The Research Of CSR-CTL On Ovarian Cancer Cell Lines In Killing Effect And Mechanism

Ovarian cancer is a common gynecoLogicaL maLignancies, the incidence of which issecond pLace in the gynecoLogicaL reproductive system tumors and It has been found thatovarian cancer with the5-year Low survivaL rate is hovering around30percent has been aserious threat to the Lives and heaLth of women, which is often found in intermediate stageor terminaL stage. Tumor cytoreductive surgery and pLatinum-based chemotherapy havehad the disease aLLeviated, but terminaL stage patients without the advanced and ideaLimpLementation of the tumor ceLLs cytoreductive surgery, continuing the Long-termchemotherapy with heavy side effects. It's a serious impact on quaLity of Life. Therefore,patients can not struggLe through, with resistance to chemotherapy. Hence there is stiLLnearLy70%reLapse rate. Therefore, it has been a research target for many schoLars totackLe the probLem, Looking for ways to overcome drug resistance and reduction ofchemotherapy side effects and dosage, increase the efficacy of security and reLiabLemethod of treatment of ovarian cancer. Immunotherapy of ovarian cancer in recent years hasbecome a new concern adjuvant therapy method by improving anti-tumor immune activity,reguLation and enhancing the human anti-tumor mechanism of immune toLerance to thesuppression and destruction of tumor ceLLs. The one hand, it's effective to kiLL residuaLcancer ceLLs in vivo inhibition of tumor ceLL recurrence and to metastasis. On the otherhand,it can improve the spontaneous anti-cancer immunity,strengthen the patient's body,reduce the chemotherapy resistance to enhance the effect of chemotherapy of ovariancancer, and uLtimateLy improve patient survivaL quaLity and proLong the time of Life.The purpose of this study is to Look for a more secure and better efficacy ofimmunotherapy to suppress and kiLL ovarian cancer ceLLs and resistant to chemotherapyresistance. Of the immunotherapy choosing the right tumor kiLLer ceLLs is a importantissue and CTL is the earLiest responders to identify tumor antigens, is aLso the mosteffective anti-tumor ceLL criticaL effect ceLL popuLations. It can eLiminate minimaLresiduaL disease,and can even make Late tumors achieved compLete remission. HER2/neuampLification or over expression in ovarian cancer with high specificity is cLoseLy reLated to poor prognosis and resistance to chemotherapy. As a noveL immune adjuvant in vivostabiLity, CpG ODN is not easy to degrade and suitabLe for mass production, and has abetter security. As an adjuvant Rg1is more effective significantLy due to the characteristicsof their chemicaL structure, spatiaL conformation of the effects. Therefore, thisexperiment wiLL treat CPGODN and Rg1jointLy as an adjuvant, combining with theHER2/neu antigen peptides to estabLish in vitro cuLture system (referred to as theCSR-CTL), and to expLore the inhibition of CSR-CTL on ovarian cancer ceLLs andreversaL mechanism of ovarian cancer.Content and experimentaL methodsFirstLy: EstabLishment and identification of the CSR-CTL of specificity, highactivity in vitro cuLture system.1. a specific antigen epitopes: Predict9amino acids of the HLA-A2restricted CTLepitopes and cLeavage sites of the HER2/neu tumor antigen by Genebank and CLCProteinWorkbench3, and SignaLP3.0softwares.2. Construction of the specific, highLy active CSR-the CTL in vitro cuLture system:①The extract PBMC ceLLs in heaLthy voLunteers. SeparateLy add up differentconcentrations of CPGODN and the HER2/neu antigen peptide to stimuLate Lymphocyteswhich wiLL be added to human ovarian cancer SKOV-3ceLL, using the MTT assay todetect common Lymphocyte stimuLation different concentrations of CPGODN andHER2/neu antigen peptide fragments and different concentrations of ginsenoside Rg1andHER2/neu antigen peptide.②The concentration of CPGODN and ginsenoside Rg1which isworked out by two experiments wiLL be combined with HER2/neu antigen peptidefragments mixed cuLture to estabLish of the CSR-CTL ceLL-free system.3.CSR-CTL cuLture system identification:(1) Get CSR-CTL ceLL into karyotypeanaLysis and experimentaL group join CPGODN, ginsenoside Rg1and HER2/neuantigen peptide fragment mixture (CSR), the controL group to join the same amount ofmedium to carry out Lymphocyte karyotype anaLysis.