摘要
第一部分再障患者和健康人骨髓CD4+T细胞增殖能力、凋亡特性及其分泌的细胞因子对脐血CD34+造血干/祖细胞影响的比较研究
目的:从细胞增殖能力、凋亡特性及其分泌的细胞因子对造血干/祖细胞的影响几方面,全面评估再障患者骨髓CD4+T细胞的生物学特性,为临床揭示再生障碍性贫血(再障)发生的免疫机制提供新线索。
方法:采用免疫磁珠分选法从再障患者和健康人群骨髓中分离出CD4+T细胞,用改良MTT法检测两者骨髓CD4+T细胞的增殖能力;用高浓度CD3单抗诱导CD4+T细胞凋亡,比较再障患者和健康人群骨髓CD4+T细胞凋亡特性的差异;以两者骨髓CD4+T细胞培养上清作用于脐血来源的CD34+造血干/祖细胞,观察脐血CD34+造血干/祖细胞CFU-GM的形成,关键Cyclin D亚型(Cyclin D3)mRNA及蛋白的表达水平。
结果:与健康人相比,再障患者的骨髓CD4+T细胞显示出明显更强的增殖能力。高浓度CD3单抗作用18h后,电镜下可见正常组和再障组均有细胞凋亡,但再障患者较正常人更多。与电镜结果一致,Annexin V-FITC流式细胞仪检测凋亡率发现再障患者早期凋亡和晚期凋亡的细胞均较正常人多。早期凋亡率:正常组(7.03±0.86)%,再障组(16.11±1.37)%;晚期凋亡率:正常组(2.07±0.42)%,再障组(8.05±0.36)%。以骨髓CD4+T细胞培养上清作用于脐血来源的CD34+造血干/祖细胞,倒置显微镜下观察结果,再障组CFU-GM为:(74.50±9.50)/104个细胞;正常组CFU-GM为:(124.25±19.80)/104个细胞,两组比较有显著性差异。RT-PCR和Western Blot分析显示,再障患者骨髓CD4+T细胞培养上清能从mRNA和蛋白水平抑制脐血CD34+造血干/祖细胞Cyclin D3的表达。
结论:再障患者骨髓CD4+T细胞在体内可能处于异常增殖、活化的状态,并且可能与再障患者骨髓的造血功能衰竭密切相关;再障患者骨髓CD4+T细胞可分泌某些可溶性的细胞因子,通过抑制造血干/祖细胞Cyclin D3的表达,从而抑制其增殖,引起造血衰竭。
第二部分再生障碍性贫血患者骨髓CD4+T细胞抑制消减杂交文库的建立
目的:分析比较再障患者与正常人骨髓CD4+T细胞的基因组变化,以期获得与再障患者骨髓CD4+ T细胞功能改变有关的基因。
方法:从再障患者和健康供髓者骨髓中分离出CD4+ T细胞,以健康供髓者骨髓CD4+T细胞作为“driver”,以再障患者骨髓CD4+T细胞作为“tester”,筛选再障患者骨髓CD4+T细胞中差异表达的基因,建立抑制消减杂交文库。
结果:我们提取的细胞总RNA进行RNA甲醛变性琼脂糖凝胶电泳显示清晰的28S、18S、5S三条带,提示所提取的RNA完整无缺。通过PCR方法对接头连接效率的分析可以看出基因特异性引物(G3PDH的3’引物)和PCR引物1的PCR产物同利用两个基因特异性引物(G3PDH的3’和5’引物)所扩增的PCR产物强度相近,说明接头连接效率较高。所建文库共检出阳性克隆110个,扩增片段在200～700bp间,与预期结果一致,PCR阳性检出率高达88%。
结论:我们利用抑制消减杂交技术成功地构建了再障患者骨髓CD4+T细胞的消减cDNA文库,为后续实验打下了基础,减少了进一步筛选、克隆再障患者骨髓CD4+细胞特异表达基因的盲目性,提高了特异性。
第三部分差异表达基因克隆序列的测定及同源性分析
目的:对所建再障患者骨髓CD4+T细胞的消减cDNA文库进行序列测定,并对测序结果进行同源性分析,以了解这些克隆所代表的基因及其可能的功能,以便对它们进行有的放矢的研究。
方法:我们根据第二部分PCR鉴定结果,选择了插入片段大小不同的10个克隆进行DNA序列全自动分析。所用测序引物为pUC57/T多克隆位点上游通用引物序列M13-47即CGCCAGGGTTTTCCCAGTCACGAC。通过Internet互联网进入http://www.ncbi.nlm.nih.gov/BLAST/,选择合适参数,实现在线DNA序列的同源性分析。
结果:在所测序的10个克隆中,发现10个差异表达的克隆代表了8个已知基因(其中2个为重复检出基因)。它们分别是:①transducin (beta)-like 1X-linked receptor 1 (TBL1XR1)(NM024665)②T cell-specific transcription factor (Tcf-1)(NM213648)③ATP-binding cassette, sub-family B (MDR/TAP),member 6(NM005689)④ARP2 actin-related protein 2 homolog (ACTR 2)(NM005722)⑤zinc finger protein 561(ZNF561)(NM152289)⑥Interferon-inducible protein 1-8U(XR019460)⑦NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6(NM002493)⑧eukaryotic translation initiation factor 3, subunit 5(NM003754)。
