WT1反义寡核苷酸对急性白血病细胞的抑制作用
摘要
前言
WT1基因首先在Wilms′瘤细胞中发现，位于11p13，全长50kb，共有10个外显子，转录52-54KDa的DNA结合蛋白。近年来，人们对它在造血系统中的作用进行了研究。发现去除鼠胚胎WT1基因后，鼠造血系统发育迟缓。WT1能调控CSF-1基因、TGF(转化生长因子)基因、bcl-2、c-myc以及维甲酸受体基因的表达，从而调节造血细胞的功能。在白血病细胞中，该基因有以下表现：1．过度表达；2．表达水平与预后呈负性相关；3．表达升高与复发有关；4．抑制G-CSF对髓前体细胞的分化作用，诱导其促进增殖作用；5．WT1在一些白血病中发生突变，不能调控细胞的生长、分化和增殖。这些现象提示WT1在白血病的发生中起癌基因作用。WT1反义寡核苷酸是与WT1基因转录本mRNA翻译起始段互补的一段18nt(nucleotide)的寡核苷酸链。
本实验目的是通过WT1反义寡核苷酸抑制WT1基因表达，观察白血病细胞形态及增殖行为的变化，进一步研究WT1基因在白血病发生发展中的作用，探讨WT1反义寡核苷酸在白血病反义治疗中的应用。
实验材料
一、主要试剂和仪器：Ficoll淋巴细胞分离液、Trizol、氯仿、异丙醇、dNTP(四种脱氧核糖核苷酸)、RNA酶抑制剂、逆转录酶、Taq酶、RPMI1640完全培养液。上述试剂均由传染病实验室提供。引物、反义寡核苷酸由宝生物公司合成。CO_2培养箱(TE-
HER WATER JACKET CO。GAS INCUBATOR）、高速冷冻离o机
（HERAEUS BIOFUGE PRIMOR）、PCR扩增仪（PE删NELMER
GENE AMP PCR SYS亚M 2400）、电泳仪（DYY－Ill型稳压稳流电
泳仪X－152℃低温冰箱门ANYO MDF－1155X－30℃低温冰箱
（SANYO eDICAL FREEZER）。
实 验 方 法
一、WTI基因阳性细胞的筛选和培养
用RT－PCR检测白血病患者骨髓单个核细胞WI’1基因，将
阳性的白血病细胞生长于含有m％胎牛血清5 链霉素和
100IU／Inl青霉素的 RPMll640培养液中，于 37℃j％CO八饱和湿
度的培养箱中传代培养。所有实验均采用对数生长期细胞。
二、反义寡核耷酸的制备
采用针对翻译起始位点的反义DNA寡核耷酸链，序列如下：
有义链 5’CAGCAAATGGGCTCCGAC3’，反义链 5’GTCGGAGC－
CCA…CCTG3’，均为硫代修饰刀PLC纯化。寡核着酸链由宝生
物公司合成。
三、反义寡核着酸处理细胞
1．2 X 10VInl和 ZX 10’／Inl的白血病细胞悬液分别接种于培
养瓶和96孔培养板中。每个培养瓶内细胞悬液总量为500ul，用
于 WTI基因表达检测；96孔培养板中每孔内加 50ul细胞悬液，用
于形态学观察以及细胞相对生存率的检测。
L不同培养方式的细胞均分 3组进行实验：AS－oh驴iner（反
义链寡核耷酸）组百一olisome（有义链寡核着酸）组和对照组，分
别加人等体积的反义链寡核耷酸、有义链寡核耷酸及培养液。细
胞初始浓度：96孔培养板中为 IX 10Vnil，培养瓶中为IX 10～ml。
寡核管酸试剂的终浓度：96孔板为15oUyInL，培养瓶中为18oU才
·二·
Inl。先在初始浓度下培养24小时，以后每24小时追加一次试剂
（每次浓度4OUgimLmLL共持续培养96小时。实验中所有加样方法
相同。
四、结果检测
1．W’TI基因的检测
96小时后收集培养瓶中各组细胞，采用RT－PCR进行WTI
基因检测。
2．细胞形态学检测
各组细胞分别制成细胞涂片，经瑞氏一姬姆萨染色。在光学
显微镜下观察其形态变化。
3．细胞生长曲线
从加人寡核耷酸起，每24小时计数96孔培养板中各组细胞
浓度，并计算细胞相对存活率：实验组细胞创对照组细胞数X
100％。
五、统计分析
：采用 SPSSll．0统计软件分析，数据以 kn表示，数据分析采
用方差分析等方法，p＜0．05认为有统计学意义。
实 验 结 果
一、WTI基因的检测
寡核着酸处理96小时后，收集各组细胞，用RT－PCR方法进
行W’YI基因检测。发现AS组WTI基因由阳性转变成阴性。S
组和对照组仍为阳性。说明反义寡核着酸有效地阻断了基因的表
达。
二、细胞形态学检测
各组细胞进行形态学比较，显微镜下AS组细胞核固缩或破
碎，细胞死亡增多。说明WTI反义寡核耷酸能促进细胞死亡。进
·3·
一步揭示WTI基因在白血病细胞中的癌基因作用。
三、细胞生长曲线
每 24小时取 AS组3组和对照组细胞做细胞计数。计算细
胞相对存活率：实验组细胁对照组细胞 X 100％。结果显示 AS
组细胞生长明显受到抑制，相对存活率低，而S组细胞无明显抑
制。说明W’I’1反义寡核管酸对白血病细胞的增殖有明显抑制作
用。
讨 论
本实验用WTI反义寡
Introduction
WT1, originally isolated from Wilms' tumor cells, is at chromosome 11 pi3, spans about 50kb and comprises ten exons as well as mRNAs of approximately 3kb. WT1 proteins - DNA binding factors, have molecular masses of 52 - 54kDa. In recent years, researchers explored its role on hematopoiesis. They have found that embryo cells lacking WT1 show deficits in hematopoietic stem cell function. WT1 can regulate the expression of the CSF-1, IGF-IR, bcl-2, c-myc and retinoic acid receptor genes. So it regulates the hematopoiesis in this way. In leukemia cells, WT1 has abnormal functions and expressions : an aberrant overexpression of wild - type WT1 in leukemia cells; an inverse correlation between