【作者】 郑敏；

【导师】 罗建明；

【作者基本信息】 广西医科大学 ， 儿科血液学， 2011， 博士

【摘要】 目的研究维生素D受体(VDR)基因FokI、BsmI、ApaI和TaqI位点多态性在广西地区儿童的分布,分析VDR基因多态性与儿童急性白血病(AL)遗传易感性之间的关系,以进一步了解AL的分子生物学机制。方法采用病例-对照的研究方法,在中国广西地区选取无血缘关系的127例AL儿童和268例健康体检儿童(作为对照组)作为研究对象,采集外周血,提取DNA,采用聚合酶链限制性片段长度多态性技术和基因测序技术,对两组儿童FokI、BsmI、ApaI和TaqI位点多态性进行分析。采用二项分布检验,对两组数据进行Hardy-Weinberg遗传平衡分析。组间频率比较采用χ2检验,以比值比(OR)及95%可信区间(CI)表示相对风险度。采用SHEsis软件进行VDR基因多态性位点连锁不平衡的计算。结果样本经遗传平衡检验表明,各基因型的观察值与期望值之间无显著性差异(P>0.05),说明所选的样本具有代表性。268例健康儿童VDR基因FokI位点ff、Ff和FF基因型频率分别为22.02%、48.13%和29.85%;f、F等位基因频率为46.08%和53.92%。BsmI位点bb、Bb和BB基因型频率分别为91.79%、7.84%和0.37%;b、B等位基因频率为95.71%、4.29%。ApaI位点aa、Aa和AA基因型频率分别为52.61%、36.57%和10.82%;a、A等位基因频率为70.90%、29.10%。TaqI位点tt、Tt和TT基因型频率分别为0%、9.70%和90.30%;t、T等位基因频率为4.85%、95.15%。结果显示广西地区健康儿童FokI、BsmI、ApaI和TaqI位点多态性分布情况与国内其它地区分布基本一致。127例AL儿童ff、Ff和FF基因型频率分别为35.43 %、43.31%和21.26%;f、F等位基因频率为57.09%、42.91%。bb、Bb和BB基因型频率分别为92.13%、7.87%和0;b、B等位基因频率为96.06%、3.94%。aa、Aa和AA基因型频率分别为48.82%、37.01%和14.17%;a、A等位基因频率为67.32%、32.68%。tt、Tt和TT基因型频率分别为0、11.81%和88.19%;t、T等位基因频率为5.91%、94.09%。FokI位点ff基因型在AL儿童的分布较健康儿童明显增高。与FF基因型相比,ff基因型患AL的风险性增加(OR=2.260,P<0.05);与F等位基因相比,携带f等位基因的个体更易罹患AL(OR=1.556,P<0.05)。BsmI、ApaI和TaqI位点多态性分布在病例组与对照组之间无显著性差异。FokI和BsmI、ApaI、TaqI位点之间不存在明显的连锁不平衡。结论中国广西地区儿童VDR基因多态性分布频率有其自身的特点。VDR基因FokI位点SNP可能是急性白血病发生的遗传易感因素之一,携带f等位基因的个体更易罹患急性白血病。VDR基因BsmI、ApaI和TaqI酶切位点的多态性可能与儿童白血病的遗传易感性无关。此结论仍需进一步行大样本实验证实。目的通过检测AL儿童VDR基因和蛋白的表达,从转录和翻译两个水平探讨VDR在AL中表达的意义,揭示VDR的表达与AL分类的可能联系。方法收集未经治疗的30例急性淋巴细胞白血病(ALL)和12例急性髓细胞白血病(AML)儿童、30例非肿瘤性血液病儿童(包括缺铁性贫血、溶血性贫血、特发性血小板减少性紫癜等,作为对照组)的骨髓标本,抽提总RNA,提取得到的RNA采用M-MLV逆转录试剂盒逆转录合成cDNA第一链,以逆转录得到的cDNA为模板,应用SYBR Green I实时荧光定量PCR方法,检测VDR基因mRNA的表达。扩增产物的特异性通过熔解曲线和琼脂糖凝胶电泳双重确认。以管家基因GAPDH基因为内参照,采用2-△△Ct法计算VDR mRNA的相对表达量,得到标化的VDR mRNA的表达。利用超敏S-P免疫细胞化学法检测了其中9例AL儿童和4例对照组骨髓细胞VDR的表达。收集未经治疗的30例ALL、10例AML和30例健康体检儿童(作为对照组)的外周血标本,提取单个核细胞(MNCs),取适量立即涂片应用免疫细胞化学法检测VDR的表达和定位情况;其余的MNCs用细胞裂解液抽提全细胞蛋白样品用于蛋白免疫印迹法检测VDR蛋白的相对表达量。用单因素方差分析比较ff、Ff和FF三种基因型VDR mRNA和蛋白表达的差异。对AL儿童VDR的表达和外周血白细胞、血小板的关系进行一元线性相关分析。结果VDR mRNA在所有检测的AL儿童和对照组儿童的骨髓中均有表达。ALL儿童VDR mRNA的表达(1.06±0.31)和AML的表达(1.13±0.34)明显低于对照组儿童的表达(3.10±0.18),组间有显著性差异(F=1701.00,P<0.01),而ALL和AML之间的表达无统计学差异(P>0.05)。经一元线性相关分析,结果显示42例AL儿童VDR mRNA的表达水平与外周血白细胞数无明显相关性(r=0.045, P=0.078),与外周血血小板数亦无明显相关性(r=0.067, P=0.675)。对42例AL儿童ff、Ff和FF三种基因型进行VDR mRNA表达的比较,VDR mRNA的表达分别为1.25±0.47(n=12),1.12±0.41(n=17),1.09±0.38(n=13),组间比较无统计学差异(F=0.489,P> 0.05)。免疫细胞化学法显示所检测样本的骨髓和外周血MNCs均表达VDR蛋白,VDR蛋白主要分布在细胞核内。被检测的9例AL儿童骨髓VDR蛋白的表达(30.80±1.56%)低于4例对照组儿童的表达(62.45±3.73%),由于样本例数太少,未作统计学分析。30例ALL儿童外周血VDR蛋白的表达(27.82±1.78%)和10例AML的表达(27.10±2.44%)明显低于30例健康儿童(59.02±3.46%),组间有显著差异(F=1150.26,P<0.01),而ALL和AML儿童中VDR蛋白的表达无统计学差异(P>0.05)。蛋白免疫印迹法亦显示相似结果:ALL儿童VDR蛋白的表达(0.299±0.071)和AML的表达(0.290±0.094)明显低于健康儿童(0.710±0.041),组间有显著差异(F=356.434,P< 0.01),而ALL和AML儿童VDR蛋白的表达无统计学差异(P>0.05)。一元线性相关分析显示40例AL儿童VDR蛋白的表达水平与外周血白细胞数无明显相关性(r=-0.133, P=0.413),与外周血血小板数亦无无直线相关关系(r=0.006,P=0.917)。病例组40例AL儿童ff、Ff和FF基因型VDR蛋白的表达分别为0.286±0.061(n=13),0.289±0.079(n=17), 0.324±0.089(n=10),组间比较无统计学意义(F=0.853,P>0.05)。结论所有检测的AL儿童的骨髓和外周血MNCs均表达VDR,考虑白血病细胞是VDR作用的靶细胞。AL儿童VDR mRNA和蛋白的表达较健康儿童明显降低,考虑VDR的表达情况可能成为AL诊断的有效指标。