| Background and ObjectiveAcute myeloblastic leukemia (AML) is a malignant disease that can threaten one's life, characterized by the rapid growth of lymphomyelocyte, and its clinic progression and prognosis have a close connection with invasive cell types, the change of gene genetics and clonal biology. Chemotherapeutics or targeted drugs can improve the complete remission (CR) of AML patients in clinical medicine, but because AML have self-renewal and proliferative stem cell groups,leukemic microclone cannot be removed in patients which possibly leads to the relapse of leukemia. Therefore, one of the goals of cancer research has been, and continues to be, the discovery of natural and synthetic products for AML prevention or treatment.AML has experienced the immunoselection and editing process of immunosurveillance, confrontation and immune escape, which makes AML cells not being killed by immune cells.NKG2D is the activated receptor in the surface of immunoctye, which can be expressed by many kinds of cells, just like NK cells, NK/T cells, Tγδcells, DC cells, IDC cells. These immunocytes can be activated through the corresponding ligand in the surface of tumor cells recognized by NKG2D molecules, express the molecules,such as TRAIL,FasL, granzyme, porforin, then induce the targeted cell apoptosis or kill them. Many researches have suggested that some drugs can up-regulated the expression of NKG2D ligand in the surface of tumor cells, and then improve the sensitivity of tumor cells killed by NK cells, which leads to effectively remove tumor cells.Snake venom has the excellent effect on the prevention and therapy of tumor. The research has shown that snake venom has the inhibitory action on human ovary cancer, carcinoma of bladder, invasive prostatic carcinoma, acute promyelocytic leukemia, etc., the mechanisms of which are related to modulate the apoptosis gene expression. Naja Naja Actra Venom Component (NNAVC), isolated from the Naja Naja Actra Venom, was chosen in the experiment because of the effect of its anti-tumor.Human acute myeloid leukemia(AML) cell line, KG1a cell was chosen as experimental subject, NNAVC and activated immune cells were used, the following would be discussed in the research:①The experimental research of leukemic model treated with NNAVC or NNAVC combined with activated immune cells in mice;②the action of proliferative inhibition and apoptosis induced by NNAVC on KG1a cell;③the molecular mechanisms related to KG1a cell apoptosis induced by NNAVC;④The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells and their synergic mechanism.The research aimed to discuss the therapeutic action of NNAVC on AML in vivo and in vitro, which will provide the theory and experimental foundation of therapy strategy for refractory and relapsed leukemia. MethodsChapter 1 The experimental research of leukemia model treated with NNAVC alone or NNAVE combined with activated immune cells in mice.The experiments were performed on Balb/c-nu mice weighing 18-22g,and Balb/c-nu mice were injected into KGla cells through vena caudalis to establish Balb/c-nu mice leukemic model, which was assessed by the morphology of peripheral blood and marrow cell, marrow primary cell culture in mice with leukemic cells, Y chromosome by FISH, membrane antigen detection, human specific antigen by immunohistochemisty, and etc. The normal Balb/c-nu mice were injected into different doses of NNAVC through intraperitoneal injection, and then the weight of mice,the change of behavior and pathological section were assessed for the side-effect of NNAVC; the survival times of leukemic model treated with NNAVC or NNAVE combined with activated immune cells were observed after KG1a leukemic model was established. Statistical analysisThe calculation was performed using SPSS 13.0 software package. The data presented as the mean±standard deviation. Comparison in experiments was performed using one-way analysis of variance (ANOVA), independent-samples t-test and repeated measurement of analysis of variance to assess the statistical significance of differences between groups. Differences with P<0.05 were considered to be significant.Chapter 2 The action of proliferative inhibition and apoptosis induced by NNAVC on KGla cellTrypan blue exclusion was used to measure the growth curve; of NNAVC on KG1a cells; KG1a cell morphological change could be seen by inverted microscope; MTT assay was used to detect the proliferative activity of KG1a cell after NNAVC treatment, and half inhibitory concentration(IC50) was