| Liver cancer is a malignant tumor, which holds high degree of malignancy and poorprognosis. Of various high incidence of liver cancer, in2012, per minute, six peoplediagnosed with cancer, where the incidence of liver cancer ranked second. In the world,China is the superpower of HCC accounted for55%of the world, and its incidence,mortality accounted for45%of the world.In China, about320,000cases of liver cancerpatients died every year. Liver cancer patients in China is not only the total number of theworld’s first, the incidence is also the first in the world. There is a lot of hepatitis B patientsin China and liver cancer mostly evolved in liver cirrhosis on the basis of high riskincidence. HCC treatment can not fundamentally improve the prognosis of liver cancer,more effective treatment methods need to be studied for the treatment of liver cancer.There has been more and more experimental data supporting the existence of cancerstem cells,the cancer stem cells are a class of tumor cells with the characteristics of stemcells in the tumor tissue, whose characters are tumor drug resistance, relapse, metastasisand proliferation are closely related,so cancer stem cells may be the key target for tumortherapy. Based on this,we design the experimental program targeted anti-cancer stem cells,which play a fundamental role in clearing tumor tissue.Cytotoxic T lymphocytes (Cytotoxic Lymphocyte, CTLs), primarily through therelease of perforin and granzyme to kill target cells, and through the Fas ligand-mediatedapoptosis of target cells. Killing specificity, continuity and efficiency, which is the primaryimmune effector cells of immune system in clearing the virus and tumor cells.Dendritic cells (Dendritic Cells, DCs) which as known is the most potentantigen-presenting cells, playing a huge role in the body’s tumor immune response. DCsare able to initial T cell to cytotoxic T lymphocytes directly, induce specific immuneresponses, which targeted killing of tumor cells. Biological vaccines in recent yearsbecome the treatment of tumors of tumor immunotherapy dendritic cells as one of the hotspots, the clinical application of data shows that DCs as a tumor vaccine have goodprospects for the treatment of cancer where prognosis can be improved.The subject in the conventional using the total RNA from tumor cells which is addedto the basis of mature DCs, the using of adenovirus type5, Ad5as vectors of P53andTERT genes introduced into the DCS cell at the same time, then mature DCs was added tothe naive T cells to induce CTLs, and in vitro screening and culture of liver cancer stemcells and detect CTLs destruction of cancer stem cells. At last we induced CTLs which anti liver cancer stem cells can be efficiently target stem cells.Showing that1.DCs are able toinduce CTLs which can kill liver cancer stem cells; introduced to DCs with virus carryingP53and TERT genes CTLs liver cancer stem cells can be further improved its activity.Cancer stem cells used in this experiment is inducted and screened from hepatoma Hep3Bcell line through a new culture medium system established before in our laboratory. Type5adenovirus vector used in the experiment is carried ITR sequences of the virus, reverseterminal repeat (Inverted Terminal Repeat, ITR) is a cis-acting elements adenovirus protein,control experiments showing that the one carries ITR The sequence of adenovirus in DCscan enhance the quantity of the protein expression and extend the expression time, so weuse adenovirus carrying ITR sequences to improve the expression of the P53and the TERTgene in DCs. The virus vector involves derived from the laboratory virus vector library.The experiment consists of the following five parts:1.Dendritic cells in vitro induction and cultureTaken normal human peripheral blood, diluted containing Ficoll in separating tube, andafter centrifugation in the lymphocyte separation above the Ficoll,there is a layer of cellswith monocyte-containing, carefully draw the cells above the Ficoll, cleaning two or threetimes with AIM-V medium dilution which was added in six-well plates at37℃,5%CO2was allowed to culture overnight, the non-adherent cells were collected for separation of Tlymphocytes, the adherent cells are added GM-CSF and IL-4and FMS kind of tyrosinekinase3(Flt3) at37℃,5%CO2for culture on day5added total RNA of liver cancerstem cells. Add viral vectors to DCs on day6, adding TNF-α to stimulat immature DCsturn to mature,then test DCs phenotype by flow cytometry, the results show that DCsphenotype of CD80expression DCs, CD83, CD86, of HLA-DR as designed.2.The choice of P53and TERT virus vectorsInfecting of DCs with adenovirus type5vector expressing green fluorescent, compareto carry ITR sequences and adenovirus vector carrying the ITR sequences infected DCsgreen fluorescent protein expression intensity and expression time differences, the resultsshow that the vector carrying ITR sequences adenoviral infected DCs at MOI=20preferably, with high expression of the protein gene.3.The production of cytotoxic T lymphocytes in vitroInduction and culturing the dendritic cells from non-adherent cells containing mainlyB-lymphocytes and T-lymphocytes by using nylon wool column filtration adsorptionmethod, this non-adherent cells adhere the pretreatment nylon wool column, since the nylon wool has great affinity of B lymphocytes, when mixed cells solution was passedthrough a nylon wool column, B lymphocytes are adsorbed, T lymphocytes can becollected through the nylon wool column and the T lymphocytes can be collected up to beinducted to CTLs.4. The production of liver cancer stem cells and CTLs in vitroThere has been establish a new type of cell culture medium for hepatoma Hep3B cellsin vitro to produce liver cancer stem cells. The T-lymphocytes and the mature DCsco-culture in vitro, which inducing T lymphocytes into the CTLs. Assaying of IFN-γsecretion by flow cytometry and the secretion of IFN-γ in proportion and strength byElispot, the results showed that a high proportion of CTLs secret IFN-γ, with high strengthand tumor cell killing activity.5. CTLs kill the liver cancer stem cells in vitroTwo kind of specific CTLs: one of which DCs do not carry the viral vector; anothercarrying viral vector. They kill liver cancer stem cells in vitro separately, throughmicroplate reader to detect cell destruction after using CCK-8kit, the results show that theCTLs induced liver cancer stem cells having killer activity, and the virus carrying P53andTERT genes infected DCs which induced CTLs and improve the activity of killing livercancer stem cells.Conclusion: We established the CTLs which can kill cancer stem cells efficiently invitro successfully, Adenovirus type5vector carrying P53and the TERT gene can enhancethe killing activity of CTLs. The program combines the dual advantage of tumor genetherapy and cell therapy, cell therapy’s efficacy can be improved to some extent, alsohighlighted the significance of liver cancer stem cell destruction in the treatment of livercancer, which is expected to become a new way for treatment of liver cancer. |
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