Cyclin E2和Survivin在急性白血病的表达及其相关性

1、Cyclin E2和Survivin在急性白血病的表达及其相关性         08-03-10 11:46:00     编辑：studa20          作者：王颖，徐世荣，林凤茹，郭晓楠，任金海  【摘要】  为了解细胞周期蛋白E2（cyclin E2）和存活蛋白（survivin）在急性白血病中基因表达及两者间的相关性，采用逆转录聚合酶链反应的方法检测

2、了84例成人急性白血病患者（16例复发患者，60例初治患者和8例持续缓解患者）和20例正常人的cyclin E2 和survivin mRNA的表达。结果表明： cyclin E2在急性白血病中表达的阳性率（70.24）高于对照组（0）；survivin在急性白血病中表达的阳性率（72.62）高于对照组（30）；在急性白血病中cyclin E2的表达与survivin的表达呈正相关性，r0.65；cyclin E2阳性组缓解率（55.81）低于cyclin E2阴性组（88.24），cyclin E2阳性组复发率（41.67）高于阴性组（20）；复发组cyclin E2表达率在三组中最高，持续

3、缓解组cyclin E2表达率在三组中最低，cyclin E2在急性髓系白血病患者表达率（59.32）低于急性淋巴细胞白血病患者的表达率（96）；与发病时白细胞计数未见有统计学意义的相关。结论：首次证实cyclin E2在急性白血病中有异常表达，且对临床预后有不良影响；cyclin E2在急性白血病中的表达与survivin有正相关，提示cyclin E2在急性白血病中有可能作为一种微小残留病变的检测指标。 【关键词】  存活蛋白As a malignant hemophaty，acute leukemia (AL)，especially，acute lymphocytic leuk

4、emia （ALL）  underwent a high relapse rate and a short disease-free  survival (DFS)，in spite of the improvement in clinical outcome  benefited from the development of hematopoietic stem cell transplantation (HSCT) and chemical therapy.Preventing relapse and prolonging DFS still is 

5、; the focus of  AL  therapy.Minimal residual disease (MRD) became an effective indication  for the foundation of post-remission therapy regulation since the routine morphological examination of the peripheral blood and  bone marrow sample from the remission patient was normal.But

6、 the fusion gene such as AML1/MTG8 and PML/RAR as  MRD marker，was only used in M2，M3，so it is valuable to search another new MRD marker for leukemia.On the other hand，RNA interference (RNAi) as a new and prospective gene therapy  for cancer has become a new focus of the study of AL  t

7、herapy1.Searching for an effective new target for a tumor-specific RNAi  is worth developing for leukemia therapy.Recently，as a type of G1 cyclin，cyclin E2 was found out to be the potential marker of tumors since it presented a positive expression in the cell lines of solid tumors and a negativ

8、e expression in proliferating normal cells2.However，there has been no report on its expression and role in AL.In order to find out the role of cyclin E2 in the progression and clinical outcome of Al patients，reverse transcription polymerase chain reaction  (RT-PCR) is adopted to test the expres

9、sion of cyclin E2 and survivin mRNA in 84 adult patients with AL，20 normal persons as controls and leukemia cell line K562.Subsequently，we investigated the relationship between the expression of cyclin E2 and the clinical progression of AL patients.The study of cyclin E2 can provide a preliminary

10、60; theoretical basis  for searching a new target of AL gene therapy and a new MRD marker.Materials and MethodsPatients enrolled84 adult inpatients with AL from April 2002 to March 2003 in  the Second Affiliated Hospital of Hebei Medical University were enrolled in this study.The diagnoses

11、 of these patients   50 males and 34 females，14-62 years old (mean of 34.1 years)were confirmed by morphological，cytochemical and immunologic marker analysis.According to the French-American-British (FAB) classification，59 patients were diagnosed as acute myelocytic leukemia (AML)，includin

12、g 2 cases of M1，12 cases of M2，21 cases of M3，11 cases of M4，11 cases of M5，1 cases of M6，1 cases of M7.25 patients were diagnosed as acute lymphocytic leukemia (ALL)   including 4 cases of L1，19 cases of L2，2 cases of L3; 60 patients with de novo AL，16 relapse cases，8 cases of continuousl

13、y complete remission (CCR)  ( time of disease-free survival3 years).20 normal persons  aged 15-60 years (mean of 35 years old) containing  11 males and 9 females served as controls.Treatments: all these patients with AML，except M3，received standard ADE regimen (cytarabine arabinoside

14、150 mg/m2  for 7 days，daunorubicin 40 mg/m2 for 3 days and etoposide 75 mg/m2 for 3 days-7 days ).Patients with M3 received all-trans-retinoic acid (20-40 mg/day) and arsenic trioxide (10 mg/day) as remission induction therapy.Patients with ALL received VITP regimen (vincristine 1.4 mg/m2 for 1