calculated; the colony forming ability of KGla in vitro was measured by methylcellulose semisolid method; flow cytometry was used to measure the influence of NNAVC on KGla cell cycle; the morphology of apoptotic cells was observed by fluorescence microscope and KGla apoptotic rate was measured by flow cytometry after KGla cell was stained with Annexin V/PI; the apoptosis of KG1a cell was further proved by DNA Ladder.Chapter 3 The effects of apoptosis-related genes and protein expression of NNAVC against KGla cellsThe mRNA expression levels of molecules related to apoptosis, such as Bcl-2,Bcl-xl,Bax,Bak,Bad,Bid,PUMA,NOXA,p53,NF-kB,etc.,were measured by RT-PCR; cytochrome C level was measured according to human cytochrome C ELISA detecting kit; expressions of Caspase-8,Caspase-9 and Caspase-3 were detected by spectrophotometric method; expressions of death receptors and decoy receptors of TRAIL were analyzed by flow cytometry.Chapter 4 The cytotoxic effect of NNAVC combined with activated immune cells on KGla cellsPeripheral blood mononuclear cells(PBMC) were separated from the healthy donors using lymphocyte separating medium, which were stimulated by cytokine to culture activated immune cells in vitro. The cytotoxic effects of NNAVC with different concentrations on PBMC and activated immune cells were measured by CCK-8 assay; activated immune cells from different healthy donors killed KGla cells by turns, the sensitivity of activated immune cells to kill KGla at different effector-to-target(E:T) cell ratios was detected by LDH cytotoxicity assay after KG1a cells were killed for different times by activated immune cells; The cytotoxic effects of NNAVC combined with activated immune cells on KG1a cells were measured by LDH releasing assay; the colony forming abilities of KGla treated with NNAVC alone or NNAVC combined with activated immune cells in vitro were measured by methylcellulose semisolid method; the expressions of NKG2D ligand in the surface of KG1a were measured by flow cytometry.ResultsChapter 1 The experimental research of leukemic model treated with NNAVC alone or NNAVE combined with activated immune cells in mice.1.1 Identification of leukemic model1.1.1 The cell morphology of leukemic modelLeukemic cells, which were similar to KG1a cells, were found in the peripheral blood after mice were injected into KG1a cells four weeks later according to the peripheral blood film once every week. The amounts of leukemic cells increased gradually with the time going.1.1.2 The primary culture originated from marrow cells in leukemic modelThe primary culture originated from marrow cells in leukemic model developed to exponential phase of growth about three to four weeks later, the cells were similar to KGla cells, could go down to the future generation steadily.1.1.3 The markers on the surface of culture cell in leukemic cellsThe CD34 and CD39 antigen expressions of normal marrow cell, KGla cell and cells in the leukemic model were measured by flow cytometry. The CD34 and CD39 antigen expressions of normal marrow cell could not be measured(CD34-CD38-,99.76%);Those of KGla cell were 32.74% (CD34+CD38-); Those of cells in the leukemic model were 83.06% (CD34+CD38-),16.72% (CD34+CD38+)。The results indicated that the model was successful because human leukemic cells were found in the marrow of leukemic model, and which were in the stem cell stage.1.1.4 The measurement of marrow culture cell chromosome in modelY chromosome could not be measured in the normal mice, sex chromosome in KG1a cells was XY, that of cells in the leukemic model was XY as well.The female mice were chosen in the experiment and KG1a cell came from a man with AML, therefore, those findings confirmed human KG1a cell existed in the model.1.1.5 The measurement of organs affected in the modelThe organs affected in the model were measured according to HE staining and immunohistochemistry assay. The research showed that leukemic cells existed in the liver, spleen, lung, kidney and other organs of model mice ,especially liver most affected; The expressions of human specific CD34 and CD45 antigens on the surface of leukemic cells were found, which suggested that the leukemic cells in the model came from human leukemic cell, not from the body of mice spontaneously.1.2 The toxic effects of NNAVC on Balb/c-nu mice1.2.1 The survival quality in miceThe mice had a good condition without restlessness, scare, apositia and no death during the experiment.1.2.2 The changes of body and organ weight in miceThe change of body