Duchenne型肌营养不良症的细胞治疗

Duchenne型肌营养不良症（Duchenne muscular dystrophy，DMD）表现为进行性肌肉萎缩，是一种致死性、遗传性神经肌肉疾病。尽管对肌肉萎缩的分子机理的研究取得了很大进展，但是仍然不能治愈，细胞治疗是很有前景的一种治疗方法。成肌细胞移植面临的主要限制是注射后细胞分布差，免疫排斥和细胞存活率低；骨髓干细胞移植需要解决横向分化的低效率问题；而肌源性干细胞看来能更有效地再生表达dystrophin的肌纤维。     

Duchenne型肌营养不良的治疗进展

进行性肌营养不良是一组遗传性肌肉变性疾病,以Duchenne型肌营养（DMD）不良最常见,目前尚无有效的治疗方法,本文从药物疗法、成肌细胞移植疗法以及基因移植疗法3方面介绍了国外近几年关于DMD的治疗进展。     

Duchenne肌营养不良症的分子遗传学

分子生物学,特别是重组DNA技术的出现,使我们对人类遗传病有了更深入的认识。这一领域的进展是极其迅速的,其中最明显的例子就是对X-连锁遗传病Duchenne肌营养不良症(DMD)的研究。用基因组探针检测携带者和产前诊断已经证明XJ1.1和pERT基因组探针,及侧翼探针如C7、754和99.6对于发现DMD携带者和产前诊断具有不可估量的价值。受累患儿血清CK水平是正常儿童的100～200倍,但用于检测携带者却不可靠,因为有1/3的肯定携带者的CK水平在正常范围。而应用RFLP进行的连锁分析具有很高的可信     

Duchenne型肌营养不良症的产前基因诊断的研究进展

Duchenne型肌营养不良症(DMD)是一种常见的X连锁隐性遗传病,本病目前无有效的治疗方法,产前诊断和选择性流产是防治本病的有效手段。DMD基因庞大,突变率高,本文在介绍DMD基因结构研究进展的基础上,综述了DMD位点部分缺失的筛选,及利用限制性片段长度多态(RFLP)分析,进行DMD产前基因诊断的研究进展。     

Duchenne型肌营养不良症的产前基因诊断研究

应用Ｄｕｃｈｅｎｎｅ型肌营养不良症（Ｄｕｃｈｅｎｎｅ　ｍｕｓｃｕｌａｒ　ｄｙｓｔｒｏｐｙ，ＤＭＤ）基因的ｃＤＮＡｓ作为探针，以限制性片段长度多态（ＲＦＬＰ）分析为策略，采用Ｓｏｕｔｈｅｒｎ分子杂交方法，成功地对１例可疑ＤＭＤ的男性胎儿及１例ＤＭＤ患儿进行基因诊断。结果显示该胎儿ＤＭＤ基因正常，而患儿存在ＤＭＤ基因缺失（缺失２．１５ｋｂ）。在基因分析前，应用聚合酶链式反应（ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ，ＰＣＲ）技术鉴定胎儿性别为男性。胎儿出生后检查结果与与产前基因诊断相吻合。为了获得高灵敏度探针，本文采用地高辛配基标记ＤＮＡ探针的方法，通过酶联免疫法，使分子杂交的ＤＮＡ检测带出现颜色反应。实验结果表明，此方法适用于基因组单拷贝ＤＮＡ顺序的检测，具有快速、安全等优越性，可以替代同位素进行推广、应用。     

外显子跳跃治疗Duchenne型肌营养不良症的研究进展

Duchenne型肌营养不良症是一种致死性肌肉疾病,抗肌萎缩蛋白基因缺陷是导致本病的原因,目前本病尚无特效的疗法.反义寡核苷酸(antisense oligonucleotides,AOs)诱导的外显子跳跃作为一种新的治疗手段具有良好的应用前景.本文主要从外显子跳跃治疗的原理、基础研究及临床研究进行综述.     