VDR的表达可能与AL分类、VDR基因型和初治时外周血象无关。目的研究不同浓度的1,25二羟基维生素D3(1,25(OH)2D3)对人白血病细胞株6T-CEM和HL-60生长、分化和凋亡的影响,并观察1,25(OH)2D3处理前后6T-CEM和HL-60细胞VDR mRNA和蛋白表达的变化,探讨其抗白血病的分子机制及用于白血病治疗的可行性。方法在体外培养条件下,用不同浓度(10-9,10-8,10-7,10-6 mol/L)的1,25(OH)2D3作用处于对数生长期的6T-CEM和HL-60。应用台盼蓝拒染法计数活细胞,甲基噻唑基四唑(MTT)比色法分析细胞增殖抑制作用,硝基四氮唑蓝(NBT)还原实验法分析HL-60细胞的分化指标,末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记染色法(TUNEL)检测细胞晚期凋亡的变化,倒置显微镜及赖-吉染色法(Wright-Giemsa)观察细胞形态学改变,运用RT FQ-PCR方法检测VDR mRNA变化,免疫细胞化学法和蛋白免疫印迹技术检测VDR蛋白表达。结果MTT法显示不同浓度的1,25(OH)2D3作用后可抑制6T-CEM及HL-60细胞的生长,呈剂量和时间依赖性。不同浓度的1,25(OH)2D3作用后可诱导HL-60细胞向成熟粒细胞分化,对6T-CEM的分化无影响。TUNEL法显示1,25(OH)2D3作用后对6T-CEM和HL-60细胞有明显的促细胞凋亡作用,凋亡率随药物浓度的增高而增高。细胞形态学观察发现药物作用后细胞表现出凋亡的形态特征。1,25(OH)2D3作用前后6T-CEM和HL-60细胞均表达VDR mRNA和蛋白,药物作用前后VDR mRNA表达改变不明显,而VDR蛋白表达上调。结论1,25(OH)2D3在一定浓度范围内可抑制6T-CEM和HL-60细胞增殖,诱导细胞凋亡。1,25(OH)2D3可诱导HL-60细胞向成熟粒细胞分化。1,25(OH)2D3可使6T-CEM和HL-60细胞VDR蛋白表达上调,但不改变VDR mRNA水平。更多还原

【Abstract】 Objective To investigate the distribution of vitamin D receptor (VDR) gene FokI, BsmI, ApaI, TaqI site gene polymorphism of children in Guangxi region.To explore the correlation between the VDR gene polymorphism and hereditary susceptibility of leukemic children. We hope it will be useful to the followed study on the relative mechanism of acute leukemia. Methods This study adopted case-control design. 127 children with acute leukemia and 268 healthy medical examination children (as controls) who had unrelated blood relationship in Guangxi region were recruited. DNA was extracted from peripheral blood sample. Polymerase chain reaction-restrictive fragment length polymerphism (PCR-RFLP) analysis technique and DNA sequencing technology were used to detect gene polymorphisms of FokI, BsmI, ApaI, TaqI locus. The Hardy -Weinberg balance of heredity in the two groups was analyzed by binomial distribution test. The difference between the frequency of VDR alleles of the two groups was analyzed by chi square test. Odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the relative risk. The linkage disequilibrium of VDR gene was analyzed by SHEsis software. Results The result of Hardy-Weinberg balance of heredity showed that there were insignificant difference between the observed value and the expected value of the frequency of FokI, BsmI, ApaI, TaqI alleles in the two groups, P>0.05. It was a declaration that the samples were representation. In the 268 healthy children, we obtained the percentage of genotypes ff, Ff and FF to be 22.02%, 48.13% and 29.85% respectively and the allelic frequencies of 46.08% and 53.92% for f and F allele. The frequency of bb, Bb and BB in the BsmI site were 91.79%, 7.84% and 0.37%. Frequencies of b and B allelic gene were 95.71% and 4.29%. The frequency of aa, Aa and AA in the ApaI site were 52.61%, 36.57% and 10.82% respectively. Frequencies of a and A allelic gene were 70.90% and 29.10%. tt, Tt and TT in the TaqI site were 0, 9.70% and 90.30% respectively. Frequencies of t and T allelic gene were 4.85% and 95.15%. The result displayed that FokI, BsmI, ApaI, TaqI site gene polymorphism of