%) experienced rebleeding during follow-up. There were significant differences between the rebleeding group and the non-rebleeding group in the proportion of patients with alcoholic cirrhosis (χ2=4.896, P=0.027), portal vein diameter (t=2.203, P=0.030), LSM(Z=-2.771, P=0.006), and SSM(t=2.678, P=0.010). The patients with alcoholic cirrhosis had a significantly higher mean number of times of bleeding than those with non-alcoholic cirrhosis (all P＜0.05). The multivariate logistic regression analysis showed that alcoholic cirrhosis (odds ratio ［OR］=5.687, 95% confidence interval ［CI］: 1.230-26.129, P=0.025), LSM(OR=1.039, 95% CI: 1.010-1.070, P=0007), and SSM(OR=1.078, 95% CI: 1.028-1.129, P=0.001) were independent risk factors for rebleeding within 5 years after treatment in EVB patients. Among the 129 patients, 45 (34.9%) died. The univariate Cox regression analysis showed that there were significant differences between the death group and the survival group in age, times of bleeding, mean arterial pressure, portal vein diameter, aspartate aminotransferase, lymphocyte percentage, and presence or absence of infection at the first time of bleeding (all P＜005). Further multivariate analysis showed that 5-year survival rate was associated with portal vein diameter (OR=1.459, 95% CI: 1056-2.014, P=0.022), age (OR=1.053, 95% CI: 1.006-1.103, P=0.026), times of bleeding (OR=1.286, 95% CI: 1.040-1.591, P=0.020), and presence or absence of infection at the first time of bleeding (OR=5.239, 95% CI: 1.750-15.641, P=0.003). ConclusionAlcoholic cirrhosis, LSM, and SSM are independent risk factors for rebleeding within 5 years in EVB patients, and age, times of bleeding, portal vein diameter, and presence or absence of infection at the first time of bleeding are associated with 5-year survival.
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Objective To prepare the vascular endothelial grow th factor receptor 2 ( VEGFR2 )/Integrinαv β3 dual‐targeted contrast ultrasound agent ,and further evaluating the physical properties ,imaging characteristics and targeted ability in v itro . Methods VEGFR2 targeted microbubble ( MBV ) ,Integrinαv β3 targeted microbubble ( MBI) and VEGFR2/Integrinαv β3 dual targeted microbubble ( MBD) were prepared by attaching VEGFR2 antibody ,Integrinαv β3 antibody and both of VEGFR2/Integrinαv β3 antibody with no targeted USphere LA respectively , using biotin‐avidin linkage method . USphere LA with no antibody attached were used as non‐targeted microbubble( MBN ) . M icrobubble′s physical properties were observed , and its stability was detected by caculating bubble′s concentration at different time points . MBD′s ultrasound imaging characteristics were evaluated by comparing its grey level of ultrasound imaging with SonoVue at the time of preparation and 3 days after preparation . To detect the targeted ability of microbubble ,different types of microbubble were added into Hepa 1‐6 and C3H10 cells ,respectively . T he above procedure was repeated using the pre‐antibody blocking test in Hepa 1‐6 cell . Results MBD had a mean size of 1 256 nm .T he concentration of microbubble in the first duration of three days was declining slowly ,and its speed was accelerated after five days of preparation . T he grey level of new prepared MBD was similar to that of SonoVue in the same concentration ( P ＝0 .113) ,while the level was higher than that of SonoVue within 3 days after preparation( P ＜0 .001) . T he number of microbubble binding to Hepea1‐6 led a tendency of MBD＞ single targeted microbubble ＞ MBN ( all P ＜0 .05 ) . T he number of C3H10 and pre‐blocked Hepa1‐6 cell attached to each group of microbubble had no statistical difference ( all P ＞0 .05) . In addition ,following the pre‐blocked precedure ,the number of Hepa 1‐6 cell attached to each group of microbubble had no statistical difference either ( all P ＞ 0 .05 ) . Conclusions VEGFR2/Integrinαv β3 dual‐targeted contrast ultrasound agent is a stable microbubble and it has excellent ultrasound imaging and targeting ability in v itro .
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Objective To explore the value of contrast-enhanced ultrasound (CEUS) in improving the diagnosis ability for xanthogranulomatous cholecystitis ( XGC ) and wall-thickening gallbladder cancer ( GBC) . Methods Forth-three patients with XGCs and 31 patients with wall-thickening GBCs proved by pathology were enrolled in this study ,the features on conventional ultrasound and CEUS were recorded ,and the preliminary diagnosis before and after CEUS were given by doctors . Results Significant differences were found in continuous gallbladder inner wall and arterial blood supplement on conventional ultrasound , 58 .1％ (25/43) had continuous inner wall and 34 .9％ (15/43) had arterial blood flow in XGCs compared to 19 .4％ (6/31) and 100％ in GBCs . On CEUS ,72 .1％ (31/43) demonstrated continuous gallbladder inner wall and 48 .8％ (21/43) had hypoechoic nodules in the wall in XGCs compared to 16 .1％ (5/31) and 19 .4％ (6/31) in GBCs ,respectively ( P <0 .05) . No significant difference was found in intra-calcification , infiltration to adjacent organs ,gallbladder stones and fast-in and fast-out enhanced pattern( P >0 .05) . The area under ROC curve was improved from 0 .701 to 0 .899 after combining with CEUS ( P < 0 .05 ) . Conclusions Conventional ultrasound combining with CEUS could help acquiring more effective ultrasonic information and may improve the differential diagnosis ability of XGCs and GBCs .