结论:用Blast软件对所测定的序列与公共数据库(Genebank non-redundant database)作DNA序列同源性比较,发现多种基因参与了再障患者骨髓CD4+T细胞的功能调节。所获得的基因包括了与细胞蛋白合成、生物氧化、信号转导、增殖调控、细胞迁移有关的基因以及功能尚未明确的基因,这些基因在再障免疫病理损伤中的作用尚未见报道。
第四部分Tcf-1基因的生物信息学分析及其在再生障碍性贫血患者骨髓CD4+T细胞中的表达鉴定
目的:对所建再障患者骨髓CD4+T细胞消减cDNA文库中筛选所得的Tcf-1基因作在线生物信息学分析,以获取该基因及其编码蛋白的初步信息;对Tcf-1基因在再障患者骨髓CD4+T细胞中的表达进行鉴定。
方法:利用网络资源作Tcf-1基因电子组织表达谱、电子基因定位分析并探究Tcf-1基因编码蛋白的基本性质及功能位点;半定量RT-PCR法分析再障患者骨髓CD4+T细胞Tcf-1 mRNA的表达水平。
结果:利用UniGene数据库进行电子表达谱分析,显示Tcf-1主要表达于胸腺组织,在未明组织、结缔组织、前列腺、脾、淋巴结、结肠、心脏、肝脏、血液等组织中也有表达。该基因定位于染色体5q31.1,编码269个氨基酸,其蛋白分子式为C1355H2108N386O378S10,分子量为30174.6,理论等电点为9.82,为亲水蛋白,不是膜蛋白,主要存在于胞核中。Tcf-1蛋白含有一个HMG-BOX核心结构域,位于154～222个氨基酸之间。与消减杂交文库结果一致,再障患者较之正常人骨髓CD4+T细胞中Tcf-1 mRNA表达水平明显上调。
结论:作为T细胞特异表达的基因,Tcf-1具有一个HMG-BOX结构域,它在细胞中以转录因子的身份发挥作用,启动或抑制靶基因的转录。该基因在正常人骨髓CD4+T细胞中表达水平低,而在再障患者骨髓CD4+T细胞中呈现明显的高表达。
第五部分Tcf-1基因沉默对再生障碍性贫血患者骨髓CD4+T细胞作用的实验研究
目的:探讨Tcf-1基因在再障患者骨髓CD4+T细胞功能改变中所起的作用。
方法:利用psiRNA-hH1neo G2质粒,构建针对Tcf-1基因的shRNA表达载体psiRNA1, psiRNA2和psiRNA3。转染T细胞性细胞株Jurkat,用半定量RT-PCR法检测siRNA对Tcf-1 mRNA表达水平的抑制作用。把筛选得到的干扰效果最显著的psiRNA2转染再障患者骨髓CD4+T细胞,用半定量RT-PCR法检测转染后再障患者Tcf-1、c-myc、CD44 mRNA表达水平的变化。
结果:不同位点的shRNA表达载体转染Jurkat细胞48h对Tcf-1 mRNA的表达均有抑制作用,其中psiRNA2的抑制作用最强,其次为psiRNA1,抑制作用最弱的是psiRNA3。用psiRNA2载体转染再障患者骨髓CD4+T细胞,结果发现转染后再障患者骨髓CD4+T细胞Tcf-1 mRNA表达受抑的同时,细胞c-myc、CD44 mRNA的表达水平也不同程度地降低,由此推测c-myc、CD44可能为Tcf-1调控的下游靶基因。
结论:Tcf-1基因在再障患者骨髓CD4+T细胞中的高表达与其功能异常密切相关,它通过调控下游靶基因如c-myc、CD44等,参与了再障患者骨髓CD4+T细胞异常增殖、活化及分泌负性造血因子病理功能的产生。以Tcf-1基因为靶点,设计治疗再障的新策略,值得进一步深入的研究下去。
PartⅠThe comparison study of bone marrow CD4~+T cells in aplastic anemia patients and healthy subjects on proliferation capablity, apoptosis feature and the impact of their secreted cytokines on hematopoietic stem/progenitor cells
Objective: To discuss the specific properties of marrow CD4~+T cells from aplastic anemia patients in respecting cell proliferation capablity, apoptosis features and the impact of their secreted cytokines on hematopoietic stem/ progenitor cells so as to provide a novel clue for the pathological mechanism of aplastic anemia.
