Lac-HSA-SPIO Enhanced Magnetic Resonance Receptor Imaging: An Experiment Study On Micro-hepatocellular Carcinoma

【objective】1.Collecting different fresh human tissues,including normal liver tissue,hepatocyte carcinoma,cholangiocellular carcinoma,haemangioma,focal nodular hyperplasia,incubated with SPIO and lactosmited human serum albumin coated SPIO(Lac-HSA-SPIO),respectively to evaluates the receptor binding capacity of Lac-HSA-SPIO and the possibility of distinguish analyse of different liver pathological changes.2.To explore primarily values of Lac-HSA-SPIO as receptor directed contrast agents for MR imaging in the rat model chemically induced by DENA and implantion VX₂ tumor.【Materials and Methods】1.In vitro to assays special distribution of Lac-HSA-SPIO as ASG receptor-directed agents in different fresh human tissue(1) Collecting different fresh human tissues:A total of 20 specimens were colleted from surgical specimens,included normal liver tissue(n=4),hepatocyte carcinoma(n=7),cholangiocellular carcinoma(n=2),haemangioma(n=2),focal nodular hyperplasia(n=5).Put specimen into -80℃refrigeratory after operation.(2)Distribution of ASG receptor in different fresh human tissues:All tissues specimens were cut into 2-4μm sections(-20℃).The sections were then thawed,air dried,and incubated for 20 seconds(37℃) with Lac-HSA-SPIO,SPIO,or buffer solution.After incubation,slides were rinsed with saline solution to remove unbound iron.Slides were then dried 24 hours at room temperature.Subsequent counterstaining for iron was performed with Perls Prussian blue stain.2.Application of Lac-HSA-SPIO in the rat model induced by DENA and VX₂ tumor.(1)In this study,25 Wistar male rats were feed freely Diethylnitrosamine(DENA, American sigma company,0.95g/ml) solution being diluted to 100ppm.After twelve weeks,the water without DENA was feed to these rats.Model of VX₂ tumor in rats:Tumor tissue with active growth characters looked like fish was obtained from the border of VX₂ tumor in breeder-rabbit.Tissue was added some physiological saline,cut into 0.5-1 mm³ with ophthalmic scissor.The rats were anaesthetized and paunched exposed the liver.One lobe of the liver was pulled lightly outside,and pierced by ophthalmic forceps.One to two pieces of tumor were put into the pierced hole.2)Animal group:14 big rats mode induced by VX₂ tumor were randomly divided into two groups of SPIO and Lac-HSA-SPIO,and 7 were of each group.12 big rats mode induced by DENA were randomly divided into two groups of SPIO and Lac-HSA-SPIO,and 7 were of each group.3) MRI scaning:MR imaging was performed with 3.0T with a section thickness of 3mm,and a field of view of 16×16cm.Pulse sequences were used as below: Tl-weighted spin echo(SE)(TR/TE:500 ms/17 ms),FGR sequence/30(Fast GRASS)(TR/TE:260 s/2.9 ms),T2-weighted fast spin echo(TR/TE:4500 ms/152 ms).T2 -weighted fast recovery fast spin echo-accelerated(FRFSE) sequence(TR 3600 ms,TE 85.8 ms),T2 -weighted FGR(TR 600 ms,TE 15 ms).Transversal,coronal and sagittal multisections images were obtained.Two kinds of SPIO models,after fifteen minutes SPIO administration(0.05mmol/kg) was injected,MR imaging was performed.Two kinds of Lac-HSA-SPIO models,After thirty minutes Lac-HSA-SPIO administration(0.05mmol/kg) was injected,MR imaging was performed.4) Observation and measure of images;Three MR doctors read MR images. Maximal diameters of lesions were measured in MR images.Average signal intensity(SI) over region-of-interest(ROI) drawn on lesions and adjacent hepatic parenchyma were measured on MR images.Background noise(N) was measured in each image and ROIs were placed adjacent background outside abdomen and ecoding direction was the same to the lesions.Signal to noise ratio(SNR),contrast to noise ratio(CNR) of lesions and liver were calculated on all images.(5)Statistical analysis:Statistical analysis was performed by using statistical package for social sciences(SPSS 12.0).The Independent-Samples T Test and Independent-Samples T Test were used.A P value of 0.05 or less was considered to indicate a significant difference.