| Part I Establishment and cell biological characteristics of enhanced green fluorescent protein (EGFP) expressing McA-RH7777cell linesPurpose:To establish and study biological characteristics of stable EGFP-expressing McA-RH7777cell lines.Materials and Methods:McA-RH7777cell lines were infected by pseudo-lentivirus with EGFP, an efficient gene transfer vehicle for tumor cells and selected by puromycin. The expressions of EGFP of the infected cells were observed by fluorescence microscopy and detected by flow cytometry. McA-RH7777cells clones expressing EGFP were isolated with cloning cylinders by trypsin/EDTA, then they were transferred and amplified by conventional culture methods. Multiple cell biological characteristics, such as cell morphological, proliferation, invasion abilities and the growth rate of tumors by subcutaneous implantation were compared between the infected and unin-fected cells.Results:McA-RH7777cell lines could be infected efficiently by pseudo-lentivirus. We obtained a stable EGFP tumor cell lines, named McA-RH7777/EGFP.180days after consecutive in viro cell passages, McA-RH7777/EGFP cell lines expressed green fluorescence, stably and intensely. There were no significant differences between McA-RH7777cells and McA-RH7777/EGFP cells in their cellbiological characteris-tics. The growth curves of tumors by subcutaneous implantation were similar. The existence of EGFP in tumor cells did not interfere on the tumorigenicity.Conclusion:McA-RH7777/EGFP cell lines can express green fluorescence stably. This cell lines could be useful for investigating the mechanisms of infilitration and metastasis on hepatocelluar carcinoma. Part ⅡEstablishment of stable enhanced green fluorescent protein expressing (EGFP) hepatocellular carcinoma metastasis model in Buffalo ratsPurpose:To establish an transplanted EGFP expressing hepatocellular carcinoma meta-stasis model in Buffalo rats.Materials and Methods:McA-RH7777/EGFP cells were subcutaneously implanted into the Buffalo rats’ right thighs. Pieces of tumor tissue were transplanted into the left lobes of the livers in30rats. The subsequent growth, invasion, metastasis of the transplanted tumors were observed and evaluated with whole-body fluorescence optical imaging system, fluorescent microscopy, MRI, CT, DSA. The tumor markers were investigated by histological analysis.Results:The tumors were detected with MRI in all rats on day7after tumor implanted. The mean survival time was45.93±5.06days in the tumor-bearing group. We demonstrated that McA-RH7777/EGFP cells maintained stalbe high-level EGFP expression during their growth in vivo. The invasion and metastasis of tumor were readily observed and accurately evaluated by fluorescent microscopy, whole-body fluorescence optical imaging system. The mean tumor volumes was12.54±3.89mm3on day7, increased to201.18±86.39mm3on day14after tumor implanted. The tumor demonstrated well-demarcated hypo-intensity signal on T2WI and hyper-intensity signal on DWI. DSA demonstrated a mass with nodular or circular tumor staining, and with enlarged or twisted feeding artery. AFP was positive. Pathologically, the tumors were approximately round or ellipse nodules. Lung metastasis can be detected in all rats at the end stage. This rat models have the similar imaging and pathological characteristics to that of human HCC.Conclusion:The EGFP tagged hepatocellular carcinoma models in Buffalo rats are superior for the detection and studying relevant patterns of hepatocellular carcinoma invasion and metastasis in vivo. These rat models are suitable for the studies about imaging diagnosis, interventional therapeutic, tumor metastasis and so on. Part IIIPreliminary study on staging of transplanted hep a to cellular carcinoma in Buffalo ratsPurpose:To establish a staging standard for transplanted hepatocellular carcinoma in Buffalo rats after studying the timing, distribution, and progression of metastasis.Materials and Methods:McA-RH7777/EGFP cells were subcutaneously implanted into the Buffalo rats’ right thighs. Pieces of tumor tissue were implanted into the left lobes of the livers in35rats. The weights and serum albumin levels of rats were measured on day7,12,18,21,26after tumor implanted. The