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Objective To explore the value of contrast-enhanced ultrasound(CEUS) on the diagnosis of undetected liver cancer.Methods The study was focused on 85 consecutive patients from Jan 2015 to Sep 2016.The patients suspected liver cancer by clinical symptoms and other imaging studies were examined for regular gray-scale ultrasound and CEUS.Results Ninety-two lesions in 85 patients were confirmed by surgery,clinic manifestation and other imaging.All lesions including invisible or poorly invisible with greyscale ultrasound were examined by CEUS with the guideline of MRI imaging.Twenty lesions were detected by grey-scale ultrasound,89 lesions were detected by CEUS.The maximum diameter of those hepatic lesions was 20 mm × 18 mm,the minimum lesions was 8 mm × 6 mm.All the lesions were isoechoic,slightly hyperechoic or hypoechoic on grey-scale sonogram with mostly indistinctive margins.After SonoVue administration,82 lesions (92.1 ％) displayed as hyper-enhanced in arterial phase and 7 lesions (7.9 ％) as iso-enhanced.Sixty-one lesions(68.5 ％) showed hypo-enhanced and 28 lesions (31.5 ％) showed iso-enhanced in portal-vein phase.Seventy-four lesions(83.1 ％) showed hypo-enhancement and 15 lesions (16.9 ％) also showed iso-enhanced in late phase.The average enhancement time began at (15.3 ±6.1)s,reached to peak at (25.2 ± 8.8)s and decreased from (95.7 ± 57.8)s.Among 89 lesions,61 lesions (68.5％) were accordanced with fast in fast out and 28 lesions (31.5％) were no fast in fast out.The percentage of no fast in fast out on CEUS in undetected liver tumor was higher than in common liver tumor,the comparative difference was statistically significant (P ＜ 0.01).Conclusions Contrast-enhanced ultrasound has a more characteristic performance in undetected liver cancer and the higher detection rate than grey scale ultrasound.
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Objective To investigate the effect and molecular mechanism of botulinum neurotoxin serotype A (BoNT/A) heavy chain on neuron regeneration. Methods Cell culture, rats, immunofluorescence, SDS-PAGE and western blot, etc. were adopted in this study to explore the alterations of histone-3 acetylation (acetyl-H3 ) by local treatment of BoNT/A heavy chain to spinal cord injury (SCI) in rats (in vivo) or by adding it into cell culture (in vitro). Meanwhile, the relevance of acetyl-H3 to neurite out-growth based on SCI and cell culture with BoNT/A heavy chain application was approached as well. Results The application of BoNT/A heavy chain to cultured Neuro-2a cells increased the level of H3 acetylation. The increase of H3 acetylation was paralleled with the growth of neuritogenesis. Also, the neuronal treatment of BoNT/A heavy chain to SCI promoted the re-growth of neuronal processes surrounding the lesions. The growth of neuronal processes was positively correlated to the level of H3 acetylation. During the periods of BoNT/A heavy chain treatment in vivo or in vitro, the increase of H3 acetylation showed two peaks. Conclusions BoNT/A heavy chain increased the H3 acetylation, which might be one of its neuritogenic mechanisms.
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Objective To evaluate the value of contrast-enhanced ultrasound (CEUS) in preoperative classification for hilar cholangiocarcinoma. Methods Forty-six patients with 46 hilar cholangiocarcinoma were diagnosed by surgical pathology in Zhongshan Hospital of Fudan University from January 2007 to April 2013. The echogenicity difference on conventinal ultrasound and CEUS were compared with chi-square test. The accuracy of conventinal ultrasound and CEUS for evaluating invaded bile duct, detective rates for portal vein invasion and displaying rate of metastatic hilar lymph nodes were compared with chi-square test or Fisher’s Exact test according to the golden standard of operative exploration. Results On CEUS, 82.6%(38/46) and 91.3%(42/46) hilar cholangiocarcinoma were hypoechoic in portal vein phase and delayed phase respectively, while 63.0%(29/46) hilar cholangiocarcinoma were isoechoic on conventinal ultrasound with vague margin. The clearly displaying rates were 37.0%(17/46), 84.8%(39/46) and 91.3%(42/46) in conventinal ultrasound, portal vein and delayed phase of CEUS and the echogenicity was signiifcantly different. The evaluation accuracy of hilar cholangiocarcinoma invading bile duct was improved from 80.4%(37/46, conventinal ultrasound) to 100%(46/46, CEUS) significantly (χ2=7.882,P=0.005). Portal vein invasion were found in 9 cases during operative exploration and the detective rates on conventinal ultrasound and CEUS were 78%(7/9) and 89%(8/9) without signiifcant difference (P=1.000). Metastatic hilar lymph nodes were found in 8 cases and the displaying rates on conventinal ultrasound and CEUS were the same (75%, 6/8) without signiifcant difference (P=1.000). Conclusions CEUS could signiifcantly improve the clearly displaying rate of hilar cholangiocarcinoma and improve the evaluation accuracy for invaded bile duct comparing with conventinal ultrasound.
