【摘要】： 目的 干細(xì)胞壁龕是干細(xì)胞生存的特定微環(huán)境,直接影響HSC的自我更新、增殖和分化。造血干細(xì)胞壁龕細(xì)胞(包括成骨細(xì)胞和內(nèi)皮細(xì)胞)均高表達(dá)富含半胱氨酸的酸性分泌型蛋白(SPARC)。本文通過分析SPARC基因缺失型小鼠體內(nèi)造血組織結(jié)構(gòu)和造血發(fā)育特點,以及外源性SPARC對造血干細(xì)胞的遷移和增殖作用,了解SPARC對造血的調(diào)控作用。 方法 1) SPARC基因缺失雜合子小鼠交配,繁育出子代后,提取鼠尾gDNA,應(yīng)用PCR方法擴(kuò)增SPARC和Neo基因；然后對擴(kuò)增產(chǎn)物進(jìn)行限制性酶切反應(yīng)；WB檢測經(jīng)PCR方法鑒定出的SPARC基因各表型小鼠脾臟組織SPARC蛋白的表達(dá)。 2)比較SPARC基因缺失純合子小鼠和野生型對照鼠骨小梁數(shù)量,脾臟的大小、結(jié)構(gòu)特點和外周血細(xì)胞分類計數(shù)；應(yīng)用流式細(xì)胞技術(shù)和LTC-IC、集落培養(yǎng)方法檢測骨髓中造血干／祖細(xì)胞的含量；體外檢測外源性SPARC對CD34+細(xì)胞的擴(kuò)增,遷移的影響。 結(jié)果 1)PCR擴(kuò)增片段經(jīng)限制性酶切反應(yīng)證實為特異性SPARC和Neo基因擴(kuò)增產(chǎn)物；經(jīng)PCR方法鑒定得到的SPARC基因純合子小鼠不能檢測到SPARC蛋白的表達(dá),而野生型小鼠可檢測到SPARC蛋白的表達(dá)； 2) SPARC基因缺失小鼠較野生型小鼠單位體積內(nèi)的骨小梁數(shù)目減少34.5%,骨小梁分離度增加65.6%；缺失鼠脾臟重量、脾臟／體重比均大于野生型對照鼠；脾臟組織切片HE染色顯示缺失鼠白髓面積明顯增大；缺失型小鼠體內(nèi)造血與野生型小鼠比較有如下差異：外周血WBC、HGB、HCT均減少；RBC減少,但無統(tǒng)計學(xué)差異；骨髓KSL (c-kit+, sca-1+, lin-)細(xì)胞的比例、KSL細(xì)胞總數(shù)均減少；長期培養(yǎng)啟動細(xì)胞(LTC-IC)明顯減少；BFU-E形成數(shù)明顯減少,CFU-GM數(shù)量無明顯差異；此外,SPARC基因缺失鼠骨髓細(xì)胞自殺率明顯低于野生型對照鼠。CD34+細(xì)胞,在有或沒有SPARC的培養(yǎng)體系中培養(yǎng)7天,加入SPARC組造血干細(xì)胞的增殖明顯增加。外源性SPARC促進(jìn)CD34+細(xì)胞遷移,加入SPARC后CD34+細(xì)胞遷移率增加了1.34倍。 結(jié)論 PCR方法可快速準(zhǔn)確鑒定出SPARC基因各表型小鼠；SPARC基因缺失型小鼠體內(nèi)存在造血組織結(jié)構(gòu)和造血發(fā)育異常,造血干/祖細(xì)胞數(shù)量減少。外源性SPARC在體外能促進(jìn)CD34+細(xì)胞增殖和遷移。
[Abstract]:Objective Stem cell niche is a specific microenvironment for stem cell survival, which directly affects self-renewal, proliferation and differentiation of HSC. Hematopoietic stem cell niche cells, including osteoblasts and endothelial cells, overexpression of cysteine-rich acidic secretory protein (SPARC). By analyzing the hematopoietic tissue structure and hematopoietic development characteristics of mice with SPARC gene deletion and the effects of exogenous SPARC on the migration and proliferation of hematopoietic stem cells, the regulation of SPARC on hematopoiesis was studied in this paper. Methods 1) SPARC gene deletion heterozygous mice were mated, then gDNA, was extracted from the tail of the mouse and amplified by PCR, and then the amplified products were digested by restriction endonuclease reaction. WB was used to detect the expression of SPARC protein in spleen tissues of SPARC gene phenotypic mice identified by PCR method. 2) the number of trabecular bone, the size and structure of spleen and the number of peripheral blood cells in homozygous mice with SPARC gene deletion were compared with those in wild type control mice. Flow cytometry and LTC-IC, colony culture were used to detect the hematopoietic stem / progenitor cells in bone marrow and the effect of exogenous SPARC on the expansion and migration of CD34 cells in vitro. Results 1) the amplified PCR fragment was confirmed to be a specific SPARC and Neo gene amplification product by restriction enzyme digestion. The homozygous mice of SPARC gene identified by PCR method could not detect the expression of SPARC protein, but the expression of SPARC protein could be detected in wild-type mice. 2) compared with wild-type mice, the number of trabeculae in mice with SPARC gene deletion decreased by 34.55.The separation degree of trabecular bone increased by 65.6.The spleen weight and spleen / body weight ratio of mice with SPARC gene deletion were higher than those of wild-type control mice. The HE staining of spleen tissue section showed that the area of white pulp of deletion mice was obviously increased. The hematopoiesis of deletion mice was different from that of wild-type mice as follows: peripheral blood WBC,HGB,HCT was decreased, RBC was decreased, but there was no statistical difference between deletion mice and wild-type mice. The percentage of KSL (c-kit, sca-1, lin-) cells and the total number of KSL cells in bone marrow were all decreased, the number of LTC-IC in long-term culture was significantly decreased, the number of BFU-E formation was obviously decreased, and the number of CFU-GM was not significantly different. In addition, the suicide rate of bone marrow cells in mice with SPARC gene deletion was significantly lower than that in wild-type control mice. The proliferation of hematopoietic stem cells increased significantly in CD34 cells cultured in culture system with or without SPARC for 7 days. Exogenous SPARC promoted the migration of CD34 cells, and the migration rate of CD34 cells increased by 1.34 times after the addition of SPARC. Conclusion the SPARC gene phenotypic mice can be identified quickly and accurately by PCR method, and the hematopoietic tissue structure and hematopoietic development are abnormal and the number of hematopoietic stem / progenitor cells is decreased in SPARC gene deletion mice. Exogenous SPARC can promote the proliferation and migration of CD34 cells in vitro.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 石敏,謝常青,盧光t;小鼠胚胎成纖維細(xì)胞條件培養(yǎng)液的蛋白質(zhì)組學(xué)初步分析[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2005年01期

，

本文編號：2386968