吴西军,姚冬静,刘庆,杨小燕,王念雪,王一慧,舒莉萍,何志旭
WU Xijun,YAO Dongjing,LIU Qing,YANG Xiaoyan,WANG Nianxue,WANG Yihui,SHU Liping,HE Zhixu

• 1:贵州医科大学组织工程与干细胞实验中心
• 2:贵州医科大学免疫学教研室
• 3:河北省人民医院儿科
• 4:贵州医科大学附院呼吸与危重症医学科

摘要(Abstract)：

目的:探讨高表达hoxb4转基因系斑马鱼胚胎定向造血发育中cdh5-mRNA的表达。方法:实验分为野生型(WT)对照组、增强绿色荧光蛋白(EGFP)对照组及高表达hoxb4实验组斑马鱼胚胎,收取定向造血阶段32、36、40、44、48 hpf的胚胎各30枚;将pCS~(2+)-cdh5重组质粒进行单酶切,用T3 RNA体外转录体系转录得到地高辛标记的cdh5基因的反义mRNA片段,以其为探针对各组胚胎进行全胚胎原位杂交,显微镜下观察32、36、40、44、48 hpf的造血组织区域cdh5-mRNA杂交信号并拍照记录灰度值。结果:32、36 hpf时高表达hoxb4实验组斑马鱼胚胎造血组织AGM区、PBI区cdh5-mRNA的表达较EGFP对照组及WT对照组增高,差异均有统计学意义(P<0.05),EGFP对照组与WT对照组差异无统计学意义(P>0.05);40、44 hpf时高表达hoxb4实验组斑马鱼胚胎造血组织PBI区cdh5-mRNA的表达较EGFP对照组及WT对照组增高,差异均有统计学意义(P<0.05),EGFP对照组与WT对照组比较,差异无统计学意义(P>0.05);48 hpf时3组斑马鱼胚胎在造血组织区域均未见cdh5-mRNA杂交信号。结论:在高表达hoxb4转基因斑马鱼胚胎中,cdh5-mRNA在定向造血阶段相应造血区域高表达。
Objective: To investigate cdh5-mRNA expression alteration in definitive hematopoiesis development of transgenic zebrafish embryos carrying high expression hoxb4 gene. Methods: Three types of zebrafish: WT control group, EGFP control group and experimental group with high expression of hoxb4. Thirty embryos at 32, 36, 40, 44 and 48 hpf were collected from each group. Antisense mRNA fragment of digoxin labeled cdh5 was transcribed from linearized pCS~(2+)-cdh5 recombinant plasmid as a template by T3 RNA transcription system, and used as a probe for whole-mount in situ hybridization in embryos. The hybridization signal of cdh5 mRNA in the hematopoietic tissue area at 32,36,40,44,48 hpf were observed and photographed under the microscope, scanned and compared the intensity of gray value of hybridization signal in different development phases of targeted hematopoietic sites. Results: The expression of cdh5 mRNA were significantly elevated in AGM area and PBI area of zebrafish embryonic hematopoietic tissue in the experimental group at 32 and 36 hpf when compared with EGFP control group and WT control group(P<0.05), while there was no difference between EGFP control group and WT control group(P>0.05). The expression of cdh5 mRNA in PBI area in the experimental group at 40 and 44 hpf were significantly elevated when compared with EGFP control group and WT control group(P<0.05), also, there was no difference between EGFP control group and WT control group(P>0.05). At 48 hpf, there was no cdh5 mRNA hybridization signal in the hematopoietic tissue of all groups. Conclusion: In hoxb4-overexpressed transgenic zebrafish embryos, cdh5-mRNA is highly expressed in the corresponding hematopoietic region of each directional hematopoiesis development phase.

关键词(KeyWords)： 基因表达;原位杂交;胚胎;造血;发育;调控
gene expression;in situ hybridization;embryo;hematopoiesis;development;regulation

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金地区项目(81760025);; 中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2017PT31042,2018PT31048);; 贵阳市科技计划项目(筑科合同201500103);; 贵州省教育厅青年成长基金[黔教合(2018)192]

作者(Author): 吴西军,姚冬静,刘庆,杨小燕,王念雪,王一慧,舒莉萍,何志旭
WU Xijun,YAO Dongjing,LIU Qing,YANG Xiaoyan,WANG Nianxue,WANG Yihui,SHU Liping,HE Zhixu

DOI: 10.19367/j.cnki.1000-2707.2019.11.002

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