本文選題：支氣管肺發(fā)育不良　切入點(diǎn)：肺表面活性物質(zhì)相關(guān)蛋白B　出處：《華中科技大學(xué)》2013年博士論文

【摘要】：目的：探討表面活性蛋白-B（surfactant protein B, SP-B）基因多態(tài)性和單體型與漢族患兒支氣管肺發(fā)育不良（Bronchopulmonary dysplasia, BPD）之間的相關(guān)性。 方法：采用聚合酶鏈反應(yīng)-限制性片段長(zhǎng)度多態(tài)性(polymerase chain reaction-restrictionfragment length polymorphism, PCR-RFLP)技術(shù)和基因測(cè)序方法，對(duì)2008年1月至2012年6月在華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬同濟(jì)醫(yī)院新生兒重癥監(jiān)護(hù)病房收治的86例BPD組與156例對(duì)照組患兒SP-B基因4個(gè)多態(tài)性位點(diǎn)rs2077079，rs1130866，rs7316及內(nèi)含子5基因型與等位基因分布情況進(jìn)行檢測(cè)，同時(shí)應(yīng)用fastPHASE軟件構(gòu)建基因單體型。 結(jié)果：內(nèi)含子5缺失型基因型與野生型基因型在BPD組與對(duì)照組之間頻率分布均有統(tǒng)計(jì)學(xué)差異（p=0.049與p=0.03）；缺失型等位基因（del）在對(duì)照組與BPD組頻率分別為4.8%與10.5%，兩組間頻率分布具有統(tǒng)計(jì)學(xué)差異（p=0.018, OR=2.314,95%CI1.135-4.209）。rs2077079多態(tài)性在BPD組與對(duì)照組的頻率分布差異有統(tǒng)計(jì)學(xué)意義（p0.05）；C等位基因在BPD組與對(duì)照組的頻率分別為45.3%和59.6%，差異有統(tǒng)計(jì)學(xué)意義（p=0.003, OR=0.56295%CI0.386-0.819）。rs1130866，rs7316基因型及等位基因頻率分布在BPD組與對(duì)照組差異均無(wú)統(tǒng)計(jì)學(xué)意義（p0.05）。單體型結(jié)果，，C-inv-C-A單體型與C-inv-T-A單體型的頻率分布在對(duì)照組均高于BPD組，且有顯著性差異（p0.05），而A-del-C-A單體型在BPD組的頻率明顯高于對(duì)照組（p=0.003）。 結(jié)論：SP-B基因內(nèi)含子5和rs2077079多態(tài)性與漢族患兒BPD相關(guān)，內(nèi)含子5等位基因del為BPD危險(xiǎn)因子，rs2077079C等位基因?yàn)锽PD保護(hù)因子；SP-B基因rs1130866和rs7316多態(tài)性與漢族患兒BPD無(wú)相關(guān)性；單體型A-del-C-A與BPD相關(guān)，四種多態(tài)性位點(diǎn)之間可能存在部分連鎖不平衡。 目的：構(gòu)建SP-B內(nèi)含子5多態(tài)性野生型及缺失型兩種真核表達(dá)質(zhì)粒pIRSE2-EGFP-SP-B-intron5-inv與pIRSE2-EGFP-SP-B-intron5-del。 方法：采用PCR方法擴(kuò)增含有內(nèi)含子5多態(tài)性的基因組DNA為目的基因，與pIRSE2-EGFP載體同時(shí)用Xho I和Hind III限制性內(nèi)切酶進(jìn)行雙酶切，酶切產(chǎn)物用T4DNA連接酶連接并轉(zhuǎn)化大腸桿菌，卡那霉素篩選陽(yáng)性菌落，采用PCR、酶切和測(cè)序方法對(duì)構(gòu)建的質(zhì)粒進(jìn)行鑒定。 結(jié)果：兩種真核表達(dá)質(zhì)粒pIRSE2-EGFP-SP-B-intron5-inv與pIRSE2-EGFP-SP-B-intron5-del含有SP-B內(nèi)含子5基因?qū)?yīng)的目的片段。 結(jié)論：成功構(gòu)建了重組質(zhì)粒pIRSE2-EGFP-SP-B-intron5-inv與pIRSE2-EGFP-SP-B-intron5-del。 目的：檢測(cè)兩種重組質(zhì)粒pIRES2-EGFP-SP-B-intron5轉(zhuǎn)染293T細(xì)胞與H441細(xì)胞后SP-B mRNA與蛋白的表達(dá)情況，探討SP-B內(nèi)含子5多態(tài)性影響SP-B基因表達(dá)的機(jī)制。 方法：采用脂質(zhì)體法將兩種重組質(zhì)粒pIRES2-EGFP-SP-B-intron5導(dǎo)入293T細(xì)胞與H441細(xì)胞中，熒光顯微鏡觀察增強(qiáng)型綠色熒光蛋白在293T細(xì)胞中表達(dá)，采用有限稀釋法挑選轉(zhuǎn)染的H441細(xì)胞單克隆；RT-PCR（reverse transcription-PCR）及western印跡檢測(cè)SP-B mRNA剪接情況以及SP-B蛋白表達(dá)。 結(jié)果：重組質(zhì)粒瞬時(shí)轉(zhuǎn)染293T細(xì)胞，轉(zhuǎn)染效率在50%以上；引物對(duì)zx與fx1的PCR產(chǎn)物中，質(zhì)粒pIRES2-EGFP-SP-B-intron5-inv轉(zhuǎn)染的293T細(xì)胞條帶約300bp，pIRES2-EGFP-SP-B-intron5-del轉(zhuǎn)染的293T細(xì)胞有兩條帶，分別為300bp與950bp左右；引物對(duì)zx與fx2的PCR產(chǎn)物在兩種質(zhì)粒轉(zhuǎn)染的293T細(xì)胞中目的條帶片段長(zhǎng)度一致約320bp；引物對(duì)zx與fx3的PCR產(chǎn)物在野生型重組質(zhì)粒轉(zhuǎn)染的293T細(xì)胞中PCR產(chǎn)物長(zhǎng)度約為730bp，而缺失型轉(zhuǎn)染的細(xì)胞中約為530bp。 SP-B中間抗體DTB檢測(cè)兩種轉(zhuǎn)染的293T細(xì)胞SP-B蛋白表達(dá)，見(jiàn)一條大約30kDa的條帶，野生型重組質(zhì)粒轉(zhuǎn)染的細(xì)胞中蛋白表達(dá)明顯高于缺失型（p0.05）；兩種質(zhì)粒轉(zhuǎn)染的H441細(xì)胞中蛋白條帶與未轉(zhuǎn)染的H441細(xì)胞條帶一致，42kDa大小SP-B前體蛋白及25kDa大小SP-B蛋白中間體，但缺失型重組質(zhì)粒轉(zhuǎn)染的細(xì)胞中蛋白表達(dá)明顯低于野生型轉(zhuǎn)染的細(xì)胞（p0.05）。 結(jié)論：本研究中SP-B內(nèi)含子5缺失型變異引起SP-B RNA剪接異常，產(chǎn)生不完全剪接的mRNA，同時(shí)也存在正常剪接的mRNA，其多態(tài)性影響正常SP-B mRNA及SP-B蛋白表達(dá)，這種機(jī)制可能與缺失型等位基因del增加BPD發(fā)病風(fēng)險(xiǎn)相關(guān)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R563

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