【Result】1.After incubation with receptor agent(SPIO),no Such blue iron deposits were seen in hepatocyte tissue.After incubation with receptor agent(Lac-HSA-SPIO), many blue iron deposits were seen in hepatocyte of normal liver tissue.Some blue iron deposits were seen in hepatocyte cytoplast in high-grade cirrhosis,but less than normal human liver tissue.No blue iron deposits were seen in cholangiocarcinoma tissue and hemangioma post-incubation.Many blue iron deposits were seen in focal nodular hyperplasia post-incubation.2.MR imaging of tumor in two big rat models(1) VX₂ tumor models:14 of 15 models of VX₂ tumor in big rats were successfully.A total of 21 tumors were detected by MR imaging.9 of then with SPIO(9 with anatomy) and 12 of them with Lac-HSA-SPIO(12 with anatomy).12 of 25 models of big rat model induced by DENA were successfully.A total of 13 tumors were detected by MR imaging.6 of then with SPIO(9 with anatomy) and 7 of them with Lac-HSA-SPIO(9 with anatomy).The minimal and maximal diameter was separately 0.2cm and 1.5cm.(2)Pathologic findingsStained by Hematoxylin-Eosin,VX2 tumor was composed mainly of squamous cell carcinoma and few of adenocarcinoma.There were seen fibrous tissues proliferation around some tumor,looked like some capsular.The hepatic structure of chemical hepatocarcinogenesis was abnormal and loose and tumor cells were obviously larger and were heterotype,and basophil of karyon was increased and had multiple or heterotypic nuclear.In HCC tissues,blood sinusoids were ecstatic and increased on cirrhotic HE staining slices.Of HCC,the structure was destroyed and the size of hepatic cells was slightly larger and some were swelling,and connective tissues increased and many inflammatory cells were in portal area.(3) SNR,CNR measurementsVX₂ tumor models:By pulse sequence of T2WI FSE,SNR of tumor post-enhanced with SPIO showed significant difference with pre-enhanced(P=0.012).By pulse sequence of T2WI FRFSE and FGR,SNR of tumor post-enhanced with Lac-HSA-SPIO showed no significant difference with pre-enhanced(P＞0.05). Tumor-liver CNR post-contrast with SPIO and Lac-HSA-SPIO showed significant differences with pre-enhanced(P＜0.05),and the difference of pre-contrast and post-contrast showed also significant differences between Lac-HSA-SPIO and SPIO(P＜0.05).DENA models:On pulse sequence of T2WIFGR,SNR of tumor after SPIO showed significant difference with that of pre-enhancement(P＜0.05).On pulse sequence of T2WI FSE,SNR of tumor after administration Lac-HSA-SPIO showed no significant difference with pre-enhanced(P＜0.05).On pulse sequence of T2WI FSE and FRFSE,SNR of tumor post-enhanced with SPIO showed no significant difference with pre-enhanced(P＞0.05).On pulse sequence of T2WI FSE, FRFSE,and FGR,SNR of tumor post-enhanced with Lac-HSA-SPIO showed no significant difference with pre-enhanced(P＞0.05).After injection of Lac-HSA-SPIO and SPIO,the CNR improved greatly compared with those plain scans respectively.The increase of CNR for Lac-HSA-SPIO was significantly different with conventional SPIO.(P＜0.05).【Conclusions】1.Our results in vitro assay demonstrated that ASG-receptor-directed agent Lac-HSA-SPIO on bind in normal human liver tissue,regeneraling nodules. Hepatocarcinoma did not show ASG-receptor activity.While primary and secondary malignant liber tumors showed on binding of ASG-receptor directed agent.which indicated that Lac-HSA-SPIO may be helpful in differentiation between malignant and benign tumors.2.Afre intra venous administration of Lac-HSA-SPIO decreased markedly T2 relaxation time of normal liver then caused brought a considerable negative enhancement effect in liver.3.Lac-HSA-SPIO improved obviously tumor-liver CNR,and which is better than that of SPIO showed significant differences.Lac-HSA-SPIO was with a better potential value than SPIO for detecte micro-hepatic lesions.