中华儿科杂志
中華兒科雜誌
중화인과잡지
Chinese Journal of Pediatrics
2015年
5期
348-354
,共7页
许永彬%陈玉冰%曾萍%陈慧珊%曾华松
許永彬%陳玉冰%曾萍%陳慧珊%曾華鬆
허영빈%진옥빙%증평%진혜산%증화송
炎症性肠病%新生儿%白细胞介素10受体α亚单位%突变
炎癥性腸病%新生兒%白細胞介素10受體α亞單位%突變
염증성장병%신생인%백세포개소10수체α아단위%돌변
Inflammatory bowel disease%Neonatal%Interleukin-10 receptor alpha subunit%Mutation
目的 对2例新生儿期发病的炎症性肠病(IBD)进行白细胞介素(IL)-10受体基因诊断,并探讨其发病机制.方法 研究对象为广州市妇女儿童医疗中心2010至2014年新生儿科和消化内科住院的2例疑诊新生儿期IBD同胞兄弟.先证者男,26 d,3.73 kg,出生后第9天出现反复发热,大便次数增多于2014年入院.先证者兄,6个月时因反复黏液脓血便5个月余,发热5d于2010年入院,8个月死亡.采用PCR方法扩增先证者及其父母和姐姐IL-10受体基因组DNA,本院儿童保健科健康体检儿童的资料作为对照组.采用反转录PCR扩增先证者IL-10受体α亚单位(IL-10RA),PCR产物进行双向序列测定,Western分析先证者IL-10RA蛋白以及IL-10刺激后磷酸化信号转导和转录激活因子3(P-STAT3)蛋白的表达.分离外周血单个核细胞(PBMC)并予脂多糖+IL-10刺激培养后酶联免疫吸附试验(ELISA)检测肿瘤坏死因子(TNF)-α水平.结果 先证者及其兄均为IBD患者,基因组测序发现IL-10RA突变位点均为c.537G＞A为同义突变,突变位点位于外显子4和内含子4连接处即CG/GT突变为CA/GT,先证者反转录PCR产物T-A克隆测序发现(c.519-537delGGTGCCGGGAAACTTCAC p.LYS173ASNfs*7),证实为剪接突变.另外一条DNA链基变位点为(c.668G＞C,p.C223S),两个突变均为新基因突变,患儿父母均为突变基因的携带者,先证者及其兄均为复合杂合隐性遗传突变引起.Western印迹分析表明先证者和正常者均可表达IL-10RA,但是IL-10RA下游功能明显缺陷,IL-10刺激后下游P-STAT3无表达.先证者PBMC予脂多糖+IL-10共刺激培养12 h后TNF-α为(2 100±356) ng/L,明显高于对照者[(200 ±50) ng/L,t=9.154,P=0.001].IL-10对炎症的负反馈调节功能严重受损.结论 IL-10受体突变能够引起婴儿早期IBD,包括新生儿溃疡性结肠炎和克罗恩病,此型常规治疗效果较差,必须尽早诊断,尽早进行造血干细胞移植.
目的 對2例新生兒期髮病的炎癥性腸病(IBD)進行白細胞介素(IL)-10受體基因診斷,併探討其髮病機製.方法 研究對象為廣州市婦女兒童醫療中心2010至2014年新生兒科和消化內科住院的2例疑診新生兒期IBD同胞兄弟.先證者男,26 d,3.73 kg,齣生後第9天齣現反複髮熱,大便次數增多于2014年入院.先證者兄,6箇月時因反複黏液膿血便5箇月餘,髮熱5d于2010年入院,8箇月死亡.採用PCR方法擴增先證者及其父母和姐姐IL-10受體基因組DNA,本院兒童保健科健康體檢兒童的資料作為對照組.採用反轉錄PCR擴增先證者IL-10受體α亞單位(IL-10RA),PCR產物進行雙嚮序列測定,Western分析先證者IL-10RA蛋白以及IL-10刺激後燐痠化信號轉導和轉錄激活因子3(P-STAT3)蛋白的錶達.分離外週血單箇覈細胞(PBMC)併予脂多糖+IL-10刺激培養後酶聯免疫吸附試驗(ELISA)檢測腫瘤壞死因子(TNF)-α水平.結果 先證者及其兄均為IBD患者,基因組測序髮現IL-10RA突變位點均為c.537G＞A為同義突變,突變位點位于外顯子4和內含子4連接處即CG/GT突變為CA/GT,先證者反轉錄PCR產物T-A剋隆測序髮現(c.519-537delGGTGCCGGGAAACTTCAC p.LYS173ASNfs*7),證實為剪接突變.另外一條DNA鏈基變位點為(c.668G＞C,p.C223S),兩箇突變均為新基因突變,患兒父母均為突變基因的攜帶者,先證者及其兄均為複閤雜閤隱性遺傳突變引起.Western印跡分析錶明先證者和正常者均可錶達IL-10RA,但是IL-10RA下遊功能明顯缺陷,IL-10刺激後下遊P-STAT3無錶達.先證者PBMC予脂多糖+IL-10共刺激培養12 h後TNF-α為(2 100±356) ng/L,明顯高于對照者[(200 ±50) ng/L,t=9.154,P=0.001].IL-10對炎癥的負反饋調節功能嚴重受損.結論 IL-10受體突變能夠引起嬰兒早期IBD,包括新生兒潰瘍性結腸炎和剋囉恩病,此型常規治療效果較差,必鬚儘早診斷,儘早進行造血榦細胞移植.
