成人型脊髓性肌萎缩症46例临床分析

目的 总结成人型脊髓性肌萎缩症(SMA_4)的临床、肌电图和病理特征。方法 收集48例经肌肉活检确诊的SMA_4病人的临床、肌电图和病理资料,对其进行回顾性分析和研究。结果 SMA_4病人平均发病年龄38.23岁,起病隐袭,肌无力和肌萎缩以四肢近端为主,进展缓慢,预后相对较好。肌电图主要表现为插入电位异常,常见纤颤波和束颤波,部分可见正相尖波,平均波幅和平均时限均显著增加,大都有多相电位增多,大力收缩都不能达到干扰相。肌活检主要表现为小群性肌萎缩,并可见同型肌君化及肌纤维代偿性肥大。结论 根据临床特征,结合肌电图和肌活检结果,可以确诊成人型脊髓性肌萎缩症。     

Survival of motor neuron protein downregulates miR-9 expression in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous absence of the survival of the motor neuron (SMN) 1 gene (SMN1), and it is the leading genetic cause of infant mortality. The severity of SMA is directly correlated with SMN protein levels in affected patients; however, the cellular regulatory mechanisms for SMN protein expression are not completely understood. In this study, we investigated the regulatory effects between SMN expression and miR-9a, a downstream noncoding small RNA. Using an inducible SMN short hairpin RNA interference (shRNAi) system in NSC 34 and human skin fibroblast cells, cellular miR-9 levels and SMN protein repression were time-dependently upregulated. Conversely, cellular miR-9 levels decreased when HeLa cells were transfected with SMN protein fused with green fluorescent protein. In SMA-like mice spinal cords and human primary skin fibroblasts isolated from patients with different degrees of SMA, human SMN exhibited a disease severity-dependent decrease, whereas cellular miR-9 levels increased. These results clearly suggested that cellular SMN proteins regulated miR-9 expression and that miR-9 expression was related to SMA severity. Thus, miR-9 may be a marker for SMA prognosis.     

Effect of Nusinersen in a late onset spinal muscular atrophy patient for 14 months: A case report

Rationale:Spinal muscular atrophy (SMA) is a genetic disorder caused by genetic defect of SMN1 gene. SMA was an untreatable disease until 2016, when Nusinersen an antisense oligonucleotide therapy was approved for treatment. We report the effect of Nusinersen in a late onset SMA for 14 months.Patient concerns:A 13-year-old boy who was diagnosed as SMA with progressive proximal limb weakness was treated with intrathecal injection of Nusinersen.Diagnosis:The patient had progressive proximal limb weakness after 2 years of age. The patient had elevated creatine kinase level and shoed neurogenic changes in the needle electromyography study. After genetic analysis, homozygous deletion in Exon 7 and 8 of SMN1 protein was found and he was diagnosed as late onset SMA.Interventions:Intrathecal Nusinersen was administered per protocol.Outcomes:After 14 months of treatment, the patient showed significant clinical improvement in the revised Hammersmith functional rating scale and 6-minute walk test.Lessons:Although there is limited data on the effect of Nusinersen in late onset SMA patients, our case adds on the effectiveness even in late onset SMA. More studies are needed to consolidate the effects and adverse events of Nusinersen in late onset SMA.     