脂肪干细胞移植对Duchenne型肌营养不良症鼠神经肌肉再生单位的影响

背景：目前,Duchenne型肌营养不良症尚无有效治疗方法,之前的研究表明基因治疗和干细胞移植治疗是可能的“治愈”方法。实验拟将两者结合起来,在动物模型上观察其疗效,并验证之前提出的神经肌肉再生单位的假说。 目的：探讨脂肪干细胞移植治疗Duchenne型肌营养不良症的有效性和可行性,观察细胞移植对肌纤维、新生血管及神经末梢的影响。 方法：体外分离培养mdx鼠脂肪干细胞,经杆状病毒基因载体进行基因修饰,用于移植治疗Duchenne型肌营养不良症模型鼠。移植后检测实验动物的血清肌酸激酶水平、肌肉病理改变及肌肉内dystrophin表达;免疫荧光检测细胞移植后血管、肌肉和神经再生情况。 结果与结论：细胞移植后,能够重建模型鼠的dystrophin表达,一定程度上减轻并逆转肌肉的病理损害,进而降低血清激酸激酶水平;此外,细胞移植后能够形成干细胞来源的肌纤维、血管内皮细胞和神经末梢。这些证据表明,脂肪干细胞移植是有希望治疗Duchenne型肌营养不良症的方法之一。     

骨髓干细胞移植治疗不同年龄mdx鼠的疗效研究

目的：研究不同年龄的Duchenne型肌营养不良鼠（mdx鼠）与骨髓干细胞移植后缺失蛋白表达的关系。 方法： 获取4-5周C57BL/6小鼠的骨髓干细胞，体外培养3 d，静脉移植到7Gy γ射线预处理的6周龄、8周龄两组各6只mdx鼠。移植12周后，对移植鼠骨骼肌dystrophin蛋白表达情况进行检测。 结果： 6周龄、8周龄两组mdx鼠，静脉移植1.2×107骨髓干细胞，3个月后，分别有16%和7%的骨骼肌纤维表达了dystrophin蛋白。 结论： 静脉移植同种、同系鼠骨髓干细胞的mdx鼠，3个月之后, 不同年龄mdx鼠骨骼肌细胞dystrophin蛋白表达的阳性率不同，幼年鼠骨髓干细胞移植有较高比率的缺失蛋白表达。     

自体骨髓间充质干细胞移植治疗杜氏型肌营养不良血清酶学变化对照研究

目的 观察骨髓间充质干细胞(BMSC)移植治疗杜氏型肌营养不良(DMD)治疗前后血清酶学变化及临床效果.方法 选择2007年6月至2009年6月解放军第四六三医院515例DMD患者,其中男性502例,女性13例;年龄1～20岁,平均年龄133岁.阳性家族史121例.经解放军第四六三医院伦理委员会同意,并征求患者本人及家...     

Carrier woman of Duchenne muscular dystrophy mimicking inflammatory myositis

Carrier woman of Duchenne muscular dystrophy (DMD) can mimic the inflammatory myositis in presenting symptoms. Two diseases should be differentiated by the clinical history, muscle biopsy and genetic study. There are few reports in which both histochemical and genetic study showed the possible link of overlapping inflammatory pathophysiology with dystrophinopathy. We report a 40-yr-old woman who presented with subacute proximal muscle weakness and high serum level of creatine kinase. She had a history of Graves' disease and fluctuation of serum liver aminotransferase without definite cause. MRI, EMG and NCV were compatible with proximal muscle myopathy. Muscle biopsy on vastus lateralis showed suspicious perifascicular atrophy and infiltration of mono-macrophage lineage cells complicating the diagnosis. Dystrophin staining showed heterogeneous diverse findings from normal to interrupted mosaic pattern. Multiple ligation probe amplification and X chromosome inactivation test confirmed DMD gene deletion mutation in exon 44 and highly skewed X inactivation.     

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans,mice, and zebrafish

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.     

