足月新生儿呼吸窘迫综合征肺表面活性物质蛋白B遗传缺陷探索性研究
发布时间：2018-07-20 16:36
【摘要】：目的评估足月新生儿呼吸窘迫综合征肺表面活性物质蛋白B（SP-B）遗传缺陷的发生频率及突变类型，，探讨足月新生儿呼吸窘迫综合征发病的遗传机制，为进一步基因治疗新生儿RDS提供科学实验依据。 方法选择无血缘关系的汉族足月新生儿RDS20例作为RDS组，汉族20例其它病例作为对照组，并在年龄、体重、性别、孕母年龄、分娩方式等方面与RDS组无统计学差异。采用ELISA技术检测支气管肺泡灌洗液的SP-B蛋白含量，免疫组化技术检测SP-B蛋白在肺组织的表达；常规DNA抽提技术提取人全血DNA，PCR及基因测序技术分析SP-B exon4的基因突变。 结果RDS组患儿BAL SP-B含量在发病第1天（20.0±8.05ng/ml）、发病第3天（21.1±8.13ng/ml）、发病第7天（20.1±7.88ng/ml）、死后30min内（19.4±8.02ng/ml）均显著低于对照组（92.8±32.21；102.4±28.76；105.6±30.41；92.0±32.71ng/ml）（P＜0.001）。RDS组患儿肺组织SP-B蛋白表达阳性细胞数（18.5±10.7）显著低于对照组（94.9±31.8）（t＝10.191，P＜0.001）。RDS组BAL SP-B蛋白含量表达水平越低及肺组织SP-B蛋白表达阳性细胞越少，则RDS胸部X线显示严重性的级别越高，即二者具有很好的依从性。两组均存在SP-B蛋白缺陷，RDS组（7例）高于对照组（1例）。exon4存在基因点突变，存在C/T、C/C、T/T3种基因型。exon4基因突变中，RDS组占7例，其中，6例为纯合C/C突变，1例为杂合C/T突变；对照组中占1例，其中，1例为杂合C/T突变，无纯合C/C突变。8对父母中的一方也存在同样的基因型。根据患者和父母的exon4基因结构综合分析，最后RDS组7例、对照组1例确定为SP-B基因遗传缺陷。基因定位在SP-B基因的1580位点，其正常的氨基酸密码子为ACT，突变后密码子为ATT或A(C/T)T，对应的氨基酸位置为131，突变后引起氨基酸的改变，由原来的苏氨酸变成了异亮氨酸（Thr131Ile）。RDS组基因突变发生频率为0.35（7/20)，对照组基因突变发生频率0.05(1/20)，RDS组基因突变发生频率较对照组增高，差异比较有统计学意义（2=3.906，P=0.048）。RDS组7例中，6例为C/C纯合突变，其发生频率为0.3（6/20），1例为C/T杂合突变，发生频率为0.05（1/20）；对照组1例为C/T杂合突变，其发生频率为0.05（1/20），无纯合突变。RDS组纯合突变发生频率（0.3）高于对照组（0），差异比较有统计学意义（2=4.902，P=0.027），提示exon41580位置基因的纯合突变与研究范围的北京市汉族NRDS发病相关联；而两组杂合突变频率相同，提示exon41580位置基因的杂合突变与研究范围的北京市汉族NRDS发病无关。 结论（1）BAL SP-B蛋白缺陷参与汉族足月NRDS发病。 （2）肺部SP-B表达减少参与汉族足月NRDS发病。 （3）汉族足月NRDS SP-B基因exon4存在基因突变，基因突变发生频率为0.35，RDS组基因突变发生频率较对照组增高，其中纯合突变发生频率为0.3，杂合突变发生频率为0.05。 （4）汉族足月NRDS SP-B基因1580位点的纯合突变与国内汉族足月儿RDS发病相关联。
[Abstract]:Objective to evaluate the frequency and mutation types of pulmonary surfactant protein B (SP-B) genetic defects in term neonates with respiratory distress syndrome (RDS), and to explore the genetic mechanism of RDS. To provide scientific experimental basis for further gene therapy of neonatal RDS. Methods 20 cases of RDS of Han nationality were selected as RDS group and 20 cases of Han nationality as control group. There was no significant difference between RDS group and RDS group in age, weight, sex, age of pregnant mother, delivery mode and so on. The SP-B protein content in bronchoalveolar lavage fluid (BALF) was detected by Elisa, the expression of SP-B protein in lung tissue was detected by immunohistochemical technique, and the gene mutation of SP-B exon4 was analyzed by conventional DNA extraction technique and gene sequencing technique. Results the levels of BALSP-B on day 1 (20.0 卤8.05ng/ml), day 3 (21.1 卤8.13ng/ml), day 7 (20.1 卤7.88ng/ml) and postmortem 30min (19.4 卤8.02ng/ml) were significantly lower than those in control group (92.8 卤32.21 卤102.4 卤28.76105.6 卤30.41). The number of SP-B protein positive cells in the lung tissue of RDS group (18.5 卤10.7) was significantly lower than that in the control group (94.9 卤31.8) (tl 10.191 P < 0.001). The lower the expression level of SP-B protein and the less SP-B protein expression in lung tissue in RDS group. RDS chest X-ray shows a higher level of severity, that is, both have good compliance. There was a point mutation in SP-B protein deficient RDS group (7 cases) than the control group (1 case). Exon4 gene mutation was found in 7 cases of C / T / C / T _ 3 genotype .exon4 gene mutation, among which 6 cases were homozygous C / C mutation 1 