weight in the experiment had significant deviation (F= 15.624, P=0.002);However, there were no significance between NNAVC-treated groups (F= 0.361, P= 0.704);The body weight in NNAVC-treated groups increased after 10 days, but comparing with negative control,that was no significant(P=0.425, P= 0.654).There were no significant in organ weight to body weight between high-dose group and low-dose group (P= 0.124, P= 0.435, P= 0.352, P= 0.403, P= 0.663).Organ weight to body weight coefficient was considered to be one of the indexes of toxic effect, so the results indicated that body and organ weight in Balb/c-nu mice treated with NNAVC were not significant.1.2.3 The change of tissue pathologyPathological changes of organs were not significant comparing with control in light microscope.1.3 The effects of survival rate and time of NNAVC and activated immune cells on model mice.1.3.1 The survival quality of miceThe mice had a good condition with normal diet during the experiment. The mice had abnormal behaviors with descending water-drinking, decreasing activity and etc. One week before death and red speckle appeared on the surface of body partially in the model, their body weight began to reduce till to death in a week.1.3.2 The survival time of mice in the modelThe medium survival time of mice in the negative control was 50 (34,66) days and died after mice were inoculated KGla cells for 52 days; That in NNAVC 0.04mg/kg and 0.08mg/kg were 43(41,45)days和61 (35,87)days; That in activated immune cells alone or combined with NNAVC 0.08mg/kg were 52(38,66) days, 71 (58,84) days, respectively. That in positive control(daunorubicin,2.5mg/kg) was 53 (39,67) days. That in activated immune cells combined with NNAVC 0.08mg/kg was longer than negative control(P=0.025), that in other groups was not significant comparing with negative control (P= 0.277, P=0.197, P= 0.341, P = 0.197); That in NNAVC 0.04mg/kg was significantly less than NNAVC 0.08mg/kg (P=0.025);That was not significant between NNAVC 0.08mg/kg and NNAVC combined with activated immune cells (P= 0.110).The results suggested that the survival time in leukemic mice was longer with the concentration of NNAVC increasing, and NNAVC combined with activated immune cells had a better effect than NNAVC alone or NNAVC combined with activated immune cells alone.1.3.3 The organs weight of NNAVC and activated immune cells on leukemic model miceThe heart weight to body weight coefficient was significant comparing with control (P=0.001),but the coefficient of liver was not significant (F=2.176, P =0.108);That of spleen in every group(negative, NNAVC low dose and high dose) was significant comparing with control (P=0.000, P=0.000, P= 0.013);That of lung and kidney in the psositive group had a increasing tendencycomparing with control (P= 0.000, P=0.011);That of kidney in activated immune cells-treated group was significant as well (P= 0.009),while other groups (P>0.05).The coefficient of spleen in activated immune cells-treated group, NNAVC combined with activated immune cells, positive control were higher comparing with negative control (P=0.000, P=0.000, P=0.000); The coefficient of kidney in activated immune cells-treated group, positive control were significant comparing with negative control (P=0.008, P=0.009)Chapter 2 The action of proliferative inhibition and apoptosis induced by NNAVC on KGla cell2.1 The influence of NNAVC on the growth curve of KGlaTrypan blue exclusion assay showed that the slope of growth curve in the low concentration group(0.625μg/ml)of NNAVC was lower than control group, that suggested NNAVC had obvious inhibition on KG1a cells; the growth of KG1a cells was so slow that the curve was almost flat when the concentration was up to 1.25μg/ml;The KGla almost died out when more than 2.5μg/ml.These results showed that NNAVC could obviously inhibit the proliferation of KGla cells in a time- and dose-dependent manner.2.2 Morphological view of KG1a cells could be seen by Zeiss inverted microscopeKG1a cells were in good condition with the integrity,smooth and lucency of cellwall; even cytoplasm; regular shape before NNAVC treatment.KGla cells began to emerge bad integrity of cellwall, irregular shape, more particles after the 6th hour of NNAVC, more cells changed with longer action time, when the action time was the 24th hour or longer, the smaller volume ,deformation,vacuole within the cell, cell pyknosis and other morphological changes were seen by inverted microscope.2.3 The cytotoxic effects of NNAVC on KGla cellsThe cytotoxic