本文選題：造血干細(xì)胞 + 淋巴系祖細(xì)胞��； 參考：《瀘州醫(yī)學(xué)院》2009年碩士論文

【摘要】： 目的:本課題主要探討人類(lèi)臍血造血干細(xì)胞( Hematopoietic Stem Cell,HSC)向淋巴系祖細(xì)胞(Colony Forming Unit-T Lymphocyte, CFU-TL)增殖分化過(guò)程中HOXC4、HOXC6、HOXB4、HOXB6基因表達(dá)的情況,并且用全反式維甲酸( all-trans-retinoic acid,ATRA)進(jìn)行干擾,以觀察在此過(guò)程中ATRA對(duì)HOX基因表達(dá)的影響。方法:1. 10例臍血標(biāo)本由本院產(chǎn)房提供,取自正常足月順產(chǎn)新生兒斷臍后的胎盤(pán)段臍血。2.實(shí)驗(yàn)分組。實(shí)驗(yàn)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(xì)胞體外培養(yǎng)技術(shù),以ATRA持續(xù)干擾人類(lèi)造血干細(xì)胞,觀察正常組、維甲酸組的人類(lèi)臍血HSC經(jīng)植物血凝素(Phytohemagglutinin, PHA-M)誘導(dǎo)后,在培養(yǎng)過(guò)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(xì)胞成分。5.在第3天、第7天、第12天分別提取各組細(xì)胞總RNA,用1%甲醛變性瓊脂糖凝膠電泳檢測(cè)RNA分子的完整性。6.通過(guò)隨機(jī)引物將各時(shí)間點(diǎn)提取到的兩組細(xì)胞總RNA逆轉(zhuǎn)錄為cDNA,采用實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(Fluorescence Quantitative Real Time Polymerize Chain Reaction, FQ-RT-PCR)進(jìn)行擴(kuò)增并檢測(cè)臍血造血干細(xì)胞向淋巴系祖細(xì)胞增殖分化過(guò)程中兩組HOXC4、HOXC6、HOXB4、HOXB6基因的表達(dá)水平。7.隨機(jī)抽取HOXC4、HOXC6、HOXB4、HOXB6基因通過(guò)電泳實(shí)驗(yàn)分別獲得其在3天、7天、12天電泳圖。8.統(tǒng)計(jì)方法:結(jié)果用DNA相對(duì)拷貝數(shù)和RNA表達(dá)相對(duì)量(2-△△Ct)表示HOX4、HOXC6、HOXB4、HOXB6基因相對(duì)表達(dá)量,采用均數(shù)加減標(biāo)準(zhǔn)差( X±S )表示HOXC4、HOXC6、HOXB4、HOXB6基因的變化情況。進(jìn)行方差齊性檢驗(yàn), 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(duì)表達(dá)量比較采用配對(duì)設(shè)計(jì)t檢驗(yàn)。由統(tǒng)計(jì)學(xué)軟件SPSS15.0完成。結(jié)果:1.集落細(xì)胞的形態(tài)學(xué)鑒定,瑞-氏染色法證明所培養(yǎng)的是淋巴系細(xì)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(wú)明顯拖尾及彌散,說(shuō)明RNA結(jié)構(gòu)完整,無(wú)明顯降解。3.逆轉(zhuǎn)錄PCR擴(kuò)增目的基因HOXC4、HOXC6、HOXB4、HOXB6和內(nèi)參照GAPDH基因的cDNA的產(chǎn)物,與DNA分子Marker相比示擴(kuò)增產(chǎn)物大小分別為:HOXC4為254個(gè)堿基(bp),HOXC6 212bp,HOXB4 138bp, HOXB6 119bp, GAPDH基因?yàn)?41bp,與預(yù)定理論值大小符合。4.人臍血造血干細(xì)胞向淋巴系祖細(xì)胞增殖分化過(guò)程中,兩組細(xì)胞的HOX基因均有規(guī)律的表達(dá),隨培養(yǎng)時(shí)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(dá)最強(qiáng)烈,第12天表達(dá)明顯降低,而HOXB4基因表達(dá)卻隨培養(yǎng)時(shí)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(diào)。結(jié)論:1. HOXC4、HOXC6、HOXB4、HOXB6基因在人臍血造血干細(xì)胞向淋巴系祖細(xì)胞增殖分化過(guò)程中均呈現(xiàn)規(guī)律表達(dá),提示HOX基因與人類(lèi)造血干細(xì)胞向淋巴系祖細(xì)胞增殖分化過(guò)程密切相關(guān)。2.人類(lèi)造血干細(xì)胞向淋巴系祖細(xì)胞增殖分化過(guò)程中, HOXC4、HOXC6、HOXB4、HOXB6基因的表達(dá)呈現(xiàn)時(shí)間上的規(guī)律性。3. ATRA能顯著上調(diào)HOXC4、HOXC6、HOXB4、HOXB6基因的表達(dá)。
[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(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)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(lèi)藥物協(xié)同誘導(dǎo)白血病細(xì)胞HOXA1基因的表達(dá)[J];中國(guó)醫(yī)師雜志;2000年04期

2 宮雁冰,鐘鳴;同源盒基因及其與細(xì)胞的分化和增殖研究進(jìn)展[J];國(guó)外醫(yī)學(xué).口腔醫(yī)學(xué)分冊(cè);2005年04期

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

4 王艷艷;徐開(kāi)林;潘秀英;;HOX基因在人類(lèi)白血病發(fā)生中的作用[J];國(guó)際輸血及血液學(xué)雜志;2006年04期

5 劉恭平,朱清華;同源異型框基因的表達(dá)和調(diào)控[J];國(guó)外醫(yī)學(xué).遺傳學(xué)分冊(cè);2000年05期

6 唐宇宏,費(fèi)小明,沈文怡,繆扣榮,崔毓桂,陸化,汪承亞;定量PCR檢測(cè)體外擴(kuò)增臍帶血造血干細(xì)胞HOXB4基因的表達(dá)[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2005年07期

7 師偉,謝超,裴雪濤;HOXB4基因及其在造血干細(xì)胞增殖分化調(diào)控中的作用[J];生理科學(xué)進(jìn)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(xì)胞增殖分化中的作用[J];實(shí)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(xì)胞的功能[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2005年02期

10 唐宇宏;費(fèi)小明;沈文怡;繆扣榮;崔毓桂;汪承亞;;臍血CD34~+細(xì)胞體外擴(kuò)增時(shí)HOXB4基因表達(dá)的變化[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2006年01期

，

本文編號(hào)：1745010