云南傣族儿童中β-地中海贫血HBB基因的变异分布特征

Objective To characterize the distribution of variation in hemoglobin beta gene ( hemoglobin beta gene, HBB )of β-thalassemia in Dai minority group in Yunnan province, and to establish a rapid and economical method for screening β-thalassemia mutation via optimizing ARMS technique.Methods DNA Sequencing was performed to detect variation in 1755 bp DNA fragment containing whole length of hemoglob beta gene ( 1606 bp) from 209 cases of Dai children with β-thalassemia. The primer design and PCR conditions of ARMS (amplification refractory mutation system ) have been optimized for detecting three frequent variations. Results Among 209 samples, there are 9 variations were detected:CD2 T＞ C (50.72%),CD26 G ＞A (35.41%),CD17 A ＞T(12.92%),IVS-Ⅱ-17 C ＞ G (12.44%), IVS-Ⅱ-16 G＞C(11.96%), ⅣS-Ⅰ-30 A＞G(9.57%) , CD6 G ＞A(9.09%) ,CD41-42 (-TCTT) (7. 18%) and CD5 T＞A(1.92%). Three mutations (CD21, CD35 and CD43) were first reported in China. There are 167, out of 209 participants (79.90%), carrying HBB gene variation. Three common mutations (CD2, CD26 and CD17 ) can be efficiently detected by combining condition of both ARMS mismatch primers and 65 ℃ of PCR annealling temperature. Conclusion The distribution of HBB gene variation in the Dai minority group in Yunnan province is different from those of other groups in China. ARMS is an effective, convenient and economical technique for rapid detection of gene mutations of β-thalassemia.     

靶向性反向半胱氨酸天冬氨酸蛋白酶3重组腺病毒诱导肝癌细胞凋亡的实验研究

目的 观察靶向性反向半胱氨酸天冬氨酸蛋白酶3(r-caspase-3)重组腺病毒体外诱导肝癌细胞凋亡的效果和特异性.方法 构建甲胎蛋白(AFP)增强子和白蛋白(ALB)启动子腺病毒载体(pAdTrack-EAFP-PALB),亚克隆r-caspase-3至载体pAdTrack-EAFP-PALP,Pme Ⅰ线性化pAdTrack-EAFP-PALB/r-caspase-3,电转化至BJ/AdEasy菌,获得重组腺病毒骨架pAdeasy-EAFP-PALB/r-caspase-3.Pac Ⅰ酶切后脂质体转染AD293细胞进行包装、扩增、获得病毒.绿色荧光蛋白(GFP)监测病毒滴度和感染效率;RT-PCR和Western印迹法检测r-caspase-3在HepG2细胞中的表达;流式细胞术检测其特异性诱导肝癌细胞凋亡的作用.结果 穿梭载体pAdTrack-EAFP-PALB/r-caspase-3酶切、测序正确.穿梭载体、pAdeasy-1载体同源重组后PCR及Pac Ⅰ酶切鉴定结果表明pAdeasy-EAFP-PALB/r-caspase-3重组成功.转染AD293细胞后可观察到GFP的表达.病毒感染HepG2细胞总DNA经RT-PCR和Western印迹均可检测到目的 基因的表达,证实Ad-EAFP-PALB/r-caspase-3病毒颗粒包装成功;靶向性r-caspase-3重组腺病毒感染各组细胞24 h后,各组凋亡指数分别为:HepG细胞48.2%、7721细胞17.7%、L-02细胞7.3%、MDA-MB-231细胞0%.结论 成功构建了靶向性Ad-EAFP-PALB/r-caspase-3重组腺病毒,体外实验表明其具有凋亡诱导靶向性,为进一步研究靶向性r-caspase-3基因治疗肝细胞肝癌及其生物学功能提供了依据.     

p53蛋白在宫颈鳞癌组织中的表达及意义

目的探讨p53蛋白在宫颈鳞癌组织中的表达及临床意义。方法应用免疫组化SP法分别对宫颈鳞癌切除标本和正常宫颈组织标本中的p53蛋白的表达状况进行检测。结果 p53蛋白在35例正常组织中不表达,在65例宫颈鳞癌组织中,p53蛋白阳性表达38例,阳性率为58.46%。癌组织中p53蛋白的表达率与正常组织比较差异有统计学意义（P〈0.05）。p53蛋白的阳性表达率与年龄及临床分期差异无统计学意义（P〉0.05）,即p53蛋白表达阳性与宫颈鳞癌患者年龄及临床分期无明显相关。p53蛋白的表达量与宫颈鳞癌的分化程度及局部淋巴结转移差异有统计学意义（P〈0.05）,即p53蛋白的表达量与病理分化程度及局部淋巴结转移有关。结论 p53蛋白的表达与宫颈鳞癌的发生发展有关,可作为宫颈鳞癌的早期诊断、治疗及预后评估的一项参考指标。     

以贫血黄疸首诊的先天性梅毒诊治分析

目的探讨不典型先天性梅毒的临床表现,诊治要点及预后。方法回顾性分析以贫血黄疸首诊的先天性梅毒临床资料,对其临床表现、相关实验室检测指标进行统计分析。结果 8例患儿均有营养不良、贫血、皮肤巩膜黄染,6例有浅表淋巴结肿大,肝脏肿大5例,脾肿大3例,皮肤出血1例,均无梅毒性天疱疮。实验室发现有不同程度低增生性贫血、血小板降低及肝功能损害,1例有骨损害,1例有神经系统受累。选用青霉素治疗,治愈5例,好转2例。结论贫血黄疸可为先天性梅毒首诊症状,及时发现,早期正规治疗是改善预后的关键。     

