足月新生兒呼吸窘迫綜合征肺表面活性物質(zhì)蛋白B遺傳缺陷探索性研究
發(fā)布時間：2018-07-20 16:36
【摘要】：目的評估足月新生兒呼吸窘迫綜合征肺表面活性物質(zhì)蛋白B（SP-B）遺傳缺陷的發(fā)生頻率及突變類型，，探討足月新生兒呼吸窘迫綜合征發(fā)病的遺傳機制，為進一步基因治療新生兒RDS提供科學實驗依據(jù)。 方法選擇無血緣關(guān)系的漢族足月新生兒RDS20例作為RDS組，漢族20例其它病例作為對照組，并在年齡、體重、性別、孕母年齡、分娩方式等方面與RDS組無統(tǒng)計學差異。采用ELISA技術(shù)檢測支氣管肺泡灌洗液的SP-B蛋白含量，免疫組化技術(shù)檢測SP-B蛋白在肺組織的表達；常規(guī)DNA抽提技術(shù)提取人全血DNA，PCR及基因測序技術(shù)分析SP-B exon4的基因突變。 結(jié)果RDS組患兒BAL SP-B含量在發(fā)病第1天（20.0±8.05ng/ml）、發(fā)病第3天（21.1±8.13ng/ml）、發(fā)病第7天（20.1±7.88ng/ml）、死后30min內(nèi)（19.4±8.02ng/ml）均顯著低于對照組（92.8±32.21；102.4±28.76；105.6±30.41；92.0±32.71ng/ml）（P＜0.001）。RDS組患兒肺組織SP-B蛋白表達陽性細胞數(shù)（18.5±10.7）顯著低于對照組（94.9±31.8）（t＝10.191，P＜0.001）。RDS組BAL SP-B蛋白含量表達水平越低及肺組織SP-B蛋白表達陽性細胞越少，則RDS胸部X線顯示嚴重性的級別越高，即二者具有很好的依從性。兩組均存在SP-B蛋白缺陷，RDS組（7例）高于對照組（1例）。exon4存在基因點突變，存在C/T、C/C、T/T3種基因型。exon4基因突變中，RDS組占7例，其中，6例為純合C/C突變，1例為雜合C/T突變；對照組中占1例，其中，1例為雜合C/T突變，無純合C/C突變。8對父母中的一方也存在同樣的基因型。根據(jù)患者和父母的exon4基因結(jié)構(gòu)綜合分析，最后RDS組7例、對照組1例確定為SP-B基因遺傳缺陷�；蚨ㄎ辉赟P-B基因的1580位點，其正常的氨基酸密碼子為ACT，突變后密碼子為ATT或A(C/T)T，對應(yīng)的氨基酸位置為131，突變后引起氨基酸的改變，由原來的蘇氨酸變成了異亮氨酸（Thr131Ile）。RDS組基因突變發(fā)生頻率為0.35（7/20)，對照組基因突變發(fā)生頻率0.05(1/20)，RDS組基因突變發(fā)生頻率較對照組增高，差異比較有統(tǒng)計學意義（2=3.906，P=0.048）。RDS組7例中，6例為C/C純合突變，其發(fā)生頻率為0.3（6/20），1例為C/T雜合突變，發(fā)生頻率為0.05（1/20）；對照組1例為C/T雜合突變，其發(fā)生頻率為0.05（1/20），無純合突變。RDS組純合突變發(fā)生頻率（0.3）高于對照組（0），差異比較有統(tǒng)計學意義（2=4.902，P=0.027），提示exon41580位置基因的純合突變與研究范圍的北京市漢族NRDS發(fā)病相關(guān)聯(lián)；而兩組雜合突變頻率相同，提示exon41580位置基因的雜合突變與研究范圍的北京市漢族NRDS發(fā)病無關(guān)。 結(jié)論（1）BAL SP-B蛋白缺陷參與漢族足月NRDS發(fā)病。 （2）肺部SP-B表達減少參與漢族足月NRDS發(fā)病。 （3）漢族足月NRDS SP-B基因exon4存在基因突變，基因突變發(fā)生頻率為0.35，RDS組基因突變發(fā)生頻率較對照組增高，其中純合突變發(fā)生頻率為0.3，雜合突變發(fā)生頻率為0.05。 （4）漢族足月NRDS SP-B基因1580位點的純合突變與國內(nèi)漢族足月兒RDS發(fā)病相關(guān)聯(lián)。
[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 鹵8.05ng/ml), day 3 (21.1 鹵8.13ng/ml), day 7 (20.1 鹵7.88ng/ml) and postmortem 30min (19.4 鹵8.02ng/ml) were significantly lower than those in control group (92.8 鹵32.21 鹵102.4 鹵28.76105.6 鹵30.41). The number of SP-B protein positive cells in the lung tissue of RDS group (18.5 鹵10.7) was significantly lower than that in the control group (94.9 鹵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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.1
本文編號：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 鹵8.05ng/ml), day 3 (21.1 鹵8.13ng/ml), day 7 (20.1 鹵7.88ng/ml) and postmortem 30min (19.4 鹵8.02ng/ml) were significantly lower than those in control group (92.8 鹵32.21 鹵102.4 鹵28.76105.6 鹵30.41). The number of SP-B protein positive cells in the lung tissue of RDS group (18.5 鹵10.7) was significantly lower than that in the control group (94.9 鹵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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.1
本文編號：2134119
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