The Study Of Anti-tumor Metastasis Effect By Transfecting PcDNA3.1-HLA-A*0201 To Highly Metastatic Lung Adenocarcinoma Cell Line(Anip973)

[Objective]Based on the high frequency of HLA-A*0201 gene in Yunnan lung cancer, human lung adenocarcinoma cell line Anip973 which with high metastatic were transfected with PcDNA3.1-HLA-A*0201, detected the differences of tumor metastatic ability between transfected Anip973 cell and untransfected Anip973 cell, with various methods in vivo and vitro. At the same time, human lung adenocarcinoma cell line Anip973 which transfected HLA-A*0201 be specifically recognized by TCR (264scTCR/multimer), to identify the superficial P53264-272/HLA-A2 complex. Explore HLA-A*0201 gene on human lung adenocarcinoma cell line Anip973 anti-tumor metaststic effect, For further study of HLA on tumor metastasis ability to lay the necessary foundation.[Method] With PCR-SSP to screen HLA-A*0201 negative human lung adenocarcinoma cell line Anip973, with PcDNA3.1-HLA-A*0201 transfected and acquired stable positively transfected cells clone by using G418. Fostered the wild P53264-272 peptide with the positively transfected cells, the binding of the positively transfected cells to wild P53264-272 peptide was confirmed by FCM. Then in vitro, through the transwell chamber invasion experiment to detect cell invasion, migration force changes, and Observe scanning electron microscope changes in cell surface structure. In vivo, compare the differentiation of tumor metastatic ability with the cells which accepted different treatment injected into nude mice. Though these methods to observe themetastatic ability of changes in Anip973 by HLA-A*0201 gene transfection.[Results]We acquired the stable cells transfected with PcDNA3.1-HLA-A*0201. The HLA-A2 expression on the surface of the transfected cells was tested by FCM and immunofluorescence staining. The recognition of TCR multimer to P53264-272/HLA-A2 complex was confirmed by FCM, the result was positive. We found that HLA-A*0201 positive human lung adenocarcinoma cell line Anip973 which invasion and migration force decreased significantly (P<0.05). And the number of cell process was reduced, pseudopodium on cell surface was shorter. In vivo, we injected HLA-A*0201 gene-positive Anip973 cells into nude mice, we found that the rate of muscle invasion and the rate of lymph node metastasis are lower, the tumor metastatic ability decline in nude mice.[Conclusion]HLA-A*0201 gene deletion or low expression is an important factor for tumor cells' escaping from human immune surveillance. Anip973 which was transfected with PcDNA3.1-HLA-A*0201 could express efficiently and presented wild P53264-272. and this P53264-272/HLA-A*0201 complex could be identified by 264scTCR/multimer, this provided the possibility that T cells which presented 264scTCR could kill the HLA-A*0201 positive expression lung cancer specifically, and to further enhance immune surveillance and reduce tumor metastasis laid the necessary foundation. In vitro, transfection of PcDNA3.1-HLA-A*0201 to HLA-A*0201 negative tumor cells could induce invasion and migration force decreased, at the same time affect the conformation of process and pseudopodium on cell surface. In vivo, we injected HLA-A*0201 gene-positive Anip973 cells into nude mice, we found that the tumor metastatic ability decline.