The Targeted-directed Probe (Hab18-SPIO) And Its Experimental Study In The Nude Mice Model Grafted With Human HCC

Hepatocellular Cacinoma (HCC) has high incidence rate in East Asia and its prognosis is very poor. It is of importent therapy significance to detect and differentiate benign lesions from malignant ones. But routine MRI or contrast-enhanced MRI can't obtain specific diagnosis of early HCCs. With the development of molecular imaging , MR immuno-imaging (MRII) attracts learner's attention. Through antigen-antibody special binding, monoclonal antibody (McAb) could lead paramagnetic element to tumor and enhance the tumor specifically. The novel McAb-conjugated MR contrast agent could obtain localizational or tumor specific diagnoses and affirm if there is metastasis in remote, any remains or recurrence after operation.This study used HCC McAb (Hab18) conjugated SPIO to prepare immunoactive targeted probe (Habl8-SPIO) which made it possible to specific diagnose of early HCCs. Objective: 1. To prepare the Target-directed probe (Habl8-SPIO); To test itsphisichemical characteristics by HPLC and electron microscope.2. To establish the nude mice model grafted with human HCC and determine the expression of memorane-associated antigen Hab18g. The in vitro binding assay of Hab18-SPIO was carried out to evaluate the immunoreactivity.3. To investigate the enhanced MR features after intravenous injection of Habl8-SPIO and to study the value of this agent in specific diagnose of early HCCs.Methods:1. The McAb Target-directed probe (Hab188-SPIO) was prepared through HCC McAb(Hab18) conjugated to SPIO using chemical method referring to Dagmar Chokeman's method, while negative McAb probe (SED-SPIO) was also prepared using the same method. These agents were tested by HPLC and electron microscope.2. The FHCC-98 cell lines were cultivated, the nude mice model grafted with human HCC was also established and expression of membrane-associated antigen Habl8g was assayed at either cell or tissue level by immuno-histochemistry. The tumor tissue was observed histo-pathologically. Five samples (Hab18, Hab18-SPIO, SED-SPIO, SED, SPIO) were assayed by flow cytomrtry (FCM) in order to evaluate the immunoactivity respectively.3. The MRII of the nude mice model grafted with human HCC was performed. SPIO was intravenousely injected into every mouse of 4 groups to block reticuloendothial system(RES) of nude mouse. After 6 hours, all mice were anaesthetized, and probes were intravenously administrated espectively to mice at an iron dose of 10mg/kg. Group A: non-contrast enhanced; Group B: Habl 8-SPIO enhanded; Group C: SPIO enhanced. Group D: SED-SPIO enhanced. Mice inother 2 groups without blocking RES were injected contrast agent after anesthesia. Group E: SPIO enhanced. Group F: Hab18-SPIO enhanced. MR T2WI was performed for every mouse after intravenously administrated Oh, 6h, 12h and 24h. Results:1. The results of the HPLC and electron microscope proved that Hab18 was conjugated SPIO effectively. The diameters of particles were between 50 and 70 ran. The binding rate of Habl8 was 97.5%.2. The nude mice model grafted with human HCC was established successfully. Both cell line and tumor tissuehighly expressed the memorane-associated antigen, Hab18. The results of the binding assay of immunoactivity of Habl8-SPIO was 96.5% in vitro.3. After blocking RES of nude mouse, the tumors at 12h and 24h of Group B (Habl 8-SPIO enhanced) appeared marked hypointense on T2WI, While the signal of other groups (Group A, C, D, E, F) didn't change after injection of contrast agent respectively .Conclusion:1. The probe targeted to membrane-associated antigen of HCC (Hab18g) has been successfully prepared , which has novel physiochemical property and high level of immunoactivity.2. With RES blocked, Hab18-SPIO has a specific enhancement effect in nude mice model grafted with FHCC-98 in vivo, and proved to be a optimal MR probe. Habl 8-SPIO will be a kind of new noninvasive specific contrast agent in early-stage diagn...