healthy children in Guangxi region was one and all with the other regions in China. In the 127 children with acute leukemia, we obtained the percentage of genotypes ff, Ff and FF to be 35.43%, 43.31% and 21.26% respectively and the allelic frequencies of 57.09% and 42.91% for f and F allele. The frequency of bb, Bb and BB in the BsmI site were 92.13%, 7.87% and 0. Frequencies of b and B allelic gene were 96.06% and 3.94%. The frequency of aa, Aa and AA in the ApaI site were 48.82%, 37.01% and 14.17% respectively. Frequencies of a and A allelic gene were 67.32% and 32.68%. tt, Tt and TT were 0, 11.81% and 88.19%. Frequencies of t and T allelic gene were 5.91% and 94.09%. Allele frequencies of the FokI polymorphism showed a significant association (χ2= 8.671, P<0.05) between the two groop. The genotype ff in the acute leukemic children was higher than that in the healthy children. The result of one way analysis showed that the subjects carrying ff genotype had 2.26 fold elevated risk for developing acute leukemia compared with the FF genotype (OR=2.260, P<0.05). Using the F allele as a reference, a significant risk of developing acute leukemia was found between the presence of f allele (OR=1.556, P<0.05). The genotype and allele frequencies of BsmI, ApaI and TaqI site were not significantly different from those in control group (P>0.05). There had no strongly apparent linkage disequilibrium between the FokI locus, BsmI locus, ApaI locus and TaqI locus. Conclusions The polymorphism frequency and distribution of this VDR gene in Guangxi population of China exhibit its own characteristics. There maybe the genetic susceptibility effect of VDR gene FokI allele in acute leukemia while the gene polymorphisms of BsmI, ApaI, TaqI enzyme digestion locus may not be related to genetic susceptibility of children acute leukemia. The individual with f allele were more easier to take AL than that with F. The conclusion must confirm by further arge sample arge sample.Objective To study the expression of VDR in children of acute leukemia and to approach the significance of VDR in the occurrence of acute leukemia from transcription and translation.To determine whether the expression is related to the classification of acute leukemia. Methods Total RNA was extracted by trizol from bone marrow cells of thirty untreated children with acute lymphoblastic leukemia (ALL), twelve untreated children with acute myeloid leukemia (AML) and thirty cases nontumorous hematologic diseases children(including iron deficiency anemia, hemolytic anemia and idiopathic thrombocytopenic purpura, et al, as controls). The first strand cDNA was acquired by reverse transcription. SYBR Green I Rea1 time fluorescent quantitative PCR (RT FQ-PCR) was used to investigate VDR mRNA expression in the three groups. The specificity of amplification production was measured by melting curves and agarose gel electrophoresis. The VDR mRNA signal was normalized to in accordance with the house-keeping gene GAPDH mRNA signal through the method of 2-△△Ct. Appropriate amount