Methods: CD4~+T cells were isolated from the bone marrow of aplastic anemia patients and healthy subjects with immunomagnetic beads sorting. Their proliferation capabilities were examined with the improved MTT method. Their properties of apoptosis induced by high concentrational CD3 monoclonal antibody were detected. The influences of CD4~+T cell culture supernatant on cord blood-source CD34+ hematopoietic stem/progenitor cells were also observed, including the impact of cell culture supernatant on cord blood CD34+ hematopoietic stem/progenitor cell CFU-GM formation and key Cyclin D subgroup (Cyclin D3) mRNA and protein expression levels.
Results: Aplastic anemia group marrow CD4 + T cells presented more enhanced reproductive activity. Evident apoptosis cells could be seen under electron microscope in both normal group and aplastic anemia group after high concentrational CD3 monoclonal antibody effecting for 18h. The apoptosis cells were more in aplastic anemia group than normal group. Apoptosis-induction quantitative detection was done with flow cytometer:the apoptosis cells in the early and advanced stage of aplastic anemia group were more than those of normal group. Apoptosis rate in early stage: normal group(7.03±0.86)%, aplastic anemia group(16.11±1.37)%; apoptosis rate in advanced stage: normal group(2.07±0.42)%, aplastic anemia group(8.05±0.36)%. The influence of marrow CD4~+ T cell culture supernatant on cord blood CD34+ hematopoietic stem/progenitor cell CFU-GM formation was CFU-GM of aplastic anemia group (74.50±9.50)/104; CFU-GM of normal group (124.25±19.80)/104, observing under an inverted microscope. A significant difference was present in the comparison between the two groups. Cord blood CD34+ cell CyclinD3 mRNA and protein expression levels were down-regulated by marrow CD4~+ T cell culture supernatant of aplastic anemia patients.
Conclusion: Aplastic anemia patients marrow CD4~+T cells were likely in an abnormally proliferative,activated state which could correlate intimately with aplastic anemia hemopoiesis damage. Aplastic anemia patient marrow CD4~+T cell could secret some soluble humoral agents that could inhibit hematopoietic stem cell proliferation-dependent Cyclin D3, accordingly its proliferation was suppressed, resulting in hemopoiesis failure.
PartⅡEstablishment of a suppressive subtractive hybridization(SSH) library of aplastic anemia bone marrow CD4~+T cells
Objective: To compare the genome changes of marrow CD4~+T cells between from aplastic anemia patients and from healthy subjects in order to find some genes to regulate functions of marrow CD4~+ T cells of aplastic anemia patients.
Methods: Marrow CD4~+ T cells were separated with magnetic bead sorting technique. With CD4~+ T cells of a typical first-visit aplastic anemia patient as“tester”and those of a normal donor as“driver”, a cDNA library was established by suppressive subtractive hybridization.
Results: The agarose gel electrophoresis clearly indicated three bands--28s, 18s and 5s, thus suggesting that total RNA of bone marrow CD4~+ T cells extracted from normal donor and aplastic anemia patient were intact. The PCR analysis indicated that the band intensities of PCR products were similar between with G3PDH 3'primer, PCR Primer l and with G3PDH 3'primer, 5'primer. The result suggested that the efficiency of adaptor ligation was rather high. 110 clones were detected by means of PCR in the established subtractive library, which contained an inserted fragment with 100～700bp respectively, and the positive detected rate was up to 88%.
Conclusion: A SSH library of marrow CD4~+ T cells of aplastic anemia was successfully established. This study provided a solid foundation for the next experiment and made it easy for screening and cloning aplastic anemia-related genes of marrow CD4~+ T cells.
PartⅢSequence and Homology analysis of differentially expressed gene clones
Objective: To analyse the sequences of the clones from the above subtracted cDNA library and the homology of the differentially expressed genes.
Methods: The 10 clones which contained different inserted fragments were picked randomly out for sequence analysis. The used sequencing primer was M13-47 (CGCCAGGGTTTTCCCAGTCACGAC). By Blast software, the obtained sequences were compared with Genebank non-redundant database.