results showed that there were reciprocal effects existing between the different concentration and time of NNAVC (F= 11.868, P= 0.000); The inhibitory rates of different NNAVC concentrations against KGla cells were statistically significant (F= 934.427, P=0.000); The inhibitory rates of two time groups of NNAVC against KGla cells were also statistically significant (F= 1074.672, P= 0.000)The growth inhibition of NNAVC against KGla cells in the same action time significantly increased between the concentration of 0.6μg/ml to 1.2μg/ml (F= 428.725, P=0.000; F= 550.172, P=0.000);The inhibitory rates of NNAVC on KG1a cells at the same concentration were significant (t=-11.549, P=0.000; t =-14.190, P= 0.000; t=-13.219, P= 0.000; t=-13.194, P= 0.000; t=-10.218, P=0.001; t=-15.272, P=0.000; t=-10.436, P= 0.000;) at the 24th and 48th hour, and higher inhibitory rate with longer action time. These findings suggested that NNAVC could obviously inhibit the proliferation of KGla cells in a time- and dose-dependent manner. The half inhibitory concentration(IC5o) of NNAVC against KG1a cells was 0.8806 (0.8654-0.8959)μg/ml at the 24th hour,0.7037 (0.6875-0.7187)μg/ml at the 48th hour。2.4 The influence of colony-forming ability of NNAVC against KGla cellsMethylcellulose semisolid method was used in the experiment, the KGla cells were dispersed on the 1st day to 2nd day, the colony was began to form on the 7th day and gradually increased on the 7th to 14th day. The morphology of colony in every group :the amount of colony forming was more in the control group, which was intensive colony; the amount of colony forming significantly decreased in the NNAVC-treated group and dead cells existing in the cells, while the colony was less and more cell debris in the high concentration group(0.8μg/ml).The amounts of colony forming in every group on the 7th and 14th day were statistically performed, and the results showed that the reciprocal effect existed between the NNAVC concentration and culture time (F=70.623, P= 0.000);The amounts of colony forming among different groups were significant(F=204.351, P = 0.000) and the culture time were also significant (F=305.877, P= 0.000)The amount of colony after the 14th day of culture was more than that on the 7th day (t=-13.784, P=0.000; t=-8.615, P=0.000; t=-6.728, P= 0.003). Different concentrations of NNAVC had an effect on colony-forming ability of KG1a cells in the designated time, and the amount of colony was significantly less inside the group (F= 34.357, P= 0.001; F=172.368, P= 0.000); The amount of colony forming in the group of NNAVC 0.8μg/ml had no significant difference comparing with in the group of NNAVC 0.7μg/ml (P=0.411, P=0.185),but the former was less than latter. These findings indicated that the proliferative ability of KG1a cells was lower in the NNAVC groups, NNAVC could inhibit the colony forming ability of KGla cells in some extent,which caused proliferative inhibition. 2.5 Cell cycle effects of NNAVC on KGlaThe proportions of KGla cells at the different stage of cell cycle in every group were significant (F= 339.698, P= 0.000; F=6.439, P= 0.032; F= 127.007, P=0.000);The proportions of cells at the G0/G1 stage at the concentration of 0.7μg/ml and 0.8μg/ml were up to 58.67% and 62.20%,respectively,which was significant comparing with the control group(52.93%,P=0.032, P= 0.002),while the proportion at the G2/M stage decreased to 5.33% and 3.13%,respectively (P= 0.000, P= 0.000).The proportion at the S stage at the concentration of 0.8μg/ml was 34.70% comparing with control group(36.90%,P= 0.012).There were no significant difference at the G0/G1 and S stage between the concentration of 0.7μg/ml group and 0.8μg/ml group (P=0.048, P= 0.079),while the proportion at the G2/M stage was significant (P=0.003),and subdiploid peak ahead of G1 stage appeared in the treated group. The results suggested that NNAVC could induce cell cycle arrest of KGla cells at the G0/G1 stage and reduce the contents at the G2/M stage to inhibit the proliferation.2.6 The observation of cell apoptosis by fluorescent microscopeKGla cells were in good condition with the integrity of cellwall and no apoptotic body emerged in the control group. The vacuole within the cell, cell pyknosis and rupture, apoptotic body appeared in the NNAVC-treated group, and more apoptotic cells emerged with higher concentrations and longer action time.2.7The apoptotic rates measurement of KGla cells treated with NNAVCThe early and late apoptotic rates increased after NNAVC treatment, and the