case was heterozygous Cr / T mutation, and 1 case in control group, and 1 case in the control group, among which 6 cases were homozygous C / C mutation, 1 case was heterozygous C / T mutation, and 1 case was in the control group. One case was a heterozygous C / T mutation and one parent had the same genotype without homozygous C / C mutation. According to the comprehensive analysis of exon4 gene structure of patients and parents, 7 cases in RDS group and 1 case in control group were identified as SP-B gene genetic defect. The normal amino acid codon is ACTand the mutation codon is ATT or A (C / T) T, the corresponding amino acid position is 131. The frequency of gene mutation was 0.35 (7 / 20) in the former Thr131Ile. RDS group and 0.05 (1 / 20) in the control group. The difference was statistically significant (2 / 3.906 / P ~ (0.048). In the RDS group, 6 cases were C / C homozygous mutation, 1 case was C / T heterozygous mutation (0. 05 (1 / 20), 1 case in the control group was a C / T heterozygosity mutation, 1 case was a C / T heterozygous mutation. The frequency of homozygous mutation was 0. 05 (1 / 20). The frequency of homozygous mutation in RDS group (0. 3) was higher than that in control group (0). The difference was statistically significant (2 / 902). It suggested that homozygous mutation of exon41580 locus gene was associated with the incidence of NRDS in Han nationality in Beijing. The frequency of heterozygous mutations in the two groups was the same, suggesting that the heterozygosity of the exon41580 locus gene was not related to the incidence of NRDS in the Han nationality in Beijing. Conclusion (1) the deficiency of BALSP-B protein is involved in the pathogenesis of full-term NRDS in Han nationality, (2) the decrease of SP-B expression in lung is involved in the pathogenesis of full-term NRDS in Han nationality. (3) there is a gene mutation in exon4 of NRDS SP-B gene in Han nationality. The frequency of gene mutation in the 0.35 RDS group was higher than that in the control group. The frequency of homozygous mutation was 0.3 and the frequency of heterozygous mutation was 0.05. (4) the homozygous mutation at locus 1580 of NRDS SP-B gene was associated with the incidence of RDS in term infants of Han nationality in China.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1
本文编号：2134119
[Abstract]:Objective to evaluate the frequency and mutation types of pulmonary surfactant protein B (SP-B) genetic defects in term neonates with respiratory distress syndrome (RDS), and to explore the genetic mechanism of RDS. To provide scientific experimental basis for further gene therapy of neonatal RDS. Methods 20 cases of RDS of Han nationality were selected as RDS group and 20 cases of Han nationality as control group. There was no significant difference between RDS group and RDS group in age, weight, sex, age of pregnant mother, delivery mode and so on. The SP-B protein content in bronchoalveolar lavage fluid (BALF) was detected by Elisa, the expression of SP-B protein in lung tissue was detected by immunohistochemical technique, and the gene mutation of SP-B exon4 was analyzed by conventional DNA extraction technique and gene sequencing technique. Results the levels of BALSP-B