脊髓损伤的最新治疗进展

脊髓损伤以后引起原发性损伤和继发性损伤导致损伤的神经组织难以修复。目前脊髓损伤的重点主要集中在减轻和延缓继发性损伤造成的伤害。本文总结了近年来在脊髓损伤治疗领域的进展包括传统的药物治疗,细胞移植和基因治疗。目前动物实验研究表明细胞移植和基因治疗在治疗脊髓损伤的中取得了可喜的成果,将在未来临床应用中发挥重要作用。     

ADA-LacZ融合基因直接注射导入小鼠皮肤组织后的表达

将ADA－LacZ融合基因经直接注射导入小鼠皮肤组织,用注射点处的皮肤组织制作冰冻切片,经组织化学方法显示：此融合基因可在皮肤成纤维细胞中表达一种具有腺苷脱氨酶（ADA）和β-半乳糖昔酶（β-gal）双重酶活性的融合蛋白,这种直接将融合基因导入体内进行表达的方式．为在基因治疗和基因疫苗研究中探索一条新的简便可行的外源基因在体内表达的途径,提供了有益的实验依据。     

骨髓间充质干细胞移植治疗大鼠脊髓损伤的神经分化研究

目的探讨骨髓间充质干细胞(BMSC)对移植脊髓损伤(SCI)模型神经再生修复的作用及其机制。方法 (1)分离原代SD大鼠BMSC;(2)体外诱导BMSC向神经分化,应用免疫荧光技术检测神经诱导分化后的BMSC神经标志Nestin、Neu N的表达;(3)运用改良的Allen撞击装置制备SD大鼠SCI模型,成年雌性SD大鼠12只,随机分组:损伤对照(n=6),BMSC细胞移植组(n=6),并选择在SCI后半小时内在蛛网膜下腔原位移植1×106 BMSC细胞注射治疗,对照组原位注射10μl PBS作为对照。每周对SCI大鼠进行BBB运动功能行为学评价。(4)在治疗后1个月处死SCI大鼠取脊髓样本进行冰冻切片检测Nestin、NeuN神经标记物表达情况,从而评判BMSC对SCI的治疗效果。计量资料结果服从正态分布、方差齐性时,采用t检验;若不服正态分布,采用KruskalWallis H秩和检验。结果 (1)分离的BMSC纯度高、生物学特征稳定。(2)BMSC在体外神经诱导环境下可分化为神经细胞,对比正常对照组,神经诱导组Nestin与Neu N的表达具有统计学差异(t=11.49、6.76,P0.05)。(3)BMSC移植治疗SCI大鼠运动行为学功能显著改善,移植组比对照组治疗5周后的BBB评分具有统计学意义(t=5.59,P0.05);损伤导致组织形态学出现脊髓白质灰质结构性损毁,神经细胞大量丢失,而BMSC移植组Nestin、GFAP与Neu N表达细胞均较损伤组差异有统计学意义(t=4.74、6.59、15.46,均P0.05)。结论 BMSC移植可促进SCI后神经细胞的存活与再生分化,在一定程度上促进SCI脊髓组织功能的恢复。     

采用质粒DNA研究人结缔组织生长因子（hCTGF）对骨损伤修复作用的研究

摘要：目的 研究人结缔组织生长因子（hCTGF）对细胞的增殖作用及其骨损伤促愈合的生物学作用。方法 利用真核基因导入系统,转染具有分化潜能的成纤维细胞,并导入骨折实验动物模型,利用分子生物学和临床骨科技术检测分析,获得数据。结果 明确了hCTGF对细胞的增殖作用,表明了hCTGF以基因治疗为手段具有修复骨损伤的生物学活性,为该因子在临床上治疗骨损伤提供新的活性因子和新的治疗方法。     

转基因DT40细胞导入早期鸡胚后的分布及绿色荧光蛋白表达的研究

目的为了研究经过基因修饰的体细胞导入到禽类胚胎以后,供体细胞及外源基因是否能在受体胚胎中成活并且外源基因是否可以长期表达。方法筛选得到稳定整合绿色荧光蛋白基因的鸡DT40细胞作为外源蛋白的运载工具,通过血管微注射的方法将其导入到于38.5℃温度条件下孵化65～70 h的鸡胚中,并将操作后的鸡胚在原孵化条件下继续孵化。在孵化的不同时期取移植了DT40细胞的嵌合体胚胎在荧光显微镜下观察荧光细胞的存活与分布情况。并通过PCR以及免疫组织化学方法检测供体细胞在受体中的位置以及绿色荧光蛋白的表达情况。结果荧光标记的DT40细胞可以存活于受体不同的组织器官中,包括：脑、心脏、肝脏等。导入胚胎的整合外源基因的DT40细胞可以存活到胚胎出雏之前,并且外源基因能够正常表达。结论可以通过此方法将外源基因导入到受体中,并使目的蛋白在受体胚胎中持续表达,为胚胎期导入外源蛋白诱导免疫耐受的研究以及将转基因细胞移植到动物体内生产目的蛋白的研究提供科学依据和技术平台。     