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ObjectiveTo investigate the diagnostic value of color Doppler ultrasound for peripheral intrahepatic cholangiocarcinoma (ICC).MethodsNinety- five patients with ICC which were confirmed histopathologically were retrospectively studied.The features on color Doppler ultrasound were analyzed.ResultsThere were 102 tumors which ranged from 20 mm× 20 mm to 130 mm× 100 mm in size.On gray-scale ultrasonic imaging,a large proportion of the tumors presented with inhomogeneous echo (n=75,73.5％), indefinite boundaries (n =70, 68.6％),and irregular shape (n=55,53.9％).On color Doppler ultrasonic imaging,most tumors had flow signals (75,73.5％) mainly in the shape of areatus,short- line and color-dot.Most flow signals had a resistant index greater than 0.6.Of the 102 tumors,thirty-seven (36.3％) were observed to produce streak hyperecho,twentynine (28.4％) slight attenuation in the rear and 22 (21.6％) unbilical depression.The coincidence rate with ultrasound was 84.2％(80/95). ConclusionsThe features of peripheral intrahepatic cholangiocarcinoma on color Doppler ultrasound are characteristic.To be able to appreciate these features is helpful to improve on the accuracy rate of ultrasound.
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Knockout is an important method for gene function study, while vector is the core of gene knockout. In order to obtain an effective vector for rapid construction of mutant and essentiality identification of the corresponding gene, we constructed a recombinant plasmid named plDM-T based on the temperature-sensitive and replication- defective plasmid plDM1 by inserting an Xcm I adapter into the EcoR I and Pst I sites of pIDM1. Digesting with Xcm I, pIDM-T can be prepared as a linear T-vector for PCR products cloning and be used to knockout the corresponding gene in the genome with insertion duplication mutagenesis. After the verification of temperature sensitivity of the replication of the plasmid, we cloned two Salmonella pullorum genes eno and ybdr into the constructed pIDM-T, and two recombinant plasmids pIDM-T_eno and pIDM-T_ybdr were identified. The recombinant plasmids were then transformed into S. pullorum strain CVCC527 and the IPC (Integration rate per cell) values were calculated. As a result, we identified the eno gene as an essential gene and the ybdr as a non-essential gene in CVCC527. We verified the correctness of recombination site in ybdr recombinant 527 clones (Sal delta ybdr) by PCR and sequencing. The pIDM-T vector can be used for gene knockout in S. pullorum, as well as the identification of essentiality of the corresponding genes, which offers an effective and rapid tool for gene function study in Salmonella.
Subject(s)
Cloning, Molecular , Gene Knockout Techniques , Genetic Vectors , Genetics , Metabolism , Plasmids , Genetics , Recombinant Proteins , Genetics , Metabolism , Recombination, Genetic , Salmonella , Classification , GeneticsABSTRACT
Objective To explore the potential value of contrast-enhanced ultrasonography (CEUS) in diagnosis of renal pelvic carcinoma. Methods The ultrasonogram of conventional ultrasound and CEUS were analyzed retrospectively in 22 patients of renal pelvic carcinoma proved pathologically. The size, echo, boundary and color flow signal of renal pelvic lesions were observed with conventional ultrasound. The enhancement modality and phase of tumors were also observed with CEUS, including wash-in and wash-out time, as well as the perfusion appearances. Results The maximum diameters ranged from 1.5 cm to 8.5 cm in 22 renal pelvic tumors. Conventional ultrasound detected flat mass in 7 tumors, irregular mass in 15 tumors; the same side hydronephrosis in 11 patients. Color Doppler flow imaging (CDFI) disclosed fairly rich flow signal of 5 tumors, a small amount of flow signal around tumor in 9 tumors and no-flow signal in the rest tumors. CEUS detected the cortical phase enhancement in all renal pelvic tumors, including synchronously enhancement in 8 tumors and delayed enhancement in 14 tumors. In peak time, hypoechogenicity compared to the normal renal cortex was showed in 18 tumors, hyperechogenicity in 3 tumors and isoechogenicity in 1 tumor. Fast wash-out in medulla phase was displayed in 20 tumors, isochronously wash-out in 1 tumor and delayed wash-out in 1 tumor. The diagnostic accuracy of the conventional ultrasound and CEUS was 63.64% (14/22) and 81.82% (18/22), respectively. Conclusion CEUS can depict blood flow supply and improve the diagnostic rate of renal pelvic carcinoma.