SP-B内含子5基因多态性在支气管肺发育不良中的机制研究

发布时间：2018-03-24 19:23

本文选题：支气管肺发育不良　切入点：肺表面活性物质相关蛋白B　出处：《华中科技大学》2013年博士论文

【摘要】：目的：探讨表面活性蛋白-B（surfactant protein B, SP-B）基因多态性和单体型与汉族患儿支气管肺发育不良（Bronchopulmonary dysplasia, BPD）之间的相关性。方法：采用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restrictionfragment length polymorphism, PCR-RFLP)技术和基因测序方法，对2008年1月至2012年6月在华中科技大学同济医学院附属同济医院新生儿重症监护病房收治的86例BPD组与156例对照组患儿SP-B基因4个多态性位点rs2077079，rs1130866，rs7316及内含子5基因型与等位基因分布情况进行检测，同时应用fastPHASE软件构建基因单体型。结果：内含子5缺失型基因型与野生型基因型在BPD组与对照组之间频率分布均有统计学差异（p=0.049与p=0.03）；缺失型等位基因（del）在对照组与BPD组频率分别为4.8%与10.5%，两组间频率分布具有统计学差异（p=0.018, OR=2.314,95%CI1.135-4.209）。rs2077079多态性在BPD组与对照组的频率分布差异有统计学意义（p0.05）；C等位基因在BPD组与对照组的频率分别为45.3%和59.6%，差异有统计学意义（p=0.003, OR=0.56295%CI0.386-0.819）。rs1130866，rs7316基因型及等位基因频率分布在BPD组与对照组差异均无统计学意义（p0.05）。单体型结果，，C-inv-C-A单体型与C-inv-T-A单体型的频率分布在对照组均高于BPD组，且有显著性差异（p0.05），而A-del-C-A单体型在BPD组的频率明显高于对照组（p=0.003）。结论：SP-B基因内含子5和rs2077079多态性与汉族患儿BPD相关，内含子5等位基因del为BPD危险因子，rs2077079C等位基因为BPD保护因子；SP-B基因rs1130866和rs7316多态性与汉族患儿BPD无相关性；单体型A-del-C-A与BPD相关，四种多态性位点之间可能存在部分连锁不平衡。目的：构建SP-B内含子5多态性野生型及缺失型两种真核表达质粒pIRSE2-EGFP-SP-B-intron5-inv与pIRSE2-EGFP-SP-B-intron5-del。方法：采用PCR方法扩增含有内含子5多态性的基因组DNA为目的基因，与pIRSE2-EGFP载体同时用Xho I和Hind III限制性内切酶进行双酶切，酶切产物用T4DNA连接酶连接并转化大肠杆菌，卡那霉素筛选阳性菌落，采用PCR、酶切和测序方法对构建的质粒进行鉴定。结果：两种真核表达质粒pIRSE2-EGFP-SP-B-intron5-inv与pIRSE2-EGFP-SP-B-intron5-del含有SP-B内含子5基因对应的目的片段。结论：成功构建了重组质粒pIRSE2-EGFP-SP-B-intron5-inv与pIRSE2-EGFP-SP-B-intron5-del。目的：检测两种重组质粒pIRES2-EGFP-SP-B-intron5转染293T细胞与H441细胞后SP-B mRNA与蛋白的表达情况，探讨SP-B内含子5多态性影响SP-B基因表达的机制。方法：采用脂质体法将两种重组质粒pIRES2-EGFP-SP-B-intron5导入293T细胞与H441细胞中，荧光显微镜观察增强型绿色荧光蛋白在293T细胞中表达，采用有限稀释法挑选转染的H441细胞单克隆；RT-PCR（reverse transcription-PCR）及western印迹检测SP-B mRNA剪接情况以及SP-B蛋白表达。结果：重组质粒瞬时转染293T细胞，转染效率在50%以上；引物对zx与fx1的PCR产物中，质粒pIRES2-EGFP-SP-B-intron5-inv转染的293T细胞条带约300bp，pIRES2-EGFP-SP-B-intron5-del转染的293T细胞有两条带，分别为300bp与950bp左右；引物对zx与fx2的PCR产物在两种质粒转染的293T细胞中目的条带片段长度一致约320bp；引物对zx与fx3的PCR产物在野生型重组质粒转染的293T细胞中PCR产物长度约为730bp，而缺失型转染的细胞中约为530bp。 SP-B中间抗体DTB检测两种转染的293T细胞SP-B蛋白表达，见一条大约30kDa的条带，野生型重组质粒转染的细胞中蛋白表达明显高于缺失型（p0.05）；两种质粒转染的H441细胞中蛋白条带与未转染的H441细胞条带一致，42kDa大小SP-B前体蛋白及25kDa大小SP-B蛋白中间体，但缺失型重组质粒转染的细胞中蛋白表达明显低于野生型转染的细胞（p0.05）。结论：本研究中SP-B内含子5缺失型变异引起SP-B RNA剪接异常，产生不完全剪接的mRNA，同时也存在正常剪接的mRNA，其多态性影响正常SP-B mRNA及SP-B蛋白表达，这种机制可能与缺失型等位基因del增加BPD发病风险相关。
[Abstract]:Objective: To explore the correlation between -B surfactant protein B (SP-B) gene polymorphism and haplotype and Bronchopulmonary dysplasia (BPD) in children with Han nationality.
Methods: using polymerase chain reaction restriction fragment length polymorphism (polymerase chain reaction-restrictionfragment length polymorphism, PCR-RFLP) technology and gene sequencing method, rs1130866 from January 2008 to June 2012 in the NICU admitted to Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology in 86 cases of BPD group and 156 cases in control group SP-B gene 4 polymorphism loci rs2077079. Detection of rs7316, and intron 5 genotype and allele distribution, and gene haplotype with fastPHASE software.
Results: the difference in frequency distribution of intron 5 gene deletion type and wild type genotype between BPD group and control group were statistically (p=0.049 and p=0.03); the allele (DEL) in the control group and BPD group frequencies were 4.8% and 10.5%, the frequency distribution between the two groups has statistical difference (p=0.018, OR=2.314,95%CI1.135-4.209).Rs2077079 polymorphism in the BPD group and the control group there were significant differences in the frequencies (P0.05); the frequency of C allele in BPD group and control group were 45.3% and 59.6%, the difference was statistically significant (p=0.003, OR,.Rs1130866, =0.56295%CI0.386-0.819) showed no significant difference with the control group in BPD rs7316 genotype and allele frequency distribution (P0.05). The C-inv-C-A haplotype, haplotype and C-inv-T-A haplotype frequencies in the control group were higher than that of group BPD, and there is a significant difference (P0.05), and A-de The frequency of l-C-A haplotype in group BPD was significantly higher than that in the control group (p=0.003).