on day 1 (20.0 卤8.05ng/ml), day 3 (21.1 卤8.13ng/ml), day 7 (20.1 卤7.88ng/ml) and postmortem 30min (19.4 卤8.02ng/ml) were significantly lower than those in control group (92.8 卤32.21 卤102.4 卤28.76105.6 卤30.41). The number of SP-B protein positive cells in the lung tissue of RDS group (18.5 卤10.7) was significantly lower than that in the control group (94.9 卤31.8) (tl 10.191 P < 0.001). The lower the expression level of SP-B protein and the less SP-B protein expression in lung tissue in RDS group. RDS chest X-ray shows a higher level of severity, that is, both have good compliance. There was a point mutation in SP-B protein deficient RDS group (7 cases) than the control group (1 case). Exon4 gene mutation was found in 7 cases of C / T / C / T _ 3 genotype .exon4 gene mutation, among which 6 cases were homozygous C / C mutation 1 case was heterozygous Cr / T mutation, and 1 case in control group, and 1 case in the control group, among which 6 cases were homozygous C / C mutation, 1 case was heterozygous C / T mutation, and 1 case was in the control group. One case was a heterozygous C / T mutation and one parent had the same genotype without homozygous C / C mutation. According to the comprehensive analysis of exon4 gene structure of patients and parents, 7 cases in RDS group and 1 case in control group were identified as SP-B gene genetic defect. The normal amino acid codon is ACTand the mutation codon is ATT or A (C / T) T, the corresponding amino acid position is 131. The frequency of gene mutation was 0.35 (7 / 20) in the former Thr131Ile. RDS group and 0.05 (1 / 20) in the control group. The difference was statistically significant (2 / 3.906 / P ~ (0.048). In the RDS group, 6 cases were C / C homozygous mutation, 1 case was C / T heterozygous mutation (0. 05 (1 / 20), 1 case in the control group was a C / T heterozygosity mutation, 1 case was a C / T heterozygous mutation. The frequency of homozygous mutation was 0. 05 (1 / 20). The frequency of homozygous mutation in RDS group (0. 3) was higher than that in control group (0). The difference was statistically significant (2 / 902). It suggested that homozygous mutation of exon41580 locus gene was associated with the incidence of NRDS in Han nationality in Beijing. The frequency of heterozygous mutations in the two groups was the same, suggesting that the heterozygosity of the exon41580 locus gene was not related to the incidence of NRDS in the Han nationality in Beijing. Conclusion (1) the deficiency of BALSP-B protein is involved in the pathogenesis of full-term NRDS in Han nationality, (2) the decrease of SP-B expression in lung is involved in the pathogenesis of full-term NRDS in Han nationality. (3) there is a gene mutation in exon4 of NRDS SP-B gene in Han nationality. The frequency of gene mutation in the 0.35 RDS group was higher than that in the control group. The frequency of homozygous mutation was 0.3 and the frequency of heterozygous mutation was 0.05. (4) the homozygous mutation at locus 1580 of NRDS SP-B gene was associated with the incidence of RDS in term infants of Han nationality in China.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1
本文编号：2134119
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