腺病毒载体的细胞导向性基因转移

腺病毒（ａｄｅｎｏｖｉｒｕｓ,Ａｄ）载体广泛应用于基因治疗,其主要特征之一是能高效地进行各种组织如肺上皮、肝、肌肉、胰岛、胆囊等的体内（ｉｎｖｉｖｏ）基因转移。然而,因其混杂的向性而引起的全身体内基因转导后治疗基因不受组织限制的表达,限制了它在基因治疗中的应用,因此希望改变腺病毒的天然向性,而使治疗基因导向转移到选择的细胞类型中。目前腺病毒载体细胞导向性的基因转移可通过（１）修饰腺病毒载体的细胞表面识别成分,限制腺病毒导向性感染选择的细胞类型;（２）腺病毒载体中导入组织特异性启动子,控制治疗基因…     

动物病毒载体与基因治疗的现状和前景(续)

动物病毒载体与基因治疗的现状和前景（续）侯云德三、甚因转移方式１。外源基因体外转移,返回体内。实际上,目前在基因治疗中常用的方法是将患者体内的淋巴细胞或肿瘤浸润淋巴细胞取出在体外培养增殖,同时将某种外源基因如ＩＬ－２、肿瘤坏死因子（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ,ＴＮＦ）等基因导入该细胞,然后再将此经基因改造的细胞重新返回人体,以达到基因治疗的目的。     

中国的遗传病基因治疗研究

随着分子生物学和基因工程技术的发展,基因转移的手段已逐趋完善。应用反转录病毒介导的基因转移已成为遗传病基因治疗的主要手段。我们以血友病B为研究对象,将人凝血因子ⅨcDNA构建在反转录病毒上,转染到各种细胞中,其中包括血友病B患者的成纤维细胞中,获得了高效的转移和表达。将这些细胞包埋在胶原中,然后腹腔移植或注射到家兔体内,获 得了高效的转移和表达。将这些细胞包埋在胶原中,然后腹腔移植或注射到家兔体内,获得了持久而稳定的表达,持续时间已超过20个月。     

大鼠实验性帕金森病的基因治疗

将稳定转染了大鼠酪氨酶羟化酶基因的大鼠成肌细胞移植于帕金森病大鼠模型模型的纹状体,进行基因治疗研究,ＲＴ－ＰＣＲ和免疫组织化学检测都证明转基因细胞可在纹状体内存活并表达ＴＨ,动物的不对称旋转行为明显改善,而且疗效可维持半年以上。     

阳离子脂质体与基因转移

阳离子脂质体是表达带正电的脂南体。它可与带负电的基因（ＤＮＡ或ＲＮＡ）形成带正电的脂－基因复合物,此复合物借静电作用吸附于细胞表面,通过与细胞膜融合或细胞内吞而将结合的基因导入细胞,并获得表达。阳离子脂质体转基因的操作简便、重复性好,杂效率高,在基因工程、物种培育和基因治疗研究等方面有广阔的应用前景。     

Human somatic cell gene therapy

The prelude to successful human somatic gene therapy, i.e. the efficient transfer and expression of a variety of human genes into target cells, has already been accomplished in several systems. Safe methods have been devised to do this using non-viral and viral vectors. Potentially therapeutic genes have been transferred into many accessible cell types, including hematopoietic cells, hepatocytes and cancer cells, in several different approaches to ex vivo gene therapy. Successful in vivo gene therapy requires improvements in tissuetargeting and new vector design, which are already being sought. Gene-transfer protocols have been approved for human use in inherited diseases, cancer and acquired disorders. Althouth the results of these trials to date have been somewhat disappointing, human somatic cell gene therapy promises to be an effective addition to the arsenal of approaches to the therapy of many human diseases in the 21st century if not sooner.     

Assessment of Glial Scar,Tissue Sparing,Behavioral Recovery and Axonal Regeneration following Acute Transplantation of Genetically Modified Human Umbilical Cord Blood Cells in a Rat Model of Spinal Cord Contusion

Objective and Methods

This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo.

Results

Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord.

Conclusion

Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.     

帕金森病裸DNA法基因治疗的实验研究

采用体内裸DNA基因治疗帕金森病(PD)取得显著疗效.将酪氨酸羟比酶(TH)基冈表达质粒与Lipofectin形成的复合物立体定位注射于PD模型鼠纹状体,显著改善了PD鼠的不对付旋转行为.免疫组化证实神经细胞表达了外源TH.     

Effective Gene Transfer into Regenerating Sciatic Nerves by Adenoviral Vectors: Potentials for Gene Therapy of Peripheral Nerve Injury

Replication defective adenoviral vectors have been demonstrated as an effective method for delivering genes into a variety of cell types and tissues both in vivo and in vitro. Transfecting genes into neuronal cells has proven to be difficult because of their lack of cell division. Since the major problem in neurological disease is the degeneration of the terminally differentiated neuronal cells, the adenoviral vectors ability to transfer genes into differentiated post-mitotic cells makes them advantageous for a gene delivery system for the nervous system. Here we showed that a replication defective recombinant adenovirus carrying the lacZ gene could infect the neuronal stem cells and even the differentiated neuronal cells derived from the central nervous system. The lacZ gene delivered into the neuronal cells was expressed efficiently. In addition, the recombinant virus also infected Schwann cells in intact and injured nerves in vivo. The expression of the lacZ gene lasted for 5 weeks, within which nerve regeneration is accomplished in the rat. Adenoviral vectors might thus be used to modulate Schwann cell gene expression for treating peripheral nerve injury or peripheral neuropathy.     