early and late apoptotic rates in the 0.7μg/ml concentration group increased to 39.63 % and 22.23%,respectively(P= 0.049, P= 0.034);those in the 0.8μg/ml concentration group were 40.73% and 41.50%(P= 0.040, P= 0.016);while no significance in the 0.6μg/ml concentration group. The early apoptotic rates between the 0.7¨g/ml and 0.8μg/ml concentration groups were not significant(P=1.000),but the late apoptotic rates were significant(P=0.021).The results confirmed that NNAVC Could induce the apoptosis of KG1a cells.2.8 DNA Ladder measurementThere was no DNA Ladder in the control group,which showed the genomic DNA；DNA Ladder was seen in the NNAVC-treated group,which consisted of 180-200bp or more.These results indicated that NNAVC could lead to the DNA fragment of KG1a cells,that further confirmed the apoptotic induction of NNAVC.Chapter 3 The effects of apoptosis-related genes and protein expression of NNAVC against KG1a cells3.1 The effects of apoptosis-related mRNA expression levels of NNAVC against KGla cellsThe results showed that the apoptosis-related genes in the different concentration of NNAVC groups,such as Bcl-2,Bcl-xL,Bax,Bid,Bad, p53,PUMA,NOXA and NF-KB,etc.,were significant,respectively(F=5.774,P= 0.021；F=20.652,P=0.000；F=9.780,P=0.005；F=6.795,P=0.014；F= 18.389,P=0.001; F=14.816,P=0.001；F=246.243,P=0.000；F=10.467, P=0.004；F=7.153,P=0.012；F=47.767,P=0.000)；The expression level of Bcl-2 gene descended comparing with control group(P=0.035,P=0.004, P= 0.028),while there were no significance among the groups(P=0.162,P=0.884, P=0.202)；The expression level of Bcl-xL gene rose up in a dose-dependent manner, especially 0.7μg/ml and 0.8μg/ml(P=0.003,P=0.018)；The expression level of Bak gene had no significant deviation in all groups(F=0.406,P=0.753)；The expression level of P53 gene,which was not expressed in normal KG 1 a cell,had obviously risen up (P=0.000,P=0.000,P=0.000),while PUMA and NOXA genes related to P53 had no change; The expression level of NF-κB gene in the concentration of 0.6μg/mland 0.7μg/ml descended (P= 0.000, P= 0.000).Taken together, NNAVC could induce the KGla cells apoptosis by modulating the balance between pro-and anti-apoptosis genes.3.2 The effects of cytochrome C of NNAVC on KGla cellsThe results showed that the expression level of cytochrome C had an up-and-down trend in the concentration of 0.7μg/ml within 12h;while that in the concentration of 0.8μg/ml rose up within 6h,and unchanged within 6h to 12h, then descended within 12h to 24h, at last that was very close between two groups. there were reciprocal effects existing between the different concentration and time of NNAVC (F= 51.868, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of cytochrome C (F= 82.331, P= 0.001;F=552.286, P=0.000); The content of cytochrome C in 0.7μg/ml group less than that in 0.8μg/ml group (P=0.018) at the 6th hour; However, the content of cytochrome C in 0.7μg/ml group more than that in 0.8μg/ml group (P=0.001; P = 0.026) at the 12th and 18th hour.3.3 The measurement of caspase-9 protein by spectrophotometric methodThere were reciprocal effects existing between the different concentration and time of NNAVC (F=23.751, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-9 protein (F= 23.982, P=0.001;F=180.774, P=0.000); The expression level of caspase-9 rose up within 12h and gradually descended after 12h; NNAVC had an effect on expression level of caspase-9 of KGla cells in a concentration-dependent manner. The results suggested that NNAVC could induce the apoptosis of KGla cells by activating caspase-9.3.4 The effects of death receptors and decoy receptors expression levels of NNAVC on KGla cellsThe expression level of death receptor 4(DR4) decreased with the NNAVC concentration increasing,and there were significant among the groups (F=170.107, P=0.000) or between groups (P≤0.005); The expression level of DR5 rose up in the concentration of 0.6μg/ml and 0.7μg/ml (P= 0.000; P=0.000); The expression level of decoy receptor 1(DcRl) had obvious significance among the groups (F=37.002, P=0.000),that in the concentration of 0.8μg/ml descended (comparing with other groups, P=0.000, P=0.000); The expression level of DcR2 decreased with the NNAVC concentration increasing, however, that in the concentration of 0.6μg/ml and 0.7μg/ml rose up (P=0.000; P=0.000)3.5 The measurement of caspase-8 protein by spectrophotometric methodThere were reciprocal effects existing between the different concentration and time of NNAVC (F=51.653, P=0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-8 (F=47.400, P = 0.000; F= 189.867, P= 0.000). The expression level of caspase-8 had a rise-and-down process in groups. These showed that NNAVC could activate the caspase-8 in a short time to initiate the apoptotic process.3.6 The effects of caspase-3 expression levelof NNAVC on KGla cellsThere were reciprocal effects existing between the different concentration and time of NNAVC (F=29.694, P= 0.000);The different concentrations and times of NNAVC had an obvious effect on the expression level of caspase-3 (F=202.507, P = 0.000; F=205.361, P= 0.000).The expression level of caspase-3 had a rise-and-down tendency in groups, and there were significant deviance in times (F= 36.236, P= 0.020; F=28.489, P= 0.025; F= 175.320, P=0.003). The expression level of caspase-3 rose up with the NNAVC concentration increasing at the 12th and 24th hour (F= 177.938, P= 0.000; F= 42.728, P= 0.000).The results indicated that NNAVC could activate the caspase-3 to induce the apoptosis of KGla cells, and caspase-3 was activated at the 6th hour,most at the 12th hour,in a dose-dependent manner.Chapter 4 The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cells4.1 The cytotoxic effects of NNAVC against PBMC and activated immune cellsThe cell viabilities of PBMC and activated immune cells were not significant difference at various concentrations of NNAVC treatment (F=4.025, P=0.062); The cytotoxic effects of cells treated with different concentration of NNAVC were significant (F=4.629, P=0.016).The toxic effect of NNAVC (0.5-2.0μg/ml) on PBMC was almost rare comparing with the control group (F= 0.626, P=0.618);There was no toxic action of NNAVC on activated immune cells when NNAVC was less than 1.0μg/ml, but toxic effect at the concentration of 2.0μg/ml was significant (P=0.003).That indicated that there were no toxic effects of NNAVC against PBMC and activated immune cells when the concentration was less than 1.0μg/ml.4.2 The cytotoxic effects of activated immune cells on KGla cellsThe results showed that the cytotoxic effects of activated immune cells on KG1a cells at the different effector-to-target(E:T) cell ratios were significant deviation (F=199.354, P=0.000),and the mean of cell activities were 50.04%,3.95 %,36.59%,23.63%,33.27% and 8.35% ,respectively. The cytotoxicity had significant deviation at the different E:T cell ratios (F=345.299, P=0.000); The cytotoxic effects of activated immune cells on targeted cells increased with the E:T cell ratios rising except for E group, there were significant deviation among the groups (F=25.711, P=0.000; F=53.466, P=0.000; F=43.224, P=0.000; F=135.884, P=0.000). The cytotoxic effects of activated immune cells on targeted cells in A to E groups had a descending tendency, which suggested that the sensitivity of KG1a cells to activated immune cells decreased after those were killed by different activated immune cells.4.3 The cytotoxic effect of NNAVC combined with activated immune cells on KG1a cellsThe cytotoxicity of NNAVC alone on KGla cells was 40.61%;while the cytotoxicity of activated immune cells alone at the E:T cell ratios of 10:1 and 20:1 on KG1a cells were 34.55% and 41.35%,respectively(P=0.414). The cytotoxicity of NNAVC combined with activated immune cells at the E:T cell ratios of 10:1 and 20:1 on KGla cells were 56.21% and 85.59%,which was significantly higher than NNAVC alone or activated immune cells alone (P= 0.018, P= 0.000). The cytotoxicity of NNAVC on activated immune cells at the E:T cell ratios of 10:1 and 20:1 was very low (4.35% and 1.33%, respectively).The results indicated that the cytotoxicity of NNAVC combined with activated immune cells on KGla cells was better than NNAVC or activated immune cells alone, but it didn't mean the synergism of NNAVC and activated immune cells.4.4 The sensitivity of KGla treated with NNAVC to activated immune cellsThe cytotoxicity of activated immune cells at the different E:T cell ratios on KG1a cells treated with NNAVC was significant (F=148.593, P=0.000); The cell activities of activated immune cells at the E:T cell ratios of 10:1 and 20:1 on targeted cells treated with NNAVC was significant (F=8.721, P=0.001). The cytotoxicity of activated immune cells at the E:T cell ratio of 10:1 on targeted cells treated with NNAVC was significant among groups (F=12.015, P=0.002); However, that at the E:T cell ratios of 20:1 was not significant among groups (F=1.177, P=0.337);The targeted cells within the group were significant (P=0.003, P= 0.010, P= 0.010, P= 0.001) at the E:T cell ratios of 