목적 대2례신생인기발병적염증성장병(IBD)진행백세포개소(IL)-10수체기인진단,병탐토기발병궤제.방법 연구대상위엄주시부녀인동의료중심2010지2014년신생인과화소화내과주원적2례의진신생인기IBD동포형제.선증자남,26 d,3.73 kg,출생후제9천출현반복발열,대편차수증다우2014년입원.선증자형,6개월시인반복점액농혈편5개월여,발열5d우2010년입원,8개월사망.채용PCR방법확증선증자급기부모화저저IL-10수체기인조DNA,본원인동보건과건강체검인동적자료작위대조조.채용반전록PCR확증선증자IL-10수체α아단위(IL-10RA),PCR산물진행쌍향서렬측정,Western분석선증자IL-10RA단백이급IL-10자격후린산화신호전도화전록격활인자3(P-STAT3)단백적표체.분리외주혈단개핵세포(PBMC)병여지다당+IL-10자격배양후매련면역흡부시험(ELISA)검측종류배사인자(TNF)-α수평.결과 선증자급기형균위IBD환자,기인조측서발현IL-10RA돌변위점균위c.537G＞A위동의돌변,돌변위점위우외현자4화내함자4련접처즉CG/GT돌변위CA/GT,선증자반전록PCR산물T-A극륭측서발현(c.519-537delGGTGCCGGGAAACTTCAC p.LYS173ASNfs*7),증실위전접돌변.령외일조DNA련기변위점위(c.668G＞C,p.C223S),량개돌변균위신기인돌변,환인부모균위돌변기인적휴대자,선증자급기형균위복합잡합은성유전돌변인기.Western인적분석표명선증자화정상자균가표체IL-10RA,단시IL-10RA하유공능명현결함,IL-10자격후하유P-STAT3무표체.선증자PBMC여지다당+IL-10공자격배양12 h후TNF-α위(2 100±356) ng/L,명현고우대조자[(200 ±50) ng/L,t=9.154,P=0.001].IL-10대염증적부반궤조절공능엄중수손.결론 IL-10수체돌변능구인기영인조기IBD,포괄신생인궤양성결장염화극라은병,차형상규치료효과교차,필수진조진단,진조진행조혈간세포이식.
Objective To explore use of interleukin-10 receptor (IL-10R) gene mutation in diagnosis and pathogenesis of neonatal inflammatory bowel disease (IBD) in 2 suspected cases.Method Two cases of sibling brothers who had suspected IBD from Guangzhou Women and Children's Medical Center Affiliated to Guangzhou Medical University during the year 2010-2014 were enrolled in the study.The proband,male,26 days old,weight 3.73 kg,presented with recurrent fever,increased stool frequency since 9 days of age,and was hospitalized at the age of 6 months in 2014.The proband's brother,male,6 months old,weight 8 kg,had repeated bloody and mucous diarrhea for more than five months,recurrent fever five days,and was hospitalized in 2010.The blood samples were collected from the children and their families for IL-10 receptor genes including IL-10 receptor α subunit (IL-10RA) and β subunit (IL-10RB) PCR amplification.Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the proband IL-10RA transcripts.Sequencing was performed on the PCR products forward and reversely.Western blot analysis was used for protein expression of the proband and normal control's IL-10RA and P-STAT3 (Tyr705) expression after IL-10 stimulation,TNF-α level was detected using Human TNF-α ELISA Kit after PBMC was cultured and stimulated.Result The proband and his brother were IBD patients.Genome sequencing showed mutation in c.537G ＞ A,namely the exon 4 and intron 4 connections changed CA/GT for CG/GT.Sequencing of the RT-PCR products and T-A clone showed that the mutation was (c.519-537del GGTGCCGGGAAACTTCAC,p.LYS173ASNfs * 7),as the splice mutation.Two gene mutations were novel mutation.The parents were the mutations carrier.Both of the children were compound heterozygous mutations in IL-10RA.The Western blot analysis showed that the patient and normal children can express IL-10RA protein,however,the function of IL-10RA had obvious defects in the patient,IL-10RA downstream signaling pathways P-STAT3 had no expression.The average level of TNF-α secreted by PBMC after LPS + IL-10 co-stimulation in patient was significantly increased as compared with control group ((2 100 ± 356)vs.(200 ± 50) ng/L,t =9.154,P =0.001),suggesting that interleukin-10-dependent negative feedback regulation is disrupted in the patient.Conclusion IL-10 receptor mutations can cause neonatal-IBD,for which common treatment effect is poor.Early diagnosis and allogeneic stem-cell transplantation performed may save the children's life.