(2) counting the number of ceLLs inthe peripheraL bLood Lymphocytes CSR in cuLtured day of1,4,7,10,13,16,19,22,25,28,31,34. and make Growth Curve.(3)CoLLect the ceLLs separateLy which aresimuLated by CPGODN,ginsenoside Rg1and HER2/neu antigen peptide fragment mixture for10days,20days and30days by fLow cytometry. AnaLyze the ceLL percentage of theLymphocyte surface phenotype LeveLs of CD3+and CD3+of CD4+and CD3+CD8+ceLLs.SecondLy: The expLoration for CSR-CTL kiLLing effect on ovarian cancer ceLLLine and mechanisms.1. CSR-CTL kiLLing effect on human ovarian cancer ceLL Lines: the experimentaLgroup considers CSR-CTL as effector ceLLs, ovarian cancer SKOV-3as target ceLLs inthe controL group with unstimuLated Lymphocytes as effector ceLLs and ovarian cancerSKOV-3as target ceLLs. Than the concentration in accordance with different efficiencytarget:5:1,10:1,20:1and40:1, to join the CSR-the CTL cuLture measured by MTTassay after48h. Then caLcuLate activity of CTL kiLLing (%).2. Inhibition of CSR-CTL on human ovarian cancer ceLL growth: in the experimentaLgroup's SKOV-3ceLLs the controL group added the medium of the CSR-CTL. MTT assaywas used respectiveLy to detect6,12,18,24,30,36,42and48hours the ceLLs were inthe waveLength of Light at492nm when the absorbance vaLue.3. CSR-CTL in human ovarian cancer effects SKOV-3ceLL cycLe and apoptosis rate:the roLe of the CSR-CTL, fLow cytometry experimentaL group and controL groupapoptosis rate and ceLL cycLe changes.4. CSR-CTL Impose on human ovarian cancer SKOV-3apoptotic protein mRNA: Usethe experimentaL group and controL group reaL-time quantitative RT-PCR method to detectthe roLe of the CSR-CTL apoptotic protein BCL-2and Caspase-3, Survivin and Smacgene5.The CSR-CTL in human ovarian cancer SKOV-3apoptotic proteins at the proteinLeveL: the roLe of the CSR-CTL is expressed by using western bLot method o detectexperimentaL group and controL group apoptotic protein BCL-2, caspase-3, Survivinand Smac protein.6.detection the infLuence of the CSR-CTL in human ovarian cancer ceLL Lines andHER2/neu and resistance-associated gene situations: the experimentaL group and controLgroup were detected by RT-PCR method before and after the CSR-the CTL roLe of HER2/neu, and GST-p, MRP, of LRP16and TOPO gene situations.7.CSR-CTL impose on human ovarian cancer ceLL Lines PI3K/AKT signaLtransduction pathway: (1) CSR-CTL imposes on protein mRNA in human ovarian cancer ceLL LinePI3K/AKT signaL transduction pathway by using reaL-time quantitative RT-PCR methodto detect CSR-the CTL processing the experimentaL group and controL group, PI3K, Aktand YB-1expression.(2) CSR-CTL of each protein in human ovarian cancer ceLL Line PI3K/AKT signaLtransduction pathway at the protein LeveL, western bLot method to detect the CSR-CTLprocessing experimentaL group and controL group of PI3K, Akt, and YB-1situations.The experimentaL resuLtsFirstLy: EstabLishment and identification of Specificity, high activity of theCSR-CTL in vitro cuLture system.1. Gain of a specific antigen epitopes.2. the estabLishment of specific, highLy active CSR-the CTL in vitro cuLture system:(1) By comparing with the controL group when the CPGODN concentration is3.75μg/mL,7.5μg/mL,15μg/mL,30μg/mL,60μg/mL, the kiLLing effect of theovarian cancer SKOV-3destruction significantLy increased (P <0.01), but when theCPGODN concentration of60μg/mL on ovarian cancer ceLLs is at30μg/mL,there is nosignificant change (P>0.05).(2)Compared with the controL group, when the concentrationof ginsenoside Rg1is3.75μg/mL,7.5μg/mL,15μg/mL,30μg/mL,60μg/mL, thekiLLing effect towards ovarian cancer SKOV-3significantLy increased (P <0.01), butwhen the concentration of ginsenoside Rg1is60μg/mL,its destruction on ovarian cancerceLLs is at30μg/mL,it takes on no significant change (P>0.05).(3)Concentrations of andginsenoside Rg1in CPGODN concentration of30μg/mL were mixed with the HER2/neuantigen peptide fragments and the mixture was added to the3days medium to changeonce each time the medium,adjusting the ceLL density cuLture in PBMC34days toestabLish the CSR-CTL ceLL-free system.3.Karyotype of the experimentaL group and controL group of nucLear research theCSR-CTL cuLture system identification:(1) through the comparison of the experimentaLgroup and controL group anaLysis of Lymphocyte karyotype,we found that mixture typeexperimentaL group which was joint with CPGODN and ginsenoside Rg1and HER2/neuantigen peptide fragment,there is no changes in the structure and the number.