脊髓性肌萎缩症的基因研究

目的 研究我国Ⅰ～Ⅳ型脊髓性肌萎缩症（SMA）患者运动神经元生存基因（SMN）及神经细胞凋亡抑制蛋白（NAIP）基因外显子的缺失情况,以探讨此两种基因与SMA表型之间的关系。方法 应用PCR法检测45例Ⅰ～Ⅳ型SMA患者、30例表型正常的SMA直系亲属及30例正常对照的SMIN基因第7、8号外显子和NAIP基因第5、6号外显子缺失情况。结论 7例Ⅰ型和Ⅱ型SMA患者中6例纯合缺失SMN基因外显子7和8,1例纯合缺失外显子7而保留外显子8;8例Ⅲ型SMA患者仅1例缺失外显子7和8,余7例均无SMN基因的缺失;成人型（Ⅳ型）SMA未检测到SMN基因缺失;45例Ⅰ～Ⅳ型SMA患者均未检测到NAIP基因外显子5和（或）6的缺失。结论 Ⅰ型、Ⅱ型SMA可通过SMN基因第7、8号外显子的检测进行确诊,Ⅲ型SMA患者SMN基因缺失率低,故通过检测SMN基因7、8外显子进行基因诊断尚需谨慎,Ⅳ型SMA未检测到SMN基因缺失,发病可能与SMN基因缺失无关;NAIP基因在SMA发病中的作用尚不清楚。     

脊髓性肌萎缩症患者神经元样细胞SMN2基因mRNA的表达

目的检测脊髓性肌萎缩症（SMA）患者神经元样细胞SMN2基因mRNA的表达。方法用PCR-RFLP的方法对SMA患者进行基因诊断,诱导其骨髓间充质干细胞分化为神经元样细胞,RT-PCR和测序检测该细胞SMN2基因mRNA的表达,所有过程与对照组进行对比研究。结果SMN2基因的扩增产物为全长转录产物fl-SMNmRNA（266bp）和转录时跳过外显子7的产物SMN△7mRNA（212bp,经测序证实缺少的54bp为外显子7的序列）;SMA患者fl-SMNmRNA的表达占总表达量的23．2％,远低于对照者（占总表达量的82．0％）;而SMN△7mRNA表达占总表达量的76．8％,高于对照者的18．0％。结论SMA患者神经元样细胞的SMN2基因转录时存在选择性剪接,即剪接时跳过外显子7,是导致其全长SMN蛋白不足的原因之一。     

弓形虫信号转导蛋白14-3-3基因的克隆与表达

目的　体外扩增弓形虫RH株信号转导蛋白1433(Toxo1433)基因编码序列,构建原核表达质粒,并表达Toxo1433。　方法　收集、纯化弓形虫RH株速殖子,提取RNA,在设计合成的引物中引入EcoRI和XhoI酶切位点。应用RTPCR扩增Toxo1433基因片段,插入原核表达质粒pET28a中,重组子双酶切、PCR和测序鉴定,转化大肠杆菌BL21并以异丙基βD硫代半乳糖苷(IPTG)诱导表达。　结果　从弓形虫RH株RNA中扩增出803bp的Toxo1433基因片段,构建重组质粒pET28a/1433;IPTG诱导,SDSPAGE显示表达产物的大小约30.7kDa,Western印迹鉴定为Toxo1433。　结论　成功地从弓形虫RH株基因组DNA中获取了1433基因,构建了pET28a/Toxo1433重组质粒,并获得高效表达。     

日本血吸虫(中国大陆株)信号蛋白14-3-3在原核细胞的高效融合表达和特性鉴定

目的　构建重组质粒pET2 8a -Sj14 - 3- 3,并在大肠杆菌中表达 ,检测表达产物的免疫活性。 方法　用亚克隆技术把Sj14 - 3- 3基因克隆至 pET2 8aT7启动子下游 ,转化大肠杆菌DH5α和BL2 1感受态细胞 ,经IPTG诱导表达 ,SDS -PAGE和Westernblot分析。结果　获得pET2 8a -Sj14 - 3- 3重组表达载体 ,SDS -PAGE和Westernblot显示Sj14 - 3- 3基因在 pET2 8a中表达的融合蛋白约为 32 4kDa ,与天然 14 - 3- 3蛋白具有相同的免疫活性。 结论　日本血吸虫信号蛋白14 - 3- 3在原核细胞中得以高效表达 ,表达产物具有免疫活性 ,为进一步研究其免疫保护作用和信号转导奠定了基础     