bone marrow cells from nine children with acute leukemia and four cases nontumorous hematologic diseases children were smeared immediately to detect the expression of VDR protein and cell localization by ultrasensitive streptavidin-biotin peroxidase (S-P) method of immunocytochemical staining. Peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood prepared of thirty untreated children with ALL, ten untreated children with AML and thirty healthy children (as controls). Appropriate amount PBMCs were smeared immediately to detect the expression of VDR protein and cell localization by immunocytochemical staining while all cell albumen extracted from the other PBMCs by cell lysate was used to detect the expression of VDR protein by Western blots analysis. The different expression of VDR mRNA and protein in the three gene types ff, Ff and Ff was compared by one-factor analysis of variance. The relationship of VDR expression and the leukocyte number and the platelet number in children with acute leukemia was analyzed by bivariate linear correlation. Results VDR mRNA was detected in all the acute leukemic children and nontumorous hematologic diseases children. The expression of VDR mRNA in children with ALL (1.06±0.31) and in children with AML (1.13±0.34) was lower significantly compared with the expression in the controls (3.10±0.18) (F=1701.00, P<0.01) while there was no significant change between children with ALL and children with AML (P>0.05). The expression lever of VDR mRNA in forty two cases children with acute leukemia had no associativity (r=0.045, P=0.078) with the leukocyte number and the platelet number (r=0.067, P=0.675) by bivariate linear correlation while there was no significant changed at the expression lever of VDR mRNA in the three gene types ff (1.25±0.47, n=12), Ff (1.12±0.41, n=17) and FF (1.09±0.38, n=13) (F=0.489, P>0.05). The results of immunocytochemistry techniques showed VDR protein was expressed in all marrow mononuclear cell and PBMNCs of each group. VDR protein was mainly localized in cell nucleus. The expression of VDR protein in nine children with AL(30.80±1.56%) was lower than that in four control subject (62.45±3.73%) but statistics analysis was not done because the sample is only a few. The expression of VDR protein in the children with ALL (27.82±1.78%) and in the children with AML (27.10±2.44%) were lower significantly compared with the expression in the control subjects (59.02±3.46%) (F=1164.47, P<0.01) while there was no significant different between the children with ALL and the children with AML (P>0.05). The result of Western blots analysis was accordance with immunocytochemistry techniques. The expression of VDR protein in children with ALL (0.299±0.071) and in children with AML (0.290±0.094) were lower significantly compared with the expression in the control subjects (0.710±0.041) (F=356.434, P<0.0) while there was no significant different between children with ALL and children with AML (P>0.05). The expression lever of VDR protein in forty cases children with acute