Effects and long‐term follow‐up of using umbilical cord blood–derived mesenchymal stromal cells in pediatric patients with severe BK virus‐associated late‐onset hemorrhagic cystitis after unrelated cord blood transplantation

This is a retrospective study to evaluate the efficacy and safety of umbilical cord blood–derived mesenchymal stromal cells (MSCs) for the treatment of pediatric patients with severe BK virus–associated late‐onset hemorrhagic cystitis (BKV‐HC) after unrelated cord blood transplantation (UCBT). Thirteen pediatric patients with severe BKV‐HC from December 2013 to December 2015 were treated with MSCs. The number of MSCs transfused in each session was 1 × 10⁶/kg once a week until the symptoms improved. The median follow‐up time was 1432 (89‐2080) days. The median frequency of MSC infusion was 2 (1‐3), with eight cured cases and five effective cases; the total efficacy rate was 100%. The copy number of urine BKV DNA was 4.43 (0.36‐56.9) ×10⁸/mL before MSC infusion and 2.67 (0‐56.3) ×10⁸/mL after MSC infusion; the difference was not significant (P = .219). There were no significant differences in the overall survival, disease‐free survival, and the incidence of relapse and acute and chronic graft‐versus‐host disease between the MSC infusion group and non‐MSC infusion group. There was also no significant difference in the cytomegalovirus, Epstein‐Barr virus (EBV), and fungal and bacterial infection rates between the two groups. Although umbilical cord blood–derived MSCs do not reduce the number of BKV DNA copies in the urine, the cells have a high efficacy rate and minimal side effects in treating severe BKV‐HC after UCBT among pediatric patients. MSCs do not affect the rates of relapse, long‐term infection, or survival of patients with leukemia.     

MKS1 基因变异所致Joubert 综合征1 例临床及基因分析

目的对Joubert 综合征进行临床特点和遗传学分析。方法回顾分析1例确诊的Joubert 综合征患儿的临床资料。结果患儿男性,3个月,双眼不追视、发育落后,四肢肌张力偏低。头颅磁共振示"磨牙征"。全外显子测序显示患儿MKS1基因c.1411_c.1412insG及c.44AG复合杂合突变。Sanger测序验证c.1411_c.1412insG移码突变来自于父亲,已有报道;c.44AG错义突变来自于母亲,未见报道,根据ACMG指南为可能致病变异。患儿确诊为Joubert综合征。结论确诊患儿为Joubert综合征,新发现c.44AG错义突变。     

儿童朗格罕细胞组织细胞增生症BRAFV600E突变对预后的影响

目的 探讨BRAFV600E突变在儿童朗格罕细胞组织细胞增生症（LCH）中的预后意义以及影响儿童LCH的预后因素。方法 回顾性纳入首都医科大学附属北京儿童医院2016年1月1日至2017年12月31日收治的初治LCH患儿,采用数字PCR方法检测患儿病灶组织中的BRAFV600E突变,分析突变与临床特征和预后的相关性,影响预后的因素。结果 140例LCH患儿纳入研究,诊断时中位年龄为2.2（0.1~15.7）岁,男89例（63.6%）,女51例。临床分型为单系统（SS）60例（42.9%）,多系统无危险器官受累（MS RO－）47例（33.6%）,多系统有危险器官受累（MS RO+）33例（23.6%）。中位随访时间34.1（0.9~50.7）月,随访期内70例（50.0%）出现进展或复发,12例（9.6%）出现持续性后遗症,死亡3例（2.1%）。总体3年无进展生存率（PFS）、无事件生存率（EFS）和总生存率（OS）分别为（48.9±4.5）%、（46.6±4.4）%和（97.8±1.2）%。①儿童LCH中BRAFV600E突变阳性率67.1%（94/140）。②BRAFV600E突变与患儿临床分型明显相关,突变阳性率MS RO+患儿（90.9%）高于MS RO－患儿（53.2%）或SS患儿（65.0%）（P=0.001）;皮肤受累患儿中突变阳性率（77.8%）高于非皮肤受累的患儿（P=0.042）;③BRAFV600E突变阳性患儿进展/复发率（57.4%）高于突变阴性患儿（34.8%）,P=0.019。3种临床分型中突变阳性和阴性患儿的预后差异均无统计学意义。④多因素分析结果表明,危险器官受累是影响LCH患儿PFS的独立预后因素（HR=2.702,P=0.003）,垂体受累（HR=3.582,P<0.001）、危险器官（HR=2.321,P=0.008）和耳部受累（HR=2.093,P=0.013）是EFS的独立预后因素。结论 LCH患儿BRAFV600E突变与危险器官受累密切相关,突变阳性患儿的进展/复发率高于突变阴性患儿,BRAFV600E不是儿童LCH的独立预后因素。     

儿童新型冠状病毒肺炎快速筛查与防控问答及专家建议

目前新型冠状病毒肺炎(novel coronavirus pneumonia,NCP)引发的疫情,已成为当前最为严重的公共卫生问题,严重威胁着人民的健康与生命。上海儿科专家近期就当前的NCP热点问题展开了线上讨论,对于儿童新型冠状病毒(2019-nCoV)感染的发病情况、流行病学特点、早期筛查要点、治疗和院内感染防护等主要问题进行深入讨论,进一步凝练了疾病发生、发展、诊疗和防控的规律,并提出相关的专家建议,供临床一线的儿科医师参考。