subsequent growth, invasion, metastasis of the implanted tumors were observed and evaluated with whole-body fluorescence optical imaging system, fluorescent microscopy. The circulating tumor cells (CTC) of the peripheral blood were detected by the flow cytometry.Results:The tumors were detected with MRI in all animals on day7after tumor im-planted. The weight couldn’t be increased obviously after the21th day. The mean tumor volumes was13.36±2.90mm3on day7,162.5±69.71mm3on day12, increased to1683.36±375.02mm3on day26after tumor implanted. With the fluorescent microscopy, micrometastasis tumors can be detected in lungs on day21after tumor implanted. Multiple small lung metastases were detected on day26. The results of circulating tumor cells (CTC) detection of the peripheral blood:no findings on day7,(8.14±1.21)/106on day12,(15.43±3.99)/106on day18after tumor implanted. The albumin levels on day26after tumor implanted were signifaicantly lower than before (P<0.05).Conclusion:The visualization of distant micrometastasis and local invasion at the single-cell level can be clearly demarcated in the EGFP tagged hepatocellular carcinoma model in Buffalo rats. The staging about hepatocellular carcinoma would be more accurately. The period from12to18days in the models may be classified as the micrometastasis stage. This staging standard might be the guideline for model selection in experimental study of hepatocellular carcinoma. Part IVThe influence between lung metastasis and transarterial embolization for hepatocellular carcinoma with micrometastasis in rat modelsPurpose:To investigate the influences between lung metastasis and transarterial emboli-zation (TAE) for hepatocellular carcinoma with micrometastasis in rat models.Materials and Methods:McA-RH7777/EGFP cells were subcutaneously implanted into the Buffalo rats’ right thighs. Pieces of tumor tissue were implanted into the left lobe of the livers in40rats. On day17after tumors implanted, tumor-bearing rats were randomly divided into four treatment groups (n=10):group A (saline, control), group B (hepatic artery ligation), group C (low dose iodized oil), group D (high dose iodized oil). Body weights, tumor volumes, lung metastasis were evaluated. All rats were sacrificed for the detection of VEGF, E-cadherin, ICAM-1by Western-blot analysis, real time PCR, immunohistochemistry. All datas were processed by SPSS16.0.Results:The results of tumor volumes ananlysis:the tumor volumes treated with high dose iodized oil were smaller than that in the other groups, P<0.05. The tumor volumes with hepatic artery ligation or low dose iodized oil were smaller than that in the group treated with saline. The rate of lung metastasis in group D was lower than other groups, the rate in hepatic artery ligation group was higher than other groups, P <0.05.The results of VEGF ananlysis:the levels of VEGF mRNA in group B (2.53±0.28), C (2.46±0.32), D (1.95±0.17) were higher than that in group A (0.93±0.29), P <0.05. The statistic differences on Western-blot, immunohistochemistry among them were similar to the results of VEGF mRNA. The increased expression of VEGF was due to the tumors hypoxia by ligation and embolization. There were no statistic differences between the group A, D on the numbers of CD31, but group B was higher than group D.The results of E-cadherin mRNA ananlysis:the levels of E-cadherin mRNA in group D (1.66±0.38) were higher than other groups, included:A (0.99±0.39), B (0.80±0.23), C (0.86±0.24), P<0.05. The statistic differences on the results for Western-blot, immunohistochemistry among them were similar to the results of E-adherin mRNA.The results of ICAM-1mRNA ananlysis:the levels of ICAM-1mRNA in group D (0.69±0.16) were lower than group A (1.09±0.19), B (1.12±0.16), C (0.97±0.23), P <0.05. The statistic differences on the results for Western-blot among them were similar to the results of ICAM-1mRNA. TAE with high dose iodized oil can inhibit the tumor growth by decreasing the expression of ICAM-1.Conclusion:TAE using high dose iodized oil for hepatocellular carcinoma with microme-tastasis can inhibit the tumor growth, decrease lung metastasis in the rat models.
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