| ForewordThe leukemia is one kind of malignant tumor of the hematological system. The same with other tumors, its generation and development have close relationship with the disorder to apoptosis. The survivin is a new member of inhibitor of apoptosis protein. It was bolted from the human genomic library by effector cell protease receptor-1 cDNA in 1997. The survivin has the function of cell cycle regulation and inhibitor of apoptosis. Up to now it was the strongest factor in inhibiting apotosis.At present, it has been discovered that, survivin expresses in the transform-ant cell lines and many human malignant tumors. The investigation about the relationship between survivin and tumorigenesis has become the hot spot in the tumor research. It is also discovered that survivin expresses in human leukemia.The expression in the drug fast cell line is obviously increased. Moreover, the masculine cases of surviving have less sensitivity to the chemotherapy compares to the negative ones. It hints that survivin is one of the important factors to determine the prognosis of hematopoietic system tumor.The formation of neogenesis micrangium is indispensable to the multiplication and the growth of the tumor cell. It is also the essential condition of the tumor invasion. Vascular endothelial growth factor promotes caryocinesis of the v-ascular endothelial cell. It is also the most important right regulating factor for angioneogenesis. It was separated from cow's normal pituitary gland follicle cell by Gonnolly in 1989. VEGF of human was first cloned from the HL-60 cell. In recent years, the relationship between VEGF and the malignant disease of hem- atopoietic system receives the attention day by day. Its expression level has become a leukemia independent prognosis factor.At present, the relevance between survivin and VEGF which express in entity tumor has been received more and more reconstruction. The experiment applies RT-PCR method to investgate the expression of survivin and VEGF genein bone marrow cells from patients with leukemia and their relationship. It is hoped that we can provide some clues for the illuminate of leukemia pathogensis.Methods1,Experimental materialCollecting bone marrow cells of 76 patients with leukemia from November, 2005 to April, 2006 in the first clinical hospital of China medical university he-matology department. There were 45 acute leukemia patients, which included 25 cases of initial treat acute myelocytic leukemia (AML), 9 cases of acute lymph-oblastic leukemia (ALL), 8 cases of complete remission and 3 cases of relapse. There were 31 chronic myelocytic leukemia patients, which included 15 cases of chronic phase, 9 cases of accelerated phase and 7 cases of blastic crisis, and 12 cases of non hematopoietic system tumor as controls.2,Experimental methodsBone marrow samples from patients with leukemia and 12 controls were u-sed for mononuclear cells (MNC) extraction. 4ml of this fresh bone marrow samples were separated by lymphocyte isolation and washed twice with phosphate buffered saline (PBS), followed by extraction of whole cell lysates, and the MN C concentration was 106cells/ml. Total RNA was extracted from MNC by Triz-olreagent.20μl transcription reaction system including total RNA 3μl, M—MLV 1μl (200U), dNTPs (10mmol/L) 2μl, RNasin 0.5μl (20U), random primer 1.5μl (90n g), 5×RT bufer 4pl was warmed for reaction at 40℃, 50min, then stoped at 99 ℃for 5min. The products was frozen at—20℃until further use.Polymerase chain reaction: 25μl PCR system including reverse transcription products 3μ1, primer 2μl, Taq DNA polymerase 0.2μl (1U), dNTPs (10mmol/L) 1.5μl, 10×PCR buffer 2.5μl, action condition as follows: 94℃35 sec, 66℃40 sec, 72℃40 sec, totally 30 cycles, 72℃7 min for survivin and P-actin to stop the treaction: 95℃60 sec, 60℃2 min, 72℃3 min, totally 35 cycles, 72℃min for VEGF to stop the reaction.The electrophoresis of 10μl PCR products was performed at 45mV for 40 min in 2% agarosegel. And the bands were viewed and photographed under UV illumination.Statistics analysis was performed using SPSS 12.0 computer software.Result1,The expression of survivin and VEGF in acute leukemiaThe rate of expression of survivin and VEGF in initial treat AL were 70.5 9% (24/34) and 55.88% (19/34), much higher than those in control (P=0.033, P=0.020). The rate of expression of survivin and VEGF in complete remission were 25.00% (2/8) and 12.5% (1/8), lower than those in initial treat AL (P=0. 038, P=0.047). In the relapse cases, the rate of expression of survivin and VE GF were 100% (3/3), much higher than those in control (P=0.044, P=0.022).2,The expression of survivin and VEGF in initial treat acute leukemiaThe rate of expression of survivin in ALL and AML were 77.78% (7/9) and 68.00% (17/25), much higher than those in control (P=0.030, P=0.032). The rate of expression of VEGF in AML was 64.00% (16/25) and higher than that in control (P=0.013). But the rate of expression of VEGF in ALL was not3,The expression of survivin and VEGF in chronic myelocytic leukemiaThe rate of expression of survivin in the CML-CP was 26.67% (4/15), and there was no difference between that and the controls (P=1.000). But in the CML-AP and CML-BC the rate of expression of survivin were 77.78% (7/9) and 85.71% (6/7), much higher than those in control (P=0.030, P=0.020) and in the CML-CP (P=0.033, P=0.020). The rates of expression of VEGF in the CML-CP, CML-AP and CML-BC were higher than those in control, the difference were significant (P=0.006, P=0.002, P=0.006).4,The correlation between expression of survivin and VEGF in leukemiaThe expression of survivin and VEGF were both positive