SPARC的造血调控作用
发布时间：2018-12-19 13:15
【摘要】： 目的 干细胞壁龛是干细胞生存的特定微环境,直接影响HSC的自我更新、增殖和分化。造血干细胞壁龛细胞(包括成骨细胞和内皮细胞)均高表达富含半胱氨酸的酸性分泌型蛋白(SPARC)。本文通过分析SPARC基因缺失型小鼠体内造血组织结构和造血发育特点,以及外源性SPARC对造血干细胞的迁移和增殖作用,了解SPARC对造血的调控作用。 方法 1) SPARC基因缺失杂合子小鼠交配,繁育出子代后,提取鼠尾gDNA,应用PCR方法扩增SPARC和Neo基因；然后对扩增产物进行限制性酶切反应；WB检测经PCR方法鉴定出的SPARC基因各表型小鼠脾脏组织SPARC蛋白的表达。 2)比较SPARC基因缺失纯合子小鼠和野生型对照鼠骨小梁数量,脾脏的大小、结构特点和外周血细胞分类计数；应用流式细胞技术和LTC-IC、集落培养方法检测骨髓中造血干／祖细胞的含量；体外检测外源性SPARC对CD34+细胞的扩增,迁移的影响。 结果 1)PCR扩增片段经限制性酶切反应证实为特异性SPARC和Neo基因扩增产物；经PCR方法鉴定得到的SPARC基因纯合子小鼠不能检测到SPARC蛋白的表达,而野生型小鼠可检测到SPARC蛋白的表达； 2) SPARC基因缺失小鼠较野生型小鼠单位体积内的骨小梁数目减少34.5%,骨小梁分离度增加65.6%；缺失鼠脾脏重量、脾脏／体重比均大于野生型对照鼠；脾脏组织切片HE染色显示缺失鼠白髓面积明显增大；缺失型小鼠体内造血与野生型小鼠比较有如下差异：外周血WBC、HGB、HCT均减少；RBC减少,但无统计学差异；骨髓KSL (c-kit+, sca-1+, lin-)细胞的比例、KSL细胞总数均减少；长期培养启动细胞(LTC-IC)明显减少；BFU-E形成数明显减少,CFU-GM数量无明显差异；此外,SPARC基因缺失鼠骨髓细胞自杀率明显低于野生型对照鼠。CD34+细胞,在有或没有SPARC的培养体系中培养7天,加入SPARC组造血干细胞的增殖明显增加。外源性SPARC促进CD34+细胞迁移,加入SPARC后CD34+细胞迁移率增加了1.34倍。 结论 PCR方法可快速准确鉴定出SPARC基因各表型小鼠；SPARC基因缺失型小鼠体内存在造血组织结构和造血发育异常,造血干/祖细胞数量减少。外源性SPARC在体外能促进CD34+细胞增殖和迁移。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2386968
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 石敏,谢常青,卢光t;小鼠胚胎成纤维细胞条件培养液的蛋白质组学初步分析[J];中南大学学报(医学版);2005年01期
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