The Study Of Mechanisms And Antisense Gene Therapy Of Immune Escape Through Fas/FasL Route In Neoplasms

The incidence and mortality rate of malignancies are rising year after year and the health and lives of human beings are threatened badly. Neoplasm and the immune system of the host influence and restrict each other in the malignant progression. Cancer cells can escape from the control of immune system through several different ways so as to keep growing. And the Fas/FasL route may be an important one.Fas was discovered as a receptor to apoptotic signals in recent years. It's expressed widely on T, B and mononuclear cells, and also on many normal tissues and some kinds of neoplasm. FasL is a death factor. Its distribution is on activated T, NK and mononuclear cells, immune privilege place such as front chamber of eye, sertoli cells of testicle. Expression of FasL can also be seen in tumors. After binding with FasL, apoptotic signals will be transduced. Thus apoptosis of the cells bearing Fas will begin till completed.Under normal circumstances cancer cells will be killed for tumor immunity when FasL of immunocompetent cells binds to Fas on them. It is reported that cancer cells are usually resistant to Fas induced apoptosis and often express FasL. Cancer cells with FasL can not only escape from cytotoxicity by immunocytes through Fas route but also kill immunocompetent cells with Fas by apoptosis inducement. That is called "Fas counterattack", which is sill a debate now in causing immune suppression.Along with advanced investigation on tumor immunology, molecular mechanisms of malignant progression and regulation of cell proliferation and differentiation, gene therapy of neoplasm has become more active in tumor control. In antisense gene therapy, antisense nucleic acids are used to combine with target genes or gene products in complementary manner in order to seal abnormal gene transcription and translation. So malignancies may be controlled for promotion of normal cell differentiation and inducement of cell apoptosis by this method.In order to make it clear, we investigate the phenomenon of "Fas counterattack" by co-culture of colon cancer cells and T lymphocytes. And we also try to cut theway of immune escape of neoplasm through Fas/FasL route by antisense gene therapy aiming at FasL.There are four parts in this research.Part I: Fas and FasL expression was detected in human colonic cancer cell lines SW620, Lovo, Colo205 and human leukemia Jurkat T lymphocyte cell line by immunocytochemistry, RT-PCR and flowcytometry. It shows that expression of FasL is high in SW620, intermediate in Colo205 and low in Lovo and Jurkat. The expression of Fas is high in Jurkat, intermediate in Colo205 and low in Lovo and SW620. So we chose SW620, which is with high FasL and low Fas, and Jurkat, which is with high Fas and low FasL. Co-culture in vitro of these two kinds of cells was done as a model for detection of Fas/FasL function. With Lovo as a control, cytotoxicity effect was measured by LDH method and apoptosis rate was detected by TUNEL and Annexin V-flowcytometry method. It is indicated that, to Jurkat cells, SW620 does have cytotoxic and apoptosis-inducing effects when compared with Lovo.Part II: We constructed a recombinant eukaryocyte expression vector of antisense FasL, plasmid pcDNA3.1-as-hFasL, from a plasmid containing human FasL cDNA. And the recombinant plasmid was identified by restrictive enzyme and sequencing analysis.Part III: SW620 was transfected with pcDNA3.l-as-h.FasL and pcDNA3.1 as control by lipofectamine. And then stable transfected cells, named as SW620(as-hFL) and SW620(pcDNA3.1), were obtained after G418 selection.Part IV: FasL expression was again detected by immunocytochemistry. Western blot, flowcytometry and RT-PCR in SW620, SW620(as-hFL) and SW620(pcDNA3.1). The results show a decrease of FasL expression after pcDNA3.1-as-hFasL, but not pcDNA3.1, transfection. Same detection of FasL function were done as in part I. And we can see the obvious reduction of cytotoxic and apoptosis-inducing effects of SW620(as-hFL) to Jurkat cells compared with controls, SW620 and SW620(pcDNA3.1).