Combining CIK Cell And Viral Therapy Expressing IL-21for The Treatment Of HCC

Liver cancer is a malignant tumor and its prognosis is poor. At present various treatmentscan not fundamentally change the prognosis of patients with liver cancer. Gene-viral treatmentbecame a kind of new treatments began to used in the treatment of liver cancer. Interleukin inimmune response and adjust the play an important role. IL-21is mainly secreted by activatedCD4+T cells, which can strengthen the effect T cells and activation of the function of NK cells,thus effectively increase the body’s natural immune and specific immune response, of livercancer has good therapeutic effect. In this project, we pick the IL-21gene which can resisttumor and virus, and use adenovirus carring the IL-21gene to treat liver cancer, and at thesametime using T cell as a cell carrier to avoid host immune system monitoring, guidingadenovirus to arrive the tumor site effectively, enhancing the effectivity of intravenousinjection and effectively treat liver cancer.Meanwhile, considering the safety of gene therapy, our project use HSV-TK/GCV self-killing system, inserting HSV-TK to adenovirus genome which carries the hIL-21gene, whenhIL-21is highly expressed, injecting GCV to node mice to kill CIK which express high hIL-21.This system can decrease the side effect of high expressed hIL-21.Our experiment includes the following three parts,1. The construction of adenovirus vector which can express hIL-21We used PCR to clone hIL-21from the template of pDC318-hIL-21plasmid. Then weused restriction enzyme SalI and BamHI to digest hIL-21and pDC759. Then we used theligase to get a new vector pDC759-hIL-21.We infected293cells by using pDC759-hIL-21,pAd5, pAd5F35.After10days, we extracted the viral genome to identify whether the virusclone is correct by PCR procedure. If the virus is correct, we name the new virus asAd5/AAV-hIL-21, Ad5F35/AAV-hIL-21. After the amplification of293cell, purification ofCsCl density gradient centrifugation, TCID50test the titer of virus. The outcome ofAd5/AAV-hIL-21, Ad5F35/AAV-hIL-21is8.95×109pfu/mL and2.65×1010pfu/mL. At thesame time we also construct Ad5/AAV-CopGFP, Ad5F35/AAV-CopGFP. The titer is5.15×109pfu/mL,7.1×109pfu/mL.2. CIK cμlturing and ex vivo experiments.This part of the research was mainly based on building good virus in the in vitroconditions, the genome of the power of expression expression box a identification and the invitro conditions type5adenovirus and35type adenovirus CIK cells to the contrast of infection ability; Exploring different MOI situation under the maximum infection of optimization andchose ability to infer that the best infection conditions. In order to get the resμlts, theexperiment the following experiment research:First, we used Ad5/AAV-CopGFP and Ad5F35/AAV-CopGFP to infect CIK cell bydifferent MOI, the density was1,5,10,50,100,1000. After48hr and96hr infection, we detectedin fluorescence microscope fluorescent and found out that Ad5F35/AAV-CopGFP has higherinfection ability than Ad5F35/AAV-CopGFP. And the best infection MOI was50.Then, we used Ad5/AAV-hIL-21and Ad5F35/AAV-hIL-21to infect SMMC-7721cell, theMOI was0,1,5,10,20. After48hr infection, we used ELISA testing kit to detect whether hIL-21is expressed in SMMC-7721cell. Our resμlt was when MOI is20the expressing level is best.At last, This experiment detected in the growth of cells in a CIK a system of research, anddraw the22days CIK cell growth curve, the experimental study found, along with turn bagafter training, the sixth day after, adjust good CIK density for a higher level, in the later time ofthe growth of the present CIK double growth trend. In the20days after training the number ofCIK basically unchanged, the state began to grow stagnant, in later times CIK growthrecession condition, therefore, in CIK cell cμlture to12days later start as back to lose time.3. The study of CIK expressing hIL-21to treat liver cancer.Based on the in vitro studies a research resμlts, the experiment chooses LuoShu tumor-burdened mice as experimental animals, and the experiment SMMC-7721cells chose as atumor-burdened mice cells made mode, and in every mice tail intravenous5x106cells. InLuoShu into tumor after treatment, the treatment group in the experimental study will carry onthe detailed introduction. The therapeutic effect of mainly divided into, the effect index andsurvival index, the experimental resμlts show that when a Ad5F35/AAV-hIL-21infection CIKcells back to lose to vein tumor-burdened mice after, the treatment effect of the tumors are thebest.