14-3-3蛋白在心肌细胞缺氧预处理中的表达及意义

目的:研究14-3-3蛋白在心肌细胞缺氧预处理中的表达及意义。方法:实验用SD新生大鼠(1～3d龄),雌雄不拘,无菌取出乳鼠心脏,经分离附着组织,胰蛋白酶消化,纯化制成心肌细胞。在培养的第4天随机分为3组:正常对照组、缺氧/复氧(A/R)组、缺氧预处理(APC)组。各组分别进行以下指标观察:①心肌细胞搏动频率;②细胞存活率(MTT法);③培养液中乳酸脱氢酶(LDH)活性;④透射电镜观察细胞超微结构;⑤Western Blotting法检测14-3-3蛋白的表达变化。RT-PCR法检测14-3-3蛋白η、σmRNA的表达变化。结果:A/R组和APC组的14-3-3蛋白表达均上调,分别是正常对照组的(3.61±0.37)倍和(5.52±0.49)倍,A/R组和APC组与正常对照组比较、A/R组与APC组比较,均P<0.01。A/R组、APC组心肌细胞14-3-3η亚型mRNA分别为正常对照组的(1.82±0.30)倍、(2.93±0.52)倍,差异均有统计学意义(P<0.01);APC组与A/R组比较,差异亦有统计学意义(P<0.01)。A/R组、APC组心肌细胞14-3-3σ亚型mRNA表达量分别为正常对照组的...     

Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.     

Reinduced Wnt signaling limits regenerative potential of sensory axons in the spinal cord following conditioning lesion

Conditioning lesion of the peripheral branch of dorsal column axons is a well-known paradigm enabling the central branch to regenerate after injury to the spinal cord. However, only a small number of regenerating axons enter grafted substrates, and they do not grow beyond the lesion. We found that conditioning lesion induces, in addition to growth-stimulating genes, related to receptor tyrosine kinase (Ryk), a potent repulsive receptor for Wnts. Wnts are expressed around the site of spinal cord injury, and we found that grafted bone marrow stromal cells secreting the Wnt inhibitors secreted frizzled-related protein 2 or Wnt inhibitory factor 1 enhanced regeneration of the central branch after peripheral conditioning lesion. Furthermore, we found that Wnt4-expressing grafts caused dramatic long-range retraction of the injured central branch of conditioned dorsal root ganglion neurons. Macrophages accumulate along the path of receding axons but not around Wnt4-expressing cells, suggesting that the retraction of dorsal column axons is not a secondary effect of increased macrophages attracted by Wnt4. Therefore, Wnt-Ryk signaling is an inhibitory force co-induced with growth-stimulating factors after conditioning lesion. Overcoming Wnt inhibition may further enhance therapies being designed on the basis of the conditioning-lesion paradigm.     

Mitral valve prolapse related to geometrical changes of the heart in cases of progressive muscular dystrophy

The significance of geometrical changes of the heart for the development of mitral valve prolapse (MVP) was studied by echocardiograms and chest x-ray films in 58 cases of progressive muscular dystrophy (PMD). The incidence of MVP was significantly higher (p less than 0.001) in cases where the thoracic spine was straight or lordotic compared with cases of kyphotic thoracic spine. The flattening of the thorax associated with deformation of the thoracic spine was correlated with the left atrial dimension and left ventricular dimension (r = 0.62, r = 0.37, respectively; p less than 0.001), and MVP developed predominantly in cases with flattened thorax and small left atrial or left ventricular dimensions. The left atrial and left ventricular dimensions were significantly smaller in cases with MVP compared to cases without MVP (p less than 0.001, p less than 0.005, respectively). When both the left atrial and the left ventricular dimension shortened to certain levels, MVP was observed in almost all cases. From these results, it was suggested that the portion from the left atrium to the left ventricle was pressed by the forward bending of the thoracic spine, and the subsequent geometrical changes of the mitral ring and the left ventricle could produce redundancy of the chorda tendinea of the mitral valve, resulting in the occurrence of MVP.