雌性小鼠骨髓移植给雄性小鼠后内源性骨髓细胞残存状态的研究

 目的:以雌性小鼠骨髓移植给雄性小鼠的方法,通过检测雄性小鼠血细胞的Y染色体来明确内源性骨髓细胞的残存状态。方法:将雌性或雄性C57BL/6小鼠作为受体,实验组用［137Cs］照射,6 h后每只经尾静脉注射供体小鼠骨髓细胞1×107。统计骨髓移植后14 d动物的存活率,并通过眶静脉采血观察外周血白细胞数量的变化,检测受体雄性小鼠体内Y染色体基因水平的变化以明确骨髓移植的效果。结果:分别用1 000、950和900 rad的照射剂量对受体小鼠进行照射后将供体小鼠的骨髓移植到受体小鼠体内,1 000和950 rad剂量时雌性受体小鼠可迅速恢复造血功能,而雄性受体小鼠则仅有48%的存活率。900 rad照射剂量骨髓移植后,雄性受体小鼠迅速恢复了造血功能,13 d后外周血白细胞计数基本恢复正常。移植后的雄性受体小鼠在5周内已检测不到外周血细胞Y染色体基因,表明雄性受体小鼠的骨髓被完全破坏,雌性供体小鼠的骨髓可完全替代受体雄性小鼠的骨髓并且发挥造血功能。结论: 在照射剂量900 rad照射后,雄性小鼠可以作为骨髓移植受体,为将来应用雄性小鼠作为骨髓移植受体动物开展有关心血管疾病的研究奠定了实验基础。     

经肾动脉自体骨髓干细胞移植治疗急性肾小管坏死的实验研究

目的：采用经肾动脉灌注移植兔自体骨髓干细胞，观察自体骨髓干细胞移植对缺血再灌注（I/R）后急性肾小管坏死肾功能的作用及其机制。方法:28只日本大耳白兔采集骨髓，分离自体骨髓干细胞，5-溴-2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine, BrdU)标记。全部实验兔建立肾脏I/R后随机分为移植组和对照组。移植组于肾血流恢复后经肾动脉灌注自体骨髓干细胞悬液，对照组同样方法注射等体积生理盐水。分别于I/R前和I/R后第1、3、5、7、14、21、28 d静脉采血，检测血清肌酐（SCr）和尿素氮（BUN），相同时点取肾组织行普通病理及BrdU免疫组化观察。结果:I/R后第1 d和第3 d 2组动物SCr、BUN均达到最高水平，以后逐渐下降；第7 d后移植组SCr及BUN水平逐渐低于对照组，到实验观察结束时的第28 d，对照组SCr和BUN分别为（135.6±32.5）μmol/L和（10.9±2.5）mmol/L，移植组SCr和BUN分别为（90.1±11.1）μmol/L和（8.0±1.5）mmol/L，2组间有显著差异（P<0.05）。普通病理观察，I/R后肾小管上皮细胞变性、坏死甚至脱落， BrdU免疫组化染色显示移植组于细胞移植后第5、7 d肾小管BrdU阳性染色并持续至实验结束，对照组阴性。结论:骨髓干细胞移植可以在一定程度上加速急性肾小管坏死后肾功能修复，自体骨髓干细胞移植是1种加速急性肾小管坏死后修复的安全而有效的方法。     

Allogeneic intra-bone marrow transplantation prevents rheumatoidarthritis in SKG/Jcl mice

The treatment of autoimmune diseases by allogeneic bone marrow transplantation remains a promising therapeutic avenue. However, more intensive studies on murine models are essential before application to a large number of human patients. In particular, the use of bone marrow transplantation to treat rheumatoid arthritis has been problematic. We have taken advantage of the SKG/Jcl mouse that develops a chronic T cell-mediated autoimmune disease that mimics rheumatoid arthritis which attempted to prevent the development of immunopathology in these mice by allogeneic bone marrow transplantation (BMT). In particular, we utilized our unique technology in which bone marrow cells (BMCs) of control C57BL/6J mice are directly injected into the bone marrow cavity in the tibia of SKG mice (intra-bone marrow [IBM]-BMT). As controls, SKG/Jcl mice were transplanted with whole BMCs from syngeneic SKG mice. Importantly, 12 months after IBM-BMT [B6 → SKG] demonstrated no evidence of arthritis, whereas the control [SKG → SKG] mice demonstrated, the expected immunopathology of a rheumatoid arthritis-like condition. Further, hematolymphoid cells in [B6 → SKG] mice were reconstituted by donor-derived cells and the percentages of Treg (Foxp3⁺/CD4⁺) cells, the percentages of receptor activator of nuclear factor-κB ligand (RANKL)⁺ cells on the CD4⁺T cells and the serum levels of tumor necrosis factor-α, interleukin-1 and interleukin-6 were normalized in the [B6 → SKG] mice. These data suggest that IBM-BMT is a viable method of immunological manipulation that suppresses the severe joint destruction and bone absorption in SKG/Jcl mice and lends further credence to the use of this methodology in humans with intractable rheumatoid arthritis.     

