| ObjectiveWith the progress in molecular biology, highly sensitive PT-PCR has been used in detecting micrometastasis of solid tumors over recent years. Messenger RNAs (mRNAs) of AFP and albumin are common markers for detecting the micrometastasis of hepatocellular carcinoma. However, since normal hepatic cells can also express AFPmRNA and albumin mRNA, the significance of these two markers is controversial in detecting the micrometastasis. Additionally, because genetic heterogeneity is present among tumor cells and the gene expression may vary from the primary lesions to the metastatic lesions, false negative results are likely to occur when micrometastasis is detected using a single marker. GPC3 is a highly cell specificly gene of hepatocarcinoma extracted in last years, the expression rate of hepatocarcinoma constitution is notable step up, there is no expression in non-carcinoma and para-carcinoma constitution,it is possible that GPC3 will become a new ideal molecular marker of hepatocarcinoma for its peculiarit. In light of these problems mentioned above, we postulate that combined detection using GPC3 and AFP multiple markers will help increase the sensitivity and specificity of detecting the micrometastasis.In this study we detected peripheral blood GPC3mRNA and AFPmRNA in rat HCC model and investigated the relationship between the results and micrometastasis of HCC in rat to provide a experimental basis for early detection of HCC. We further assessed the feasibility of detecting the micrometastasis using multiple markers, investigated the changes of AFPmRNA and GPC3mRNA in the disease course of HCC in rat and their relations to the micrometastasis.MethodsThe animals were divided into the experimental group and the control group. Rat HCC model was established using diethylnitrosamine. A total of 120 Wistar rats ( 102 in the experimental group and 18 in the control group ) were included and their body weight was 150 ± 20g. 2 %a diethylnitrosamine solution was administered through intubation in oropharynx, 0.9 ml daily and for successive five days in a week. From the 13th week on, the drug was administered for a succession of 3 d and discontinued since the 18th week. Rats in the control group were fed with isovolumic saline. From the 10th week on, rats were decapitated and 10ml of peripheral blood were collected and anticoagulated with heparin at different time points. The specimens included: single nodule of HCC, 25 cases; HCC with 2-5 nodules, 20; HCC with more than 5 nodules, 18; HCC with obvious metastatic lesions, 12. The controls were healthy rats. Total RNA was extracted from peripheral blood. Expression of GPC3mRNA and AFPmRNA in peripheral blood was detected using nested PCR following reverse transcription.Results1. Establishment of the HCC model75 rats developed HCC following induction with 2%o diethylnitrosamine, with a rate of carcinogenesis of 73.53 %. The lesions were single nodule of HCC in 25 rats, HCC with 2-5 nodules in 20 rats, HCC with more than 5 nodules in 18 rats, and HCC with obvious metastatic lesions in 12.2. Quality control of total RNA of peripheral blood5ug of total RNA was subjected to denaturing gel electrophoresis. Three bands, 28s, 18s and 5s, were obtained and the optical density of the 28s band was two times that of the 18 s, a result that confirmed the high quality of RNA.3. Detection of micrometastasis using peripheral blood 3.1 Detection of AFPmRNA and GPC3mRNAIn successfully established rat HCC models, the positive rate of GPC3mRNA in 25 rats with single nodule of HCC was 32%, that of AFPmRNA was 28%; the positive rate of GPC3mRNA in 20 rats with 2-5 nodules of HCC was 45%, that of AFPmRNA was 35%;the positive rate of GPC3mRNA in 18 rats with more than 5 nodules of HCC was 72.2%, that of AFPmRNA was 61.1%;the positive rate of GPC3mRNA in 12 rats with metastatic lesions of HCC was 83.3%, that of AFPmRNA was 75%. There is no expression of GPC3 in 18 control rats, that of AFPmRNA was 11.1%.3.2 The relative of double-marker combined detection and micrometastasis With increased possibility of metastasis, the positive rate of GPC3mRNA and AFPmRNA significantly increased in peripheral blood from rat with HCC over time (p<0.05), indicating that GPC3 and AFP can be think as molecul marker of metastasis. Meanwhile, the positive rate of GPC3mRNA was significantly higher than that of AFPmRNA (p<0.05) . The total positive rate of each group was higher than that using a single marker.Combined detection of .them significantly increased the positive rate of detection. GPC3 was not detected in these 18 healthy rats, but the positive rate of AFP was 11.1% in them, demonstrating that the sensitivity and specificity of GPC3 is higher than that of AFP.Conclusions1. Following induction with diethylnitrosamine, 75 rats developed HCC, with a rate of carcinogenesis of 73.53% (75/102) and arate of mortality of 7.84%(8/102). Therefore, the establishment of animal model was successful.2. Using AFPmRNA or GPC3mRNA as the marker, nested reverse transcription PCR is feasible for detecting the micrometastasis of HCC through peripheral blood. Combined detection of hepatic cell-specific AFPmRNA and cancer cell-specific GPC3mRNA helps increase the sensitivity and specificity of detecting the micrometastasis of HCC through peripheral blood.
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