| Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, has been found to be a crucial components in tumor growth, progression and metastasis. The mechanisms by which neovascularization stimulates tumor progression are the delivery of the nutrients and oxygen necessary for tumor growth. Intratumoral microvessel density (MVD) was a significant predictor of a poor prognosis of about all solid tumors.ANG, a Mr 14,200 protein originally isolated from medium conditioned by HT-29 human colon adenocarcinoma cells and subsequently from normal serum , is one of the most potent angiogenic factors in vivo .Although its physicochemical properties and mechanisms of receptor binding have been characterized in detail, little is known about its role in neovascularization, particularly in tumor growth and metastasis. Previous experiments in which a monoclonal antibody against human ANG was shown to suppress the growth of human adenocarcinoma in nude mice by reducing tumor neovascularization support the idea of an important function of ANG in angiogenesis . Nothing is known, however, about ANG expression and function during esophageal Carcinoma development.In this study, we and studied the effect on the biological behavior and angiogenesis of esophageal carcinoma .The effect and feasibility of a esophageal carcinoma treatment based on inhibition of ANG gene expression by RNAi technology will be studied. The main work is described as follows: 1.Analyse of ANG expression by immunohistochemistry and RT-PCR assay.Intratumoral microvessel density (MVD) and expression of ANG protein were studied by immonohistochemical staining. We found that the over-expression of ANG protein was positively correlated with high MVD and both were significantly associated with histopathological grading and lymph node metastasis in patients with pancreatic carcinoma. ANG mRNA isoforms were determined in 11 patients with esophageal carcinoma and 2 human esophageal carcinoma cell lines (TE-1 and EC9706) by reverse transcriptase-polymerase chain reaction (RT-PCR). ANG mRNA was detected in all two human esophageal carcinoma cell lines and ANG were identified as the predominant species produced in esophageal carcinoma cells. 2.Effect of inhibition of ANG gene expression by RNAi technology on biological behaviorofTE-1 cell in vitro.Northern blot analyses were performed to determine the level of endogenous ANG mRNA expression. TE-1 cells were inhibited of ANG gene expression by RNAi technology and then the totel cellular RNA was isolated at different times (l~5day) after RNAi. Maximal down-regulation of the endogenous ANG mRNA was observed between 3 and 5 days after infection. ELISA analyses were performed to determine the amounts of secretary ANG proteins in conditioned media collected 3> 5^ 7 and 9 days. Results suggest secretary ANG protein levels were significantly dropped at 5 days and the effects of RNAi on ANG protein secretion was maximal at 7 days after infection .Our result indicated that the exogenous ANG expression were efficiently inhibited. 3.1nfluence of tumor growth in nude mice by transduction of recombinantThe TE-1 cells (5 X 106/mouse) were injected s.c. into the cervix of the nude mouse . By 7 days, visible and palpable s.c nodules had developed at all injection sites. Intratumoral treatment with serum-free medium or 109 PFU of Ad-Hl-AngshRNA or Ad-LacZ(six mice/group) was started and repeated every other day for a total of four times. Tumor sizes were measured with calipers every other day. Four weeks after treatment, the mice were sacrificed and their tumors were excised for routine histological examination ^ HE staining and immunohistochemistral staining of FVIII-related antigen ANG> PCNA. Tumor cell apoptosis was detected by TUNEL method. Results: Inhibition of ANG gene expression by RNAi technology can suppress the growth of tumors derived from TE-1 cells and MVTh the expression of ANG protein and PCNA significantly decrease. Increase of tumor cell apoptosis was observed by RNAi treatment.. Conclusion:1. ANG is over-expressed in human esophageal carcinoma and this over-expressed is associated with its generation > progress and metastasis.2. Inhibition of ANG gene expression by RNAi technology in vitro can efficiently down-regulates ANG mRNA and secretory ANG protein.3. Inhibition of ANG gene expression by RNAi technology significantly inhabits tumor growth of nude mice with reduction in the amount of microvessles. Inhibition of ANG gene may be induce a reduction of the expression level of PCNA and an increase of the tumor cell apoptosis.
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