WT1 expression levels and prognosis; an increase in WT1 expression levels at relapse; inhibition of differentiation and induction of proliferation myeloid progenitor cells in response to G - CSF stimulation; Some leukemias have mutations in WT1 gene, which produce truncated proteins with ruinated zinc - finger domain. Due to the absence of zinc -finger domain, WT1 protein can not regular the target genes and control the development , the proliferation and the division of the cells. So did its negative control on itself. These phenomenon show that WT1 gene is a tumor gene during the development of the leukemia.
WT1 gene antisense oligonucleotides complement with original transcript part of WT1 mRNA, which contain 18 nucleotides.
The object of this experiment is to inhibit the expression of WT1 gene by antisense oligonucleotides , to observe the changes of morphology and proliferation behavior of leukemia cells, to research the role of the WT1 in the genesis and development of leukemia and discuss the application of antisense oligonucleotides on bone marrow depletion and antisense therapy.
Materials
Main materials and equipment
Ficoll lymphocyte separative liquid , Trizol, chloroform, dNTP, RNA AMV , RPMI1640 , Taq DNA polymerase are provided from Infectious Disease Experiment Lab. Primers and oligonucleotides are purchased from biological company. CO2 gas incubator, -152 C medical freezer, PCR equipment.
Methods
WT1 gene assay and cell culture
Leukemia cells , expressing the WT1 gene , drawn out of patients' bone marrow , were cultured in RPM1 1640 which contain 10% heat-inacted fetal bovine serum, 50ug/ml streptomycin and 100IU/ ml penicillin and incubated at 37 C in 5% CO2 atmosphere. The cells in the logarithmic growth phase are used in experiment.
Antisense oligonucleotides preparation
Design the antisense oligonucleotide to complement with the original sites for translation. The sequence is followed: sense strand:5' CAGCAAATGGGCTCCGAC3'; antisense strand:5'GTCGGAGC-
CCATTTGCCTG3', which were modified with sulfration and purified with HPLC.
Cells treatment with antisense oligonucelotide
Leukemia cells, are divided into three groups and separately cultured with antisense oligonucleotides, sense oligonucleotides and RP-MI1640. We add the reagents with 40ug/mL per 24 hours until 96 hours.
Assays for WT1 gene expression
Collecting cells of each group after 96 hours and detecting the expression of WT1 gene by RT - PCR .
Assays for the changes of morphology
Dyeing the cells before and after experiment observing the morphological changes under the microscope.
Assays for the proliferation of cells
Counting the cells per 24hours and computing the relative survival percent : amount of experiment group cells/control group cells x 100%.
