造血干细胞向淋巴系祖细胞增殖分化过程中同源盒基因表达的研究

发布时间：2018-04-13 15:03

本文选题：造血干细胞 + 淋巴系祖细胞　；参考：《泸州医学院》2009年硕士论文

【摘要】： 目的:本课题主要探讨人类脐血造血干细胞( Hematopoietic Stem Cell,HSC)向淋巴系祖细胞(Colony Forming Unit-T Lymphocyte, CFU-TL)增殖分化过程中HOXC4、HOXC6、HOXB4、HOXB6基因表达的情况,并且用全反式维甲酸( all-trans-retinoic acid,ATRA)进行干扰,以观察在此过程中ATRA对HOX基因表达的影响。方法:1. 10例脐血标本由本院产房提供,取自正常足月顺产新生儿断脐后的胎盘段脐血。2.实验分组。实验分2组:(1)正常组(normal):不加全反式维甲酸,代之等量1640培养液加入基本培养体系。(2)维甲酸组(all-trans-retinoic acid,ATRA组):加入ATRA稀释液0.1ml,终浓度6×10-8mol/l。3.采用造血干/祖细胞体外培养技术,以ATRA持续干扰人类造血干细胞,观察正常组、维甲酸组的人类脐血HSC经植物血凝素(Phytohemagglutinin, PHA-M)诱导后,在培养过程第3天,7天和12天的CFU-TL集落生成情况。4.采用瑞氏染色法鉴定CFU-TL集落细胞成分。5.在第3天、第7天、第12天分别提取各组细胞总RNA,用1%甲醛变性琼脂糖凝胶电泳检测RNA分子的完整性。6.通过随机引物将各时间点提取到的两组细胞总RNA逆转录为cDNA,采用实时荧光定量聚合酶链式反应(Fluorescence Quantitative Real Time Polymerize Chain Reaction, FQ-RT-PCR)进行扩增并检测脐血造血干细胞向淋巴系祖细胞增殖分化过程中两组HOXC4、HOXC6、HOXB4、HOXB6基因的表达水平。7.随机抽取HOXC4、HOXC6、HOXB4、HOXB6基因通过电泳实验分别获得其在3天、7天、12天电泳图。8.统计方法:结果用DNA相对拷贝数和RNA表达相对量(2-△△Ct)表示HOX4、HOXC6、HOXB4、HOXB6基因相对表达量,采用均数加减标准差( X±S )表示HOXC4、HOXC6、HOXB4、HOXB6基因的变化情况。进行方差齐性检验, ONE—WAY方差分析,组间均数两两比较用LSD法。两组间基因相对表达量比较采用配对设计t检验。由统计学软件SPSS15.0完成。结果:1.集落细胞的形态学鉴定,瑞-氏染色法证明所培养的是淋巴系细胞。2. 1%甲醛变性琼脂糖凝胶电泳显示RNA电泳图的5s,18s,28s三条带型整齐,无明显拖尾及弥散,说明RNA结构完整,无明显降解。3.逆转录PCR扩增目的基因HOXC4、HOXC6、HOXB4、HOXB6和内参照GAPDH基因的cDNA的产物,与DNA分子Marker相比示扩增产物大小分别为:HOXC4为254个碱基(bp),HOXC6 212bp,HOXB4 138bp, HOXB6 119bp, GAPDH基因为141bp,与预定理论值大小符合。4.人脐血造血干细胞向淋巴系祖细胞增殖分化过程中,两组细胞的HOX基因均有规律的表达,随培养时间推移,正常组、ATRA组HOXC4、HOXC6、HOXB6基因均在增殖分化的第7天表达最强烈,第12天表达明显降低,而HOXB4基因表达却随培养时间推移逐渐降低。5.与正常组比较,HOXC4、HOXC6、HOXB4、HOXB6基因均受ATRA(6×10-8mol/l)的上调。结论:1. HOXC4、HOXC6、HOXB4、HOXB6基因在人脐血造血干细胞向淋巴系祖细胞增殖分化过程中均呈现规律表达,提示HOX基因与人类造血干细胞向淋巴系祖细胞增殖分化过程密切相关。2.人类造血干细胞向淋巴系祖细胞增殖分化过程中, HOXC4、HOXC6、HOXB4、HOXB6基因的表达呈现时间上的规律性。3. ATRA能显著上调HOXC4、HOXC6、HOXB4、HOXB6基因的表达。
[Abstract]:Objective: This paper mainly discusses the human umbilical cord blood hematopoietic stem cells (Hematopoietic Stem Cell, HSC) to lymphoid progenitor cells (Colony Forming Unit-T Lymphocyte, HOXC4, CFU-TL) in the differentiation and proliferation of HOXC6, HOXB4, HOXB6 gene expression, and the use of all trans retinoic acid (all-trans-retinoic, acid, ATRA) interference effect in order to observe the ATRA expression of HOX gene in this process. Methods: 1.10 cases of umbilical cord blood samples were provided by delivery room in our hospital, from normal full-term newborn umbilical cord blood placenta segment.2. experimental groups after cutting the umbilical cord. The experiment was divided into 2 groups: (1): normal group (normal) with all trans retinoic acid, generation the same amount of 1640 medium added basic culture system. (2) retinoic acid group (all-trans-retinoic acid, ATRA group): adding ATRA dilution 0.1ml, final concentration of 6 * 10-8mol/l.3. with hematopoietic stem / progenitor cells cultured by ATRA interference technology, continuous human hematopoiesis Stem cells, observe the normal group, retinoic acid group of human umbilical cord blood HSC by phytohemagglutinin (Phytohemagglutinin, PHA-M) after induction in culture for third days, 7 days and 12 days of CFU-TL colony forming.4. by Wright's staining to identify CFU-TL colony cells.5. in third days, seventh days, Twelfth days the total RNA were extracted from the cells