Results: These 10 differentially expressed gene clones represented 8 known genes including 2 repeatedly detected ones. They were①transducin (beta)-like 1X-linked receptor 1 (TBL1XR1) ( NM024665 )②T cell-specific transcription factor (Tcf-1)(NM213648)③ATP-binding cassette, sub-family B (MDR/TAP),member 6(NM005689)④ARP2 actin-related protein 2 homolog (ACTR 2)(NM005722)⑤zinc finger protein 561(ZNF561)(NM152289)⑥Interferon-inducible protein 1-8U(XR019460)⑦NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6(NM002493)⑧eukaryotic translation initiation factor 3, subunit 5(NM003754).
Conclusion: The clones of this library were selectively sequenced and analyzed by Blast. Some genes were found that they could take part in regulating the functions of marrow CD4~+ T cells from aplastic anemia patients. The recognized genes included some genes connected with protein synthesis, biology oxidation, signal transduction, proliferation regulation, cell migration and some gene whose function was unclear. Up to the present, the immune injury effects of the above genes in aplastic anemia have not been reported.
PartⅣBioinformatics analysis of Tcf-1 gene and identification of Tcf-1 gene expression in marrow CD4~+ T cells from aplastic anemia patients
Objective: To complete the bioinformatics analysis of Tcf-1 gene from the subtracted cDNA library to obtain its information of gene and protein. To identify Tcf-1 gene expression in marrow CD4~+ T cells from aplastic anemia patients.
Methods: The expression profile and gene location of Tcf-1 gene and basal characters and functional domains of Tcf-1 protein were analysed through Internet databases. Expression of Tcf-1 mRNA in marrow CD4~+ T cells from aplastic anemia patients was assayed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR).
Results: Tcf-1 gene was mainly expressed in thymus, it was also in the uncharacterized tissue, connective tissue, prostate, spleen, lymph node, colon, heart, liver, blood and so on. It was located chromosome 5q31.1. Its protein consisted of 269 aa, and its molecular formula was C1355H2108N386O378S10. The molecular weight was 30174.6 and theoretical isoeleetrie point was 9.82. Tcf-1 protein was a hydrophilic protein, and was not a membrane protein. It lied mainly in cell nuclear. Tcf-1 protein had an HMG-BOX domain that was located between 154 and 222 aa. As a result of semi-quantitative RT-PCR, compared with the normal donors, Tcf-1 gene was up-expressed in marrow CD4~+ T cells of patients with aplastic anemia.
Conclusion: As T-cell special expression gene, Tcf-1 had an HMG-BOX domain which made it be cognized as a transcription factor. Tcf-1 mRNA expression level of marrow CD4~+ T cells from aplastic anemia patients was markedly higher than that of normal people.
PartⅤThe effects of Tcf-1 gene silence on marrow CD4~+ T cells from aplastic anemia patients
Objective: To discuss the effects of Tcf-1 gene silence on marrow CD4~+ T cells from aplastic anemia patients.
Methods: The shRNA expression vectors psiRNA1, psiRNA2 and psiRNA3 were constructed with psiRNA-hH1neo G2 plasmid. After they were transfected into Jurkat cells, the expression level of Tcf-1 mRNA in Jurkat cells was assayed by semi-quantitative reverse-transcription polymerase chain reaction. After the selected psiRNA2 were transfected into marrow CD4~+ T cells from aplastic anemia patients, the expression levels of Tcf-1, c-myc and CD44 mRNA in marrow CD4~+ T cells from aplastic anemia patients was detected by semi-quantitative RT-PCR.
Results: Three plasmid-derived siRNAs could effectively reduce the expression level of Tcf-1 mRNA in transfected Jurkat cells. The plasmid-derived siRNA psiRNA2 was found to be the most effective inhibitor of Tcf-1 expression. After the selected psiRNA2 were transfected into marrow CD4~+ T cells from aplastic anemia patients, the expression levels of Tcf-1, c-myc and CD44 mRNA in marrow CD4~+ T cells from aplastic anemia patients were reduced to different extents.
Conclusion: The up-expression of Tcf-1 gene could be closely related to abnormal functions of marrow CD4~+ T cells from aplastic anemia patients. It could regulate downstream target genes such as c-myc and CD44 to take part in the mechanisms of over-proliferation, activation and excreting certain soluble humoral agents which could inhibit hematopoietic stem cell proliferation of marrow CD4~+ T cells from aplastic anemia patients. It was worthwhile to carry out the study of the new therapy against Tcf-1 gene in aplastic anemia.
引文
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