in 18 cases of A L. The positive coincidence was 75.00% (18/24). The expression of VEGF in survivin(+) and surviving(-) were 75.00% (18/24) and 10.00% (1/10), the difference was significant (P=0.001).The expression of survivin and VEGF were both positive in 16 cases of CML. The positive coincidence was 64.00% (16/25). The expression of survivin inVEGF(+) and VEGF(-) were 64.00% (16/25) and 16.67% (1/6). The difference was not significant (P=0.067).DiscussionThe apoptosis is also called programmed cell death. It is one of the important protection mechanisms which the normal somatic cells maintenance stable. Apoptosis receives the influence from the promote factor and inhibit factor. Up to now, survivin is the strongest anti-apoptosis. In vitro experiment indicated that, survivin play the role to inhibit apoptosis and the activeness of caspases. At the same time, survivin also indirectly inhibit spindle hydrolysis through the combination with the spindle textile- fiber. It is advantageous for protection the integrity of the fission cell, then inhibit apoptosis. In addition, survivin can also c ombine with the cyclical element dependence protein activating enzyme Cdk4, causes the massive cells unlimited multiplication finally. Therefore, if survivin expresses excessively, it can increase the cell multiplication, reduce apoptosis. The balance between cell multiplication and apoptosis will be broken, finally the tumor would occurrenc.It has been discovered that survivin expressed excessively in the tumor. The experiment demonstrates that the rate of expression of survivin in initial treat-acute leukemia is mach higher than that in the control. It prompts that survivin is possible concerned with the pathogenesis of acute leukemia. The rate of expression of survivin ALL is higher than that in AML, but the differentce was not significant. The rate of the expression of survivin in complete remission descends, and it advances in the relapse cases. It prompts that surviving has relationship with the prognosis of acute leukemia. Obviously, the examination of survivin gene is advantageous to the clinical diagnosis and the curative effect appraisal of acute leukemia.This experiment discovers that, the expression of survivin in CML is connected with the staging. There is no difference between the rate of expression of survivin in the CML-CP and in the controls. But in the CML-AP and CML-BC, the rate of expression of survivin were much higher than those in controland in the CML-CP. Obviously, along with the progression of the course of CML,the expression rate of survivin elevates obviously. It prompts that surviving possiblyhas some kind of relation with the cell differentiation.But the accurate mechanism still wait for further study.The tumor cell growth and the number increase rely on the new vessel formation. The short term research indicated that malignancy in hematological system also existed vassular neogenesis. In recent years, the related literature indicated that the majority of malignant cell in hematonosis expressed VEGF obviously. This experiment demonstrates that the rate of the expression of VEGF in in- itial treat AL is much higher than that in control and complete remission. In the relapse cases, the expression rate of VEGF gene advances obviously, much higher than that in controls. Therefore, the examination of VEGF is helpful to understand the condition and judge the prognosis.This experimental result showed that, the expression of VEGF gene in CML is obviously higher than that in controls. But the difference among CML-CP, CML-AP and CML-BC was not significant. It prompts that there was no relationship between the expression of VEGF and clinical stage of patients with CML. In recent years the researches confirm that, it is unable to examine surviving in the normal capillary endothelium cell, but in the granulation its expression step up obviously. VEGF may up-regulation the expression of survivin to promote vascular endothlial cell multiplication. This experiment demonstrated thet the expression of survivin and VEGF were both positive in 18 cases of AL. The positive coincidence was 75.00%. The expression of VEGF in surviving(+) and survivin(—) were 75.00% and 10.00%. The difference was significant. It prompted that the expression of survivin correlated with that of VEGF in AL. But the expression of survivin and VEGF were both positive in 16 cases of CML. The positive coincidence was 64.00%. The expression of survivin in VEGF(+) and V EGF(—) were 64.00% and 16.67%, the difference was not significant. It prompted that the expression of survivin does not correlate with that of VEGF in CML. This diversity was possibly because of distinct pathogenesis between AL and CML. At present, the investigation about the relationship between the expression of surviving and VEGF in patients with CML has not been reported. Therefor we still wait for the further research.ConclusionSurvivin and VEGF express excessively in initial treat and recidivism patients with acute lymphoblastic leukemia. The rates of expression of them descend in complete remission. Survivin and VEGF participate in the nosogenesis and t- he development of acute leukemia, and it is helpful prognosis decision.The expression of survivin in chronic myelocytic leukemia is connected with the staging. It prompts that surviving possibly has some kinds of relation with the cell differentiation.The expression of survivin correlated with that of VEGF in initial treat acute leukemia. Survivin plays certain role on promoting the proliferation of vascular endothelial cell.
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