of each group, with the integrity of.6. RNA detection 1% formaldehyde denaturing agarose gel electrophoresis by the random primers at different time points to extract two groups of cells total RNA reverse transcription cDNA, using real-time fluorescence quantitative polymerase chain reaction (Fluorescence Quantitative Real Time Polymerize Chain Reaction, FQ-RT-PCR) were amplified and detected in the two groups umbilical cord blood hematopoietic stem cells to lymphoid progenitor cell proliferation and differentiation in the process of HOXC4, HOXC6, HOXB4,.7. expression levels were randomly selected from HOXC4, HOXB6 gene of HOXC6, HOXB4, HOXB6 gene by electrophoresis. The test respectively in 3 days, 7 days, 12 days of electrophoresis of.8. statistical method: the results of DNA relative copy number and RNA expression relative amount (delta 2- Ct) HOX4, HOXC6, HOXB4, the relative expression of HOXB6 gene, using the mean and standard deviation (X + S) HOXC4, HOXC6 HOXB4, the changes of HOXB6 gene. The homogeneity of variance test, analysis of variance between groups ONE and WAY, were pairwise comparison with LSD method. The relative expression of genes between the two groups were compared using paired t test. By statistical software SPSS15.0. Results: the morphological identification of 1. colony cells, Wright's stain the proof is the cultivation of the lymphoid cell.2. 1% formaldehyde denaturing agarose gel electrophoresis showed that RNA electrophoresis of 5S, 18S, 28s and three bands which had no obvious tailing and diffusion, RNA structural integrity, no amplification of target gene HOXC4, obvious degradation of.3. reverse transcriptase PCR HOXC6, HOXB4, GAPD and HOXB6 in the reference The product of H gene of cDNA, compared with DNA Marker showed molecular size of PCR products were HOXC4 254 base pairs (BP), HOXC6 212bp, HOXB4 138bp, HOXB6 119bp, GAPDH gene is 141bp, with a predetermined value conforms to the.4. theory of human cord blood hematopoietic stem cells to lymphoid progenitor cell proliferation and differentiation the expression of HOX gene, had regular cells in two groups, with the incubation time, the normal group, ATRA group, HOXC4, HOXC6, HOXB6 gene in the proliferation and differentiation of seventh days is most strongly expressed, Twelfth days expression decreased significantly, while the expression of HOXB4 gene with the training time of.5. decreased compared with the normal group, HOXC4 HOXC6, HOXB4, HOXB6, ATRA genes were up-regulated (6 x 10-8mol/l). Conclusion: 1. HOXC4, HOXC6, HOXB4, HOXB6 gene in cells to lymphoid progenitor cell proliferation and differentiation in the process of present regular expression in human umbilical cord blood hematopoietic stem, suggesting that the HOX gene and human hematopoietic stem cells Cell proliferation and differentiation process is closely related to the proliferation of lymphoid progenitor cells. The expression of HOXC4, HOXC6, HOXB4 and HOXB6 genes is regular in time during the proliferation and differentiation of.2. hematopoietic stem cells to lymphoid progenitor cells..3. ATRA can significantly increase HOXC4, HOXC6, HOXB4 and HOXB6 gene expression.

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】