Conclusion: SP-B gene intron 5 and rs2077079 polymorphism and were related to BPD, intron 5 del allele is the risk factor of BPD, rs2077079C allele of BPD protective factor; there is no correlation between SP-B gene rs1130866 and rs7316 polymorphism and haplotype A-del-C-A in children with BPD; and BPD, part of the chain is not possible the balance between the four polymorphic loci.
Objective: to construct SP-B intron 5 polymorphic wild type and two eukaryotic expression plasmid pIRSE2-EGFP-SP-B-intron5-inv and pIRSE2-EGFP-SP-B-intron5-del.
Methods: using the method of PCR amplification of intron 5 polymorphism of the genomic DNA gene with pIRSE2-EGFP vector, I and Hind with Xho III restriction endonuclease digested enzyme products were connected and transformed into Escherichia coli with T4DNA ligase, kanamycin screening positive colonies, by PCR, enzyme digestion and sequencing method for identification of plasmid.
Results: two eukaryotic expression plasmids pIRSE2-EGFP-SP-B-intron5-inv and pIRSE2-EGFP-SP-B-intron5-del contain the target fragment corresponding to the 5 gene of the intron of SP-B.
Conclusion: recombinant plasmid pIRSE2-EGFP-SP-B-intron5-inv and pIRSE2-EGFP-SP-B-intron5-del. were successfully constructed.
Objective: to detect the expression of SP-B mRNA and protein after transfection of two recombinant plasmid pIRES2-EGFP-SP-B-intron5 into 293T cells and H441 cells, and to explore the mechanism of SP-B intron 5 polymorphism affecting SP-B gene expression.
Methods: using Lipofectamine two recombinant plasmid pIRES2-EGFP-SP-B-intron5 was transfected into 293T cells and H441 cells, enhanced green fluorescent protein expression in 293T cells was observed by fluorescence microscope, H441 cells transfected with monoclonal selection using limited dilution method; RT-PCR (reverse transcription-PCR) expression and Western blot detection of SP-B mRNA and SP-B protein splicing.
Results: the recombinant plasmid was transfected to 293T cells, the transfection efficiency was above 50%; ZX and FX1 primers for PCR products, about 300bp of 293T cells with transfection of plasmid pIRES2-EGFP-SP-B-intron5-inv pIRES2-EGFP-SP-B-intron5-del transfected 293T cells with two bands, respectively 300bp and 950bp; 293T cells with FX2 primer pairs ZX PCR products in two kinds of plasmid transfected target bands in fragment length consistent about 320bp; PCR product length is about 730bp in 293T cells and fx3 ZX primers for PCR products in the wild type recombinant plasmid, and the deletion of transfected cells was about two of 293T cells transfected with expression of SP-B protein 530bp. SP-B intermediate antibody detection DTB see, a band of about 30kDa, was significantly higher than that of wild type protein expression deletion recombinant plasmid transfected cells (P0.05); two kinds of plasmid transfected H441 cells, protein bands and not turn The H441 cell bands were consistent, 42kDa size, SP-B precursor protein and 25kDa size SP-B protein intermediates, but the protein expression in transfected cells was significantly lower than that in wild type transfected cells (P0.05).
Conclusion: in this study 5 SP-B intron deletion by SP-B RNA splicing, produce incomplete splicing of mRNA, there are also normal splicing of mRNA, affecting the normal SP-B mRNA polymorphism and SP-B protein expression increased, this mechanism may be related to the allele del related to BPD risk.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563

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