Identification of gene coexpression modules,hub genes,and pathways related to spinal cord injury using integrated bioinformatics methods

Spinal cord injury (SCI) is characterized by dramatic neurons loss and axonal regeneration suppression. The underlying mechanism associated with SCI-induced immune suppression is still unclear. Weighted gene coexpression network analysis (WGCNA) is now widely applied for the identification of the coexpressed modules, hub genes, and pathways associated with clinic traits of diseases. We performed this study to identify hub genes associated with SCI development. Gene Expression Omnibus (GEO) data sets GSE45006 and GSE20907 were downloaded and the significant correlativity and connectivity between them were detected using WGCNA. Three significant consensus modules, including 567 eigengenes, were identified from the master GSE45006 data following the preconditions of approximate scale-free topology for WGCNA. Further bioinformatics analysis showed these eigengenes were involved in inflammatory and immune responses in SCI. Three hub genes Rac2, Itgb2, and Tyrobp and one pathway “natural killer cell-mediated cytotoxicity” were identified following short time-series expression miner, protein-protein interaction network, and functional enrichment analysis. Gradually upregulated expression patterns of Rac2, Itgb2, and Tyrobp genes at 0, 3, 7, and 14 days after SCI were confirmed based on GSE45006 and GSE20907 data set. Finally, we found that Rac2, Itgb2, and Tyrobp genes might take crucial roles in SCI development through the “natural killer cell–mediated cytotoxicity” pathway.     

Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates

Background

The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.

Methods

Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34⁺ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells.

Results

Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes.

Conclusions

Low levels of GFP‐transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Gene therapy of severe combined immunodeficiencies

Recent advances in gene transfer in human hematopoietic cells, combined with a better understanding of the genetic aspects of several immunodeficiencies, has offered new opportunities in the domain of gene therapy. Severe combined immunodeficiency (SCID) appear to represent a good model for the application of gene therapy, combining an expected selective advantage for transduced cells, an absence of immunological response to the vector and/or the therapeutic transgene, together with accessibility to hematopoietic stem cells (HSC). Ex vivo retroviral transduction of a therapeutic transgene in HSC prior to transplantation appears to be a particularly effective and long‐lasting means of restoring the expression of a mutated gene in the lymphoid lineage. Furthermore, encouraging therapeutic benefits as a result of a gene therapy protocol for the treatment of X‐linked severe combined immunodeficiencies (SCID‐X1) invites many questions as to the reasons for this therapeutic benefit. This review outlines the results that have been achieved in gene therapy for SCID‐X1, ADA‐SCID as well as other types of SCID, and discusses the possible relationship between the physiopathology of each disease and the success of relevant trials. Copyright © 2001 John Wiley & Sons, Ltd.     

Polymer based cardiovascular gene therapy

Therapeutic angiogenesis is a new potential treatment in cardiovascular disease. It is performed by the delivery of the angiogenic agents (protein, gene). Most important consideration for gene therapy is the construction of an effective therapeutic gene. Currently, VEGF is the most effective therapeutic gene for the neovascularization. We constructed the hypoxia-regulated VEGF plasmid using the Epo enhancer and RTP801 promoter. The efficiency of the pEpo-SV-VEGF and pRTP801-VEGF were evaluated by various methodsin vitro andin vivo. The results suggested that the hypoxia-inducible VEGF gene therapy system is effective and safe, which may be useful for the gene therapy of ischemic heart disease. Development of a safe and efficient gene carrier is another main requirement for successful gene therapy. Although viralbased gene delivery is currently the most effective way to transfer genes to cells, nonviral vectors are increasingly being considered forin vivo gene delivery. The advantages of nonviral gene therapy are lack of specific immunogenecity, simplicity of use, and ease of large-scale production. In addition, the simple conjugation of a targeting moiety to nonviral gene carrier can facilitate tissue-targeting gene delivery. We have developed two new gene carrier systems, TerplexDNA and WSLP (water soluble lipopolymer). These two are efficient carrier to ischemic myocardium and has low toxicity and high transfection efficiency. So it may allow for application ofin vivo gene therapy in the treatment of heart disease.     

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin‐containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross‐talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus‐mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication‐Competent Avian sarcoma‐leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back‐transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)⁺Neo^r+‐electroporated melanocytes from Hypeco nera were selectively grown in G418‐supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS‐mOrange, mOrange⁺ keratinocytes and transplanted EGFP⁺ melanocytes were frequently juxtaposed each other. High‐resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.     

17β‐Estradiol Protects Human Eyelid‐Derived Adipose Stem Cells against Cytotoxicity and Increases Transplanted Cell Survival in Spinal Cord injury

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b‐estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose‐derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)‐induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂‐induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.