10:1 and 20:1.The results showed that the cytotoxicity of activated immune cells on targeted cells treated with NNAVC was stronger with higher E:T cell ratio, KG1a treated with NNAVC to activated immune cells was still very sensitive.4.5 The colony-forming abilities of NNAVC alone or combined with activated immune cells on KGla cellsThe colony-forming amounts of NNAVC alone or combined with activated immune cells on KG1a cells were significant (F= 108.614, P=0.000) after KG1a was inoculated for 7 days; The different concentrations of NNAVC were significant as well (F=270.741, P=0.000); There were reciprocal effects existing between the different concentrations and treatment strategy of NNAVC (F=9.956, P= 0.003) The inhibitory effect of colony-forming abilities of NNAVC alone on KGla cells was significantly worse than the NNAVC combined with activated immune cells group (t=9.141, P=0.001; t=8.273, P=0.001); The colony-forming amounts significantly decreased with the concentration of NNAVC increasing (F=58.886, P = 0.000; F=426.629, P= 0.000)The colony-forming amounts of NNAVC alone or combined with activated immune cells on KG1a cells were significant (F= 26.059, P=0.000) after KG1a was inoculated for 14 days; The different concentrations of NNAVC were significant as well (F= 174.366, P=0.000); There were not reciprocal effects existing between the different concentrations and treatment stategy of NNAVC (F=2.623, P=0.114). The inhibitory effect of colony-forming abilities of NNAVC alone on KGla cells was significantly worse than the NNAVC combined with activated immune cells group (t=5.822, P=0.004; t=6.554, P=0.003); The colony-forming amounts significantly decreased with the concentration of NNAVC increasing (F=49.972, P = 0.000; F=167.300, P= 0.000). The colony-forming amounts of NNAVC combined with activated immune cells on KGla cells less than NNAVC alone, which suggested that NNAVC combined with activated immune cells could decrease the amount of colony by killing the colony-forming cells of KG1a.4.6 The effects of NNAVC on expressions of NKG2D ligand on the surface of KGla cellsThe expressions of MICA,MICB,ULBP1,ULBP2 and ULBP3 in KGla cells treated with NNAVC were significant between groups (F=3654.504, P=0.000; F = 978.748, P= 0.000;F= 1328.553, P=0.000;F=455.271,P= 0.000;F=223.263, P= 0.000); The expressions of ULBP3 in KGla cells treated with 0.6μg/ml NNAVC significantly rose up (P=0.003),however, those of other ligands were significantly less than control group (P=0.000, P=0.000, P=0.000, P=0.000); The expressions of ULBP1,ULBP2 and ULBP3 except for MICB in KGla cells treated with 0.7μg/ml NNAVC significantly rose up comparing with control (P= 0.000, P=0.000, P=0.000).Those treated with 0.8μg/ml NNAVC were as well (P= 0.000, P=0.001, P=0.000); The expressions of ULBP1,ULBP2 and ULBP3 were significant between groups (P= 0.000).The results indicated that ULBP1,ULBP2 and ULBP3 could be the target of NNAVC in KG1a cells.Conclusion1. NNAVC can obviously inhibit the proliferation of KGla cells by inducing apoptosis, cell cycle arrest of KG1a cells at the G0/G1 stage and the inhibition of colony-forming cells.2. NNAVC could upregulated the expression of Bax, Bad, p53 and NOXA at mRNA level, and downregulated the expression of Bcl-2 and NF-KB.3. NNAVC could induce cytochrome-c released into the endochylema, activate the caspase-9 and caspase-3, the intrinsic apoptosis pathway was involved in the action of NNAVC to KG1a.4. At the range of effective concentration of NNAVC to KG1a cells,the cytotoxicity of NNAVC on activated immune cells was less than on KG1a cells.5. The KG1a cells that are survived after NNAVC's effection could be killed by activated immune cells.6. NNAVC can combine with activated immune cells to kill KGla cells whether co-incubated or in one-after-another way.7. NNAVC(0.08mg/kg) combined with activated immune cells can increase the survival time of hu-Balb/c-nu-KG1a leukemic mice.Innovation of our researchOur research firstly discovered the fact that NNAVC combined with activated immune cells could be used to treat acute myeloid leukemia, and NNAVC had no side effect on activated immune cells when they killed leukemic cells.Values of our researchOur research demonstrated NNAVC combined with activated immune cells could kill the KGla cells more effectively and treat hu-Balb/c-nu-KGla leukemic mice,whose survival times were increased.The research provided a new insight for the the therapy of AML and longer survival time of patients.
|
PDF Full Text Request