(2) by thedrawing of the growth curve of CSR-CTL ceLLs, we can see that, Lymphocytes mixed with CPGODN, ginsenoside Rg1and HER2/neu antigen peptide fragment ceLL numberwas significantLy increased (P <0.01) from7thday cuLturing, to19thday has reached apLateau.(3) Through fLow cytometry anaLysis, Lymphocyte immune phenotype changesafter joining CPGODN, ginsenoside Rg1and HER2/neu antigen peptide fragmentmixture---CD3+, CD3+CD4+, CD3+CD8+ceLLs significantLy increased (P<0.05)..Second:the expLoration of CSR-CTL kiLLing effect on ovarian cancer ceLL Lineand mechanisms1.CSR-CTL kiLLing effect on human ovarian cancer ceLL Lines: regardLess of theefficiency target designed the concentration for5:1,10:1,20:1or40:1, CTL activity of theexperimentaL group were obviousLy higher than that of the controL group (P <0.01).2, The inhibition of CSR-CTL on human ovarian cancer ceLL growth: theexperimentaL group ceLLs grew into the downward trend, whiLe an upward trend in thecontroL group in the cuLture time of48h.. At the same cuLturing time the ceLLs number ofexperimentaL groups decreased significantLy (P <0.01).3. the infLuence of CSR-CTL on human ovarian cancer SKOV-3ceLL cycLe andapoptosis rate: During the monitoring of fLow cytometry, the apoptosis rate of theexperimentaL group was significantLy higher than the controL group (P <0.01). TheexperimentaL group G1and S phase ceLLs increased significantLy (P <0.01), and G2phase ceLLs were significantLy reduced (P <0.01).4.Effect ofCSR-CTL on human ovarian cancer SKOV-3apoptotic protein mRNAexpression of apoptotic protein Caspase-3and Smac gene: using reaL-time quantitativeRT-PCR method to detect the roLe of the CSR-CTL and we found that in the experimentaLgroup and controL group Caspase-3and Smac gene expressions both rose,but BCL-2andSurvivin down reguLated.5. the effect towards CSR-CTL expression and controL of human ovarian cancerSKOV-3apoptotic proteins in the protein LeveL: detect apoptotic protein Caspase-3andSmac by using western bLot method in the experimentaL group. The resuLt is thatapoptotic protein Caspase-3and Smac group increase, and BCL-2and Survivin downreguLated.6. Detection HER2/neu and resistance-associated gene expressions of the CSR-CTL inhuman ovarian cancer ceLL Lines: the expressions of the HER2/neu and GST-p, MRP and of LRP16and TOPO genes were down-reguLated by RT-PCR method to compare theexperimentaL group with controL group.7.the impose of CSR-CTL on human ovarian cancer ceLL Lines PI3K/AKT signaLtransduction pathway:(1) detect PI3K, Akt, and YB-1genes expression before and afterthe CSR-CTL was treated in ovarian cancer by using the reaL-time quantitative RT-PCRmethod to compare the CSR-CTL processing experimentaL group with the controL group.PI3K, Akt and YB-1down towards reguLated.(2)Detect PI3K, Akt and YB-1proteinexpression before and after the protein LeveLs test in the CSR-CTL was treated ovariancancer ceLLs. By western bLot method CSR-CTL we processed the experimentaL group tocompared with controL group. PI3K, Akt and YB-1down towards reguLated.ConcLusion1. Combine two new types of immunoadjuvant CPGODN and the ginseng saponinswith Rg1HER2/neu immune fragments to set up the CSR-CTL in vitro proLiferationsystem.2. CSR-CTL has a highLy antigen-specific destruction and significant inhibitiontowards SKOV-3in vitro on ovarian cancer.3. CSR-CTL can destroy ovarian cancer SKOV-3normaL ceLL cycLe, which wiLLbe stuck in the S phase. In the meanwhiLe CSR-CTL can reguLate a variety of apoptoticgenes and induce ovarian cancer SKOV-3apoptosis.4. CSR-CTL can inhibit a variety of resistance genes in ovarian cancer to effectSKOV-3of PI3K/Akt pathway-reLated protein expression and to inhibit of PI3K/Aktpathway, so as to pLay the roLe of anti-tumor and reversing resistance to chemotherapy.This study estabLished the CSR-CTL in vitro mass cuLture system and confirmed thattheir proLiferative capacity and the advantages of a high degree of antigen-specific andsignificant cytotoxicity.It provided the experimentaL basis for the CSR-CTL to achievespecific and efficient anti-ovarian cancer and effective reversaL of ovarian cancer. It openedup new design ideas and methods for the treatment of ovarian cancer with importantscientific and practicaL significance.