15、 day of each week，iphosphamide 1.2 g/m2  for 3 days of each week，pirarabicin 20 mg/m2 for 3 days of each 2 weeks，prednisone 1 mg/kg  for 14 days and then gradually decrement until the end of regimen that lasts for about 28 days).Cells and cell cultureBone marrow samples from 84 patients wi

16、th AL and 20 controls were used for mononuclear cells (MNC) extraction.4-5 ml of this fresh heparinized bone marrow samples were separated by Ficoll-Hypaque (Pharmacia Biotech，Uppsala，Sweden) and washed twice with phosphate-buffered saline (PBS)，followed by extraction of whole cell lysates，and the M

17、NC concentration was 106 cells/ml.Leukemia cells amounted to over 80% in bone marrow samples from  84  AL patients  assesed by Wright s staining，and  then the MNCs were frozen after centrifugation and stored at-80 until further use.K562 cell line was obtained from the Institute o

18、f Hematology，Chinese Academy of Medical Sciences.Cells were maintained at 37 in a humidified  atmosphere containing 5% CO2.Exponentially growing suspension cultures of leukemia cells were propagated by reseeding at  5×105 cells/ml every 3-5 days，in RPMI 1640 medium supplemented with 1

19、0% fetal bovine serum，50 U/ml penicillin G，and 50 g/ml streptomycin sulfate.K562 cells were harvested at logarithmic growth phase，separated by Ficoll-Hypaque and washed twice with PBS，then frozen after centrifugation and stored at-80 until use.RNA extraction  Total RNA was extracted from MNC an

20、d K562 cell by TRIZOL reagent (Gibco)，according to the recommendations of the manufacturer. The concentration and purity of total RNA was determined by UV spectrophotometry，The ratio of A260/A280 reached 1.8-2. The electrophoresis pattern on 0.1% agarose gel stained by ethidium bromide  was use

21、d for determining the  integrity of total RNA  showing  two bands of 18S and 28S in the gel.Total RNA was frozen at-80 until further use.Semi-quantitative RT-PCR Reverse transcription reaction  20 l  reverse transcription reaction system including total RNA 1 g，M-MLV（Pr

22、omega）200 U，dNTPs （Promega）2 mmol/L 4 l，RNasin(Promega) 20  U，random primer （Promega）0.5 g，5×RT buffer 4 l（Tris-HCI 250 mmol/L，pH 8.3，KCl  375 mmol/L，MgCl2 25 mmol/L，DTT 50 mmol/L） was warmed  for reaction at 37，60 min，then stop at  95 for 5 min.The products was frozen at -2

23、0 until further use. Polymerase chain reaction  25 l PCR system including reverse transcription products 2 l，oligonucleotide primer 25 pmol，Taq DNA polymerase (Huamei）1 U，dNTPs （Promega）0.2 mmol/L，10×PCR buffer 2.5 l (MgCl2 15 mmol/L，pH 8.4，Tris-HCl  100 mmol/L，KCl 500 mmol/L，BAS 20 g

24、/ml），reaction condition asfollows: 94 40 sec，55 40 sec，72 60 sec，totally 29 cycles for cyclin E2 and  -actin; 94 30 sec，55 30 sec，72 60 sec，totally 35 cycles，72 10 min for survivin to stop the reaction.For each set of PCR reactions，parallel   reaction using double-distilled water 

25、; instead of the cDNA template solution  as a negative control to assure the quality of the PCR.PCR primers were synthesized  by Cybersyn.Survivin: sense primer: 5-TTGGCAGGTGCCTGT TGAAT-3，antisense  primer: 5-AGCCAGTCCCC CACAGCAT-3.The am plified product should be 465 bp3. cyclin E2 p

26、rimer was designed with oligo 6.2 according to gene gi3885975:  sense primer 5-TG GCTTTTAGAGGTATGTGAA-3，antisense primer 5-TAATGAATCAATGGCTAGAAT-3 product  426 bp，-actin sense  primer 5-TCATCACCATTGGCAAT GAG-3，antisense  primer 5-CACTGTGTTGGCGTA CAGGT-3，product 155 bp。PCR analysi

27、s of productsThe electrophoresis of 10 l PCR products was performed  at 85 V for 90 min in 2% agarose gel contained  0.5 mg/L ethidium bromide.And the bands were viewed and photographed under UV illumination. The transcripts were estimated  by the optical density ratio of cyclin E2 or

28、 survivin to  -actin.The ratio of  positive standard was larger than  0.15.Statistical analysisThe data was registered as mean±SD.Difference  between different groups were evaluated by the chi-square test，correction for continuity chi-square test，and exact probabilities in 2

29、×2 table.Correlation test: analysis of Pearson correlation，P values of less than 0.05 was considerd statistically significant.Statistical analysis was performed using SPSS 10.0 computer software.ResultsExpression of cyclin E2 and survivin mRNA in K562 cells  The expression of both cyclin E2 and survivin in the K562 c