AxCA-脑源性神经营养因子转染大鼠损伤坐骨神经后基因表达的检测

目的观察携带小鼠脑源性神经营养因子（BDNF）cDNA表达片段的非依赖辅助复制缺陷型重组腺病毒载体（AxCA）-BDNF转染大鼠坐骨神经后基因表达情况。方法成年大鼠随机分成A组（坐骨神经缺损＋硅胶管＋AxCA-BDNF原液8μl）；B组（坐骨神经缺损＋硅胶管＋BDNF溶液8μl）；C组（坐骨神经缺损＋硅胶管＋空白病毒稀释液8μl）三组。应用原位杂交和免疫组织化学检测方法，从BDNF mRNA和蛋白水平，进行坐骨神经损伤后BDNF基因表达的定性和半定量分析测定。结果伤后腺病毒介导的BDNF基因转移组，在3、7、14d、1个月，4个时相点，近、远端神经干和脊髓（L3-6）中BDNF mRNA水平均远远高于其他两组，BDNF的水平也远远高于作为对照的单纯硅胶管套接组。结论通过腺病毒介导转染的BDNF基因在大鼠坐骨神经SCs内得到了有效表达，并通过轴突逆行转运到了相应的脊髓神经元。     

重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后基因表达的检测

目的观察携带小鼠BDNFcDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠坐骨神经后基因表达情况。方法成年大鼠随机分成A组（坐骨神经缺损加硅胶管加AxCA-BDNF原液8μl）；B组（坐骨神经缺损加硅胶管加BDNF溶液8μl）和C组（坐骨神经缺损加硅胶管加空白病毒稀释液8μl）等三组。应用原位杂交和免疫组化等手段，从BDNFmRNA和蛋白水平，进行坐骨神经损伤后BDNF基因表达的定性和半定量分析测定。结果伤后腺病毒介导的BDNF基因转移组，在3、7、14d和1个月4个时相点，近、远端神经干和脊神经（L3-6）中BDNFmRNA水平均远远高于其它两组，BDNF的水平也远远高于作为对照的单纯硅胶管套接组。结论通过腺病毒介导转染的BDNF基因在大鼠坐骨神经许旺细胞内得到了有效表达，并通过轴突逆行转运到了相应的脊髓神经元。     

神经干细胞移植对大鼠脊髓损伤后BDNF与GAP-43基因表达的影响

目的：研究海马源性神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后生长相关蛋白43(GAP-43)及脑源性神经营养因子(BDNF)基因表达的影响，探讨神经干细胞移植修复大鼠脊髓损伤的机制。方法：NSCs提取自新生胎鼠的海马区，经过培养及鉴定。实验分为3组：NSCs移植组、DMEM填充组、正常对照组。大鼠SCI后第7d移植NSCs，应用RT-PCR法观察NSCs移植后，大鼠脊髓损伤区GAP-43和BDNF基因表达的变化。结果：NSCs移植组较单纯损伤组明显增强了GAP-43mRNA与BDNFmRNA的表达。结论：NSCs移植后改变脊髓损伤区的微环境，上调BDNFmRNA，促进GAP-43mRNA的表达，是修复脊髓损伤的机制之一。     

骨髓基质干细胞移植对大鼠脊髓损伤后BDNF与GAP-43基因表达的影响

[目的]研究骨髓基质干细胞(mesenchymal stem cells,MSCs)移植对大鼠脊髓损伤(SCI)后生长相关蛋白43(GAP-43)及脑源性神经营养因子(BDNF)基因表达的影响,探讨骨髓基质干细胞移植修复大鼠脊髓损伤的机制.[方法]大鼠SCI后第7 d移植MSCs,应用RT-PCR法观察MSCs移植后,大鼠脊髓损伤区GAP-43和BDNF基因表达的变化.[结果]MSCs移植组较单纯损伤组明显增强了GAP-43 mRNA、BDNFmRNA的表达.[结论]MSCs移植后改变脊髓损伤区的微环境,上调BDNFmRNA,促进GAP-43 mRNA的表达,是修复脊髓损伤的机制之一.     

神经干细胞移植对大鼠脊髓损伤后BDNF与GAP-43基因表达的影响

目的:研究海马源性神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后生长相关蛋白43(GAP-43)及脑源性神经营养因子(BDNF)基因表达的影响,探讨神经干细胞移植修复大鼠脊髓损伤的机制.方法:NSCs提取自新生胎鼠的海马区,经过培养及鉴定.实验分为3组:NSCs移植组、DMEM填充组、正常对照组.大鼠SCI后第7d移植NSCs,应用RTPCR法观察NSCs移植后,大鼠脊髓损伤区GAP-43和BDNF基因表达的变化.结果:NSCs移植组较单纯损伤组明显增强了GAP-43mRNA与BDNFmRNA的表达.结论:NSCs移植后改变脊髓损伤区的微环境,上调BDNFmRNA,促进GAP-43mRNA的表达,是修复脊髓损伤的机制之一.     

腺病毒介导的脑源性神经营养因子基因治疗大鼠神经根损伤

目的：观察腺病毒介导的脑源性神经营养因子(BDNF)基因脊髓内转移对神经根损伤后的治疗效果。方法：制作大鼠神经根切断吻合模型，分为两组：治疗组通过显微注射法在立体定位仪上将2μl重组腺病毒BDNF载体(AxCA—BDNF)直接注入大鼠神经根损伤部相应的脊髓腹角；损伤组注射病毒缓冲液。术后观察霍乱毒素-辣根过氧化物酶(CT-HRP)逆行标记的神经元数目及坐骨神经功能指数(SFI)的改变。结果：与损伤组比较，治疗组神经根伤后CT—HRP标记神经元的数目明显增加，坐骨神经功能指数(SFI)的恢复率明显升高。结论：AxCA—BDNF基因治疗对大鼠脊髓水平的神经根修复是一种有效的方法。     

脑源性神经生长因子转基因细胞移植和大剂量甲基强的松龙对脊髓损伤后细胞凋亡的影响

目的：探讨大鼠脊髓损伤后腺病