本文選題：造血干細胞 + 淋巴系祖細胞��； 參考：《瀘州醫(yī)學院》2009年碩士論文

【摘要】： 目的:本課題主要探討人類臍血造血干細胞( Hematopoietic Stem Cell,HSC)向淋巴系祖細胞(Colony Forming Unit-T Lymphocyte, CFU-TL)增殖分化過程中HOXC4、HOXC6、HOXB4、HOXB6基因表達的情況,并且用全反式維甲酸( all-trans-retinoic acid,ATRA)進行干擾,以觀察在此過程中ATRA對HOX基因表達的影響。方法:1. 10例臍血標本由本院產房提供,取自正常足月順產新生兒斷臍后的胎盤段臍血。2.實驗分組。實驗分2組:(1)正常組(normal):不加全反式維甲酸,代之等量1640培養(yǎng)液加入基本培養(yǎng)體系。(2)維甲酸組(all-trans-retinoic acid,ATRA組):加入ATRA稀釋液0.1ml,終濃度6×10-8mol/l。3.采用造血干/祖細胞體外培養(yǎng)技術,以ATRA持續(xù)干擾人類造血干細胞,觀察正常組、維甲酸組的人類臍血HSC經(jīng)植物血凝素(Phytohemagglutinin, PHA-M)誘導后,在培養(yǎng)過程第3天,7天和12天的CFU-TL集落生成情況。4.采用瑞氏染色法鑒定CFU-TL集落細胞成分。5.在第3天、第7天、第12天分別提取各組細胞總RNA,用1%甲醛變性瓊脂糖凝膠電泳檢測RNA分子的完整性。6.通過隨機引物將各時間點提取到的兩組細胞總RNA逆轉錄為cDNA,采用實時熒光定量聚合酶鏈式反應(Fluorescence Quantitative Real Time Polymerize Chain Reaction, FQ-RT-PCR)進行擴增并檢測臍血造血干細胞向淋巴系祖細胞增殖分化過程中兩組HOXC4、HOXC6、HOXB4、HOXB6基因的表達水平。7.隨機抽取HOXC4、HOXC6、HOXB4、HOXB6基因通過電泳實驗分別獲得其在3天、7天、12天電泳圖。8.統(tǒng)計方法:結果用DNA相對拷貝數(shù)和RNA表達相對量(2-△△Ct)表示HOX4、HOXC6、HOXB4、HOXB6基因相對表達量,采用均數(shù)加減標準差( X±S )表示HOXC4、HOXC6、HOXB4、HOXB6基因的變化情況。進行方差齊性檢驗, ONE—WAY方差分析,組間均數(shù)兩兩比較用LSD法。兩組間基因相對表達量比較采用配對設計t檢驗。由統(tǒng)計學軟件SPSS15.0完成。結果:1.集落細胞的形態(tài)學鑒定,瑞-氏染色法證明所培養(yǎng)的是淋巴系細胞。2. 1%甲醛變性瓊脂糖凝膠電泳顯示RNA電泳圖的5s,18s,28s三條帶型整齊,無明顯拖尾及彌散,說明RNA結構完整,無明顯降解。3.逆轉錄PCR擴增目的基因HOXC4、HOXC6、HOXB4、HOXB6和內參照GAPDH基因的cDNA的產物,與DNA分子Marker相比示擴增產物大小分別為:HOXC4為254個堿基(bp),HOXC6 212bp,HOXB4 138bp, HOXB6 119bp, GAPDH基因為141bp,與預定理論值大小符合。4.人臍血造血干細胞向淋巴系祖細胞增殖分化過程中,兩組細胞的HOX基因均有規(guī)律的表達,隨培養(yǎng)時間推移,正常組、ATRA組HOXC4、HOXC6、HOXB6基因均在增殖分化的第7天表達最強烈,第12天表達明顯降低,而HOXB4基因表達卻隨培養(yǎng)時間推移逐漸降低。5.與正常組比較,HOXC4、HOXC6、HOXB4、HOXB6基因均受ATRA(6×10-8mol/l)的上調。結論:1. HOXC4、HOXC6、HOXB4、HOXB6基因在人臍血造血干細胞向淋巴系祖細胞增殖分化過程中均呈現(xiàn)規(guī)律表達,提示HOX基因與人類造血干細胞向淋巴系祖細胞增殖分化過程密切相關。2.人類造血干細胞向淋巴系祖細胞增殖分化過程中, HOXC4、HOXC6、HOXB4、HOXB6基因的表達呈現(xiàn)時間上的規(guī)律性。3. ATRA能顯著上調HOXC4、HOXC6、HOXB4、HOXB6基因的表達。
[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.

【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【參考文獻】

相關期刊論文 前10條

1 王吉偉,陳漢春,田菁燕,劉新發(fā),羅賽群,曾海濤;兩種維甲類藥物協(xié)同誘導白血病細胞HOXA1基因的表達[J];中國醫(yī)師雜志;2000年04期

2 宮雁冰,鐘鳴;同源盒基因及其與細胞的分化和增殖研究進展[J];國外醫(yī)學.口腔醫(yī)學分冊;2005年04期

3 王晴;同源盒基因家族在造血調控中的作用[J];國外醫(yī)學.輸血及血液學分冊;2002年06期

4 王艷艷;徐開林;潘秀英;;HOX基因在人類白血病發(fā)生中的作用[J];國際輸血及血液學雜志;2006年04期

5 劉恭平,朱清華;同源異型框基因的表達和調控[J];國外醫(yī)學.遺傳學分冊;2000年05期

6 唐宇宏,費小明,沈文怡,繆扣榮,崔毓桂,陸化,汪承亞;定量PCR檢測體外擴增臍帶血造血干細胞HOXB4基因的表達[J];南京醫(yī)科大學學報(自然科學版);2005年07期

7 師偉,謝超,裴雪濤;HOXB4基因及其在造血干細胞增殖分化調控中的作用[J];生理科學進展;2004年01期

8 馮靜喬;劉文君;;同源盒基因B簇在造血干/祖細胞增殖分化中的作用[J];實用兒科臨床雜志;2006年11期

9 唐宇宏,汪承亞;HOXB家族基因與造血干/祖細胞的功能[J];中國實驗血液學雜志;2005年02期

10 唐宇宏;費小明;沈文怡;繆扣榮;崔毓桂;汪承亞;;臍血CD34~+細胞體外擴增時HOXB4基因表達的變化[J];中國實驗血液學雜志;2006年01期

，

本文編號：1745010