NKT cells,Treg, and their interactions in bone marrow transplantation

Bone marrow transplantation (BMT) is a potentially curative treatment for patients with leukemia and lymphoma. Tumor eradication is promoted by the anti‐tumor activity of donor T cells contained in the transplant; however, donor T cells also mediate the serious side effect of graft‐versus‐host disease (GVHD). Separation of GVHD from graft anti‐tumor activity is an important goal of research in improving transplant outcome. One approach is to take advantage of the immunomodulatory activity of regulatory NKT cells and CD4⁺CD25⁺ Treg of host and/or donor origin. Both host and donor NKT cells and donor Treg are able to prevent GVHD in murine models. In this review, we summarize the mechanisms of NKT cell‐ and Treg‐mediated protection against GVHD in mice while maintaining graft anti‐tumor activity. In addition, we also examine the interactions between NKT cells and Treg in the context of BMT, and integrate the data from murine experimental models with the observations made in humans.     

Immunohistological skin alterations in allogeneic bone marrow transplantation

Summary Skin biopsies of 26 patients with leukemia and seven patients with aplastic anemia were investigated before and at different stages after allogeneic bone marrow transplantation (BMT) to establish the immunological criteria which distinguish skin alterations during normal reconstitution from dermal lesions mediated by graft-versushost disease (GvHD). Of the 33 patients studied 27 presented with clinically diagnosed acute and/or chronic GvHD, one patient died of bone marrow rejection. Immunohistological analysis of the respective skin biopsies with selected monoclonal antibodies against human leukocyte antigens (HLA) and differentiation antigens of the lympho-hematopoietic cells revealed low dermal mononuclear cell counts with phenotypically normal constituents in five cases with uncomplicated reconstitution post-grafting. In contrast, increased dermal cellular infiltrates predominantly consisting of Lyt 3⁺, OKT 8⁺ T-lymphocytes, as well as of a large number of Ia-like (immune response associated = HLA-D) determinant⁺ monocytes/macrophages were observed in all patients with active acute/chronic GvH reactivity. As sign of activation simultaneous expression of HLA-D region products was also found on a subset of the invading OKT 8⁺ T-lymphocytes. Progression of GvHD was associated with additional surface staining of keratinocytes for Ia-like determinants. Loss of Ia-like determinant⁺, OKT 6⁺ dentritic epithelial cells in all leukemic patients, as well as in patients with aplastic anemia with or without GvHD suggested damage of Langerhans cells due to the previous radiotherapy and/or specific immunological destruction. In patients with fatal outcome of GvHD prolonged reduction of these dentritic epithelial cells seemed to be indicative of impaired immune reconstitution or bone marrow dysfunction. Thus immunopathological features of skin GvHR may enable early recognition and prognostic evaluation of this disease possibly allowing more effective therapy.Abbreviations GvHD graft-versus-host disease - GvHR graft-versus-host reactivity - SAA severe aplastic anemia - TBI total body irradiation - BMT bone marrow transplantation - HLA human leukocyte antigens - Ia-like antigens Immune-response-associated antigens - PBS phosphate buffered saline - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate Supported by DFG Sonderforschungsbereich 120, Projekt A 2 and B 1Dedicated to Prof. Dr. Dr. h.c. H.E. Bock on the occasion of his 80^th birthday