leukemia had no associativity with the leukocyte number (r=-0.133, P=0.413) and the platelet number (r=0.006, P=0.917) by bivariate linear correlation while there was no significant changed at the expression lever of VDR protein in the three gene types ff (0.286±0.061, n=13), Ff (0.289±0.079, n=17) and FF (0.324±0.089, n=10) ((F=0.853, P>0.05). Conclusions VDR was expressed in all the acute leukemic children and we get such a conclusion that leukemic cells were one of the target cells of VDR. The expression of VDR mRNA and VDR protein in acute leukemia children was significantly lower than that in control subjects. VDR expression might be a effective monitoring index in diagnosing of leukemia. The expression of VDR may be had no associativity with classification, genotype and peripheral blood index. Objective To investigate the possible mechanism of inducing apoptosis of 1,25-Dihydroxyvitamin D3 (1,25(OH)2 D3 ) and to determine the therapeutic potential of 1,25(OH)2D3 in leukemia, we observed the effects of inhibition, differentiation, apoptosis and the expression of VDR gene and protein on human leukemia cell lines 6T-CEM and HL-60 induced by 1,25(OH)2D3 at different concentrations. Methods Leukemia cell lines 6T-CEM and HL-60 in exponential growth phase were treated by 1,25(OH)2 D3 with different concentration of 10-9, 10-8, 10-7 and 10-6 mol/L in vitro. Viable cells were counted by Trypan blue exclusion. Methy thiazoly tetrazolium (MTT) assay was used to evaluate the cell proliferation. The differentiation of HL-60 cells was detected by nitro blue tetrazolium (NBT) reduction test. The cells late apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining assays (TUNEL). Cell morphology was observed by inverted microscope and Wright-Giemsa staining method. The expression of VDR mRNA of each group was analyzed by RT FQ-PCR. The expressions of VDR protein were detectied by immunocytochemistry technique and Western blot analysis. Results MTT assay showed that 1, 25(OH)2D3 could significantly inhibit the proliferation of 6T-CEM and HL-60 cells in a dose-dependent and time-dependent manner. 1,25(OH)2 D3 could remarkably induce HL-60 cells to differentiate towards mature granulocyte while there was not change in 6T-CEM cells. The result of TUNEL indicated 1,25(OH)2D3 with different concentration could enhanced cell apoptosis rate in a dose-dependent manner. The observation of cell morphology showed the cells treated by 1,25(OH)2D3 had morphologic characteristics of apoptosis. VDR mRNA and protein were expressed in the all 6T-CEM and HL-60 cells before and after the action of 1,25(OH)2D3. VDR protein expression was up-regulated after drug action while VDR mRNA expression was no change. Conclusions 1,25(OH)2D3 at a certain concentration range can significantly inhibit the proliferation and induce apoptosis in 6T-CEM and HL-60 cells. 1,25(OH)2D3 can induce HL-60 cells to differentiate towards mature granulocyte. 1, 25(OH)2D3 can increase expression of VDR protein in 6T-CEM and HL-60 cells but not VDR mRNA...更多还原