Result
1. The expression of WT1 gene
Collecting each group cells after treatment with antisense oligonu-cleotids for 96 hours, we detected the WT gene by RT - PCR. The result is that WT1cDNA in AS group was disappeared, while that in both S and control groups could still be detected. We can conclude that this antisense oligonucleotide can inhibit the expression of WT1 gene effectively.
2. Assays for the morphological changes
The cells after treatment with antisense oligonucleotide were dead a lot. This phenomena shows tha
WT1基因首先在Wilms′瘤细胞中发现，位于11p13，全长50kb，共有10个外显子，转录52-54KDa的DNA结合蛋白。近年来，人们对它在造血系统中的作用进行了研究。发现去除鼠胚胎WT1基因后，鼠造血系统发育迟缓。WT1能调控CSF-1基因、TGF(转化生长因子)基因、bcl-2、c-myc以及维甲酸受体基因的表达，从而调节造血细胞的功能。在白血病细胞中，该基因有以下表现：1．过度表达；2．表达水平与预后呈负性相关；3．表达升高与复发有关；4．抑制G-CSF对髓前体细胞的分化作用，诱导其促进增殖作用；5．WT1在一些白血病中发生突变，不能调控细胞的生长、分化和增殖。这些现象提示WT1在白血病的发生中起癌基因作用。WT1反义寡核苷酸是与WT1基因转录本mRNA翻译起始段互补的一段18nt(nucleotide)的寡核苷酸链。
本实验目的是通过WT1反义寡核苷酸抑制WT1基因表达，观察白血病细胞形态及增殖行为的变化，进一步研究WT1基因在白血病发生发展中的作用，探讨WT1反义寡核苷酸在白血病反义治疗中的应用。
实验材料
一、主要试剂和仪器：Ficoll淋巴细胞分离液、Trizol、氯仿、异丙醇、dNTP(四种脱氧核糖核苷酸)、RNA酶抑制剂、逆转录酶、Taq酶、RPMI1640完全培养液。上述试剂均由传染病实验室提供。引物、反义寡核苷酸由宝生物公司合成。CO_2培养箱(TE-
HER WATER JACKET CO。GAS INCUBATOR）、高速冷冻离o机
（HERAEUS BIOFUGE PRIMOR）、PCR扩增仪（PE删NELMER
GENE AMP PCR SYS亚M 2400）、电泳仪（DYY－Ill型稳压稳流电
泳仪X－152℃低温冰箱门ANYO MDF－1155X－30℃低温冰箱
（SANYO eDICAL FREEZER）。
实 验 方 法
一、WTI基因阳性细胞的筛选和培养
用RT－PCR检测白血病患者骨髓单个核细胞WI’1基因，将
阳性的白血病细胞生长于含有m％胎牛血清5 链霉素和
100IU／Inl青霉素的 RPMll640培养液中，于 37℃j％CO八饱和湿
度的培养箱中传代培养。所有实验均采用对数生长期细胞。
二、反义寡核耷酸的制备
采用针对翻译起始位点的反义DNA寡核耷酸链，序列如下：
有义链 5’CAGCAAATGGGCTCCGAC3’，反义链 5’GTCGGAGC－
CCA…CCTG3’，均为硫代修饰刀PLC纯化。寡核着酸链由宝生
物公司合成。
三、反义寡核着酸处理细胞
1．2 X 10VInl和 ZX 10’／Inl的白血病细胞悬液分别接种于培
养瓶和96孔培养板中。每个培养瓶内细胞悬液总量为500ul，用
于 WTI基因表达检测；96孔培养板中每孔内加 50ul细胞悬液，用
于形态学观察以及细胞相对生存率的检测。
L不同培养方式的细胞均分 3组进行实验：AS－oh驴iner（反
义链寡核耷酸）组百一olisome（有义链寡核着酸）组和对照组，分
别加人等体积的反义链寡核耷酸、有义链寡核耷酸及培养液。细
胞初始浓度：96孔培养板中为 IX 10Vnil，培养瓶中为IX 10～ml。
寡核管酸试剂的终浓度：96孔板为15oUyInL，培养瓶中为18oU才
·二·
Inl。先在初始浓度下培养24小时，以后每24小时追加一次试剂
（每次浓度4OUgimLmLL共持续培养96小时。实验中所有加样方法
相同。
四、结果检测
1．W’TI基因的检测
96小时后收集培养瓶中各组细胞，采用RT－PCR进行WTI
基因检测。
2．细胞形态学检测
各组细胞分别制成细胞涂片，经瑞氏一姬姆萨染色。在光学
显微镜下观察其形态变化。
3．细胞生长曲线
从加人寡核耷酸起，每24小时计数96孔培养板中各组细胞
浓度，并计算细胞相对存活率：实验组细胞创对照组细胞数X
100％。
五、统计分析
：采用 SPSSll．0统计软件分析，数据以 kn表示，数据分析采
用方差分析等方法，p＜0．05认为有统计学意义。
实 验 结 果
一、WTI基因的检测
寡核着酸处理96小时后，收集各组细胞，用RT－PCR方法进
行W’YI基因检测。发现AS组WTI基因由阳性转变成阴性。S
组和对照组仍为阳性。说明反义寡核着酸有效地阻断了基因的表
达。
二、细胞形态学检测
各组细胞进行形态学比较，显微镜下AS组细胞核固缩或破
碎，细胞死亡增多。说明WTI反义寡核耷酸能促进细胞死亡。进
·3·
一步揭示WTI基因在白血病细胞中的癌基因作用。
三、细胞生长曲线
每 24小时取 AS组3组和对照组细胞做细胞计数。计算细
胞相对存活率：实验组细胁对照组细胞 X 100％。结果显示 AS
组细胞生长明显受到抑制，相对存活率低，而S组细胞无明显抑
制。说明W’I’1反义寡核管酸对白血病细胞的增殖有明显抑制作
用。
讨 论
本实验用WTI反义寡
Introduction
WT1, originally isolated from Wilms' tumor cells, is at chromosome 11 pi3, spans about 50kb and comprises ten exons as well as mRNAs of approximately 3kb. WT1 proteins - DNA binding factors, have molecular masses of 52 - 54kDa. In recent years, researchers explored its role on hematopoiesis. They have found that embryo cells lacking WT1 show deficits in hematopoietic stem cell function. WT1 can regulate the expression of the CSF-1, IGF-IR, bcl-2, c-myc and retinoic acid receptor genes. So it regulates the hematopoiesis in this way. In leukemia cells, WT1 has abnormal functions and expressions : an aberrant overexpression of wild - type WT1 in leukemia cells; an inverse correlation between WT1 expression levels and prognosis; an increase in WT1 expression levels at relapse; inhibition of differentiation and induction of proliferation myeloid progenitor cells in response to G - CSF stimulation; Some leukemias have mutations in WT1 gene, which produce truncated proteins with ruinated zinc - finger domain. Due to the absence of zinc -finger domain, WT1 protein can not regular the target genes and control the development , the proliferation and the division of the cells. So did its negative control on itself. These phenomenon show that WT1 gene is a tumor gene during the development of the leukemia.
WT1 gene antisense oligonucleotides complement with original transcript part of WT1 mRNA, which contain 18 nucleotides.
The object of this experiment is to inhibit the expression of WT1 gene by antisense oligonucleotides , to observe the changes of morphology and proliferation behavior of leukemia cells, to research the role of the WT1 in the genesis and development of leukemia and discuss the application of antisense oligonucleotides on bone marrow depletion and antisense therapy.
Materials
Main materials and equipment
Ficoll lymphocyte separative liquid , Trizol, chloroform, dNTP, RNA AMV , RPMI1640 , Taq DNA polymerase are provided from Infectious Disease Experiment Lab. Primers and oligonucleotides are purchased from biological company. CO2 gas incubator, -152 C medical freezer, PCR equipment.
Methods
WT1 gene assay and cell culture
Leukemia cells , expressing the WT1 gene , drawn out of patients' bone marrow , were cultured in RPM1 1640 which contain 10% heat-inacted fetal bovine serum, 50ug/ml streptomycin and 100IU/ ml penicillin and incubated at 37 C in 5% CO2 atmosphere. The cells in the logarithmic growth phase are used in experiment.
Antisense oligonucleotides preparation
Design the antisense oligonucleotide to complement with the original sites for translation. The sequence is followed: sense strand:5' CAGCAAATGGGCTCCGAC3'; antisense strand:5'GTCGGAGC-
CCATTTGCCTG3', which were modified with sulfration and purified with HPLC.
Cells treatment with antisense oligonucelotide
Leukemia cells, are divided into three groups and separately cultured with antisense oligonucleotides, sense oligonucleotides and RP-MI1640. We add the reagents with 40ug/mL per 24 hours until 96 hours.
Assays for WT1 gene expression
Collecting cells of each group after 96 hours and detecting the expression of WT1 gene by RT - PCR .
Assays for the changes of morphology
Dyeing the cells before and after experiment observing the morphological changes under the microscope.
Assays for the proliferation of cells
Counting the cells per 24hours and computing the relative survival percent : amount of experiment group cells/control group cells x 100%.
Result
1. The expression of WT1 gene
Collecting each group cells after treatment with antisense oligonu-cleotids for 96 hours, we detected the WT gene by RT - PCR. The result is that WT1cDNA in AS group was disappeared, while that in both S and control groups could still be detected. We can conclude that this antisense oligonucleotide can inhibit the expression of WT1 gene effectively.
2. Assays for the morphological changes
The cells after treatment with antisense oligonucleotide were dead a lot. This phenomena shows tha
引文
1. Sean Bong Lee and Daniel A. Haberl Wilms Tumor and the WT1 Gene Experimental Cell Research 264, 74-99 (2001)
2. Alberta JA, Springett GM, Rayburn H, Natoli TA, Loring J, Kreidberg JA, Housman D. Role of the WT1 tumor suppressor in mufine hematopoiesis. Blood. 2003 Apr 1 ;101(7) :2570-4
3. Akihiro Tsuboi a, Yoshihiro Oka a, Hiroyasu Ogawa a, Olga A Elisseeva a, etc. Constitutive expression of the Wilms' tumor gene WT1 inhibits the differentiation of myeloid progenitor cells but promotes theirproliferation in response to granulocyte -colony stimulating factor( G - CSF). Leukemia Research 23 (1999) 499-505
4. Volkhern Scharnhorst. Alex J. Vander Eb. Aart G. Jochemsen. WT1protein: functions in growth and differentiation. Gene 273 (2001) 141-161
5. Loeb DM, Sukumar S. The role of WT1 in oncogenesis: tumor suppressor or oncogene? Int J Hematol. 2002 Aug; 76 (2): 117-26. Review.
6. Ellisen LW. Regulation of gene expression by WT1 in development and tumorigenesis. Int J Hematol 2002 Aug;76(2) :110-6
7. Algar E. A review of the Wilms' tumor 1 gene (WT1) and its role in hematopoiesis and leukemia. J Hematother Stem Cell Res. 2002 Aug;11(4) :589-99. Review.
8. Reuven Agami. RNAi and related mechanisms and their potential use for therapy Current Opinion in Chemical Biology 2002, 6:829-834
9. Hosen N, Sonoda Y, Oji Y, Kimura T, Minamiguchi H, Tamaki
H, Kawakami M, Very low frequencies Of human normal CD34 + haematopoietic progenitor cells express the Wilms' tumout gene WT1 at levels similar to those in leukaemia. Br J Haematol. 2002 Feb;116(2) :409-20
2. Alberta JA, Springett GM, Rayburn H, Natoli TA, Loring J, Kreidberg JA, Housman D. Role of the WT1 tumor suppressor in mufine hematopoiesis. Blood. 2003 Apr 1 ;101(7) :2570-4
3. Akihiro Tsuboi a, Yoshihiro Oka a, Hiroyasu Ogawa a, Olga A Elisseeva a, etc. Constitutive expression of the Wilms' tumor gene WT1 inhibits the differentiation of myeloid progenitor cells but promotes theirproliferation in response to granulocyte -colony stimulating factor( G - CSF). Leukemia Research 23 (1999) 499-505
4. Volkhern Scharnhorst. Alex J. Vander Eb. Aart G. Jochemsen. WT1protein: functions in growth and differentiation. Gene 273 (2001) 141-161
5. Loeb DM, Sukumar S. The role of WT1 in oncogenesis: tumor suppressor or oncogene? Int J Hematol. 2002 Aug; 76 (2): 117-26. Review.
6. Ellisen LW. Regulation of gene expression by WT1 in development and tumorigenesis. Int J Hematol 2002 Aug;76(2) :110-6
7. Algar E. A review of the Wilms' tumor 1 gene (WT1) and its role in hematopoiesis and leukemia. J Hematother Stem Cell Res. 2002 Aug;11(4) :589-99. Review.
8. Reuven Agami. RNAi and related mechanisms and their potential use for therapy Current Opinion in Chemical Biology 2002, 6:829-834
9. Hosen N, Sonoda Y, Oji Y, Kimura T, Minamiguchi H, Tamaki
H, Kawakami M, Very low frequencies Of human normal CD34 + haematopoietic progenitor cells express the Wilms' tumout gene WT1 at levels similar to those in leukaemia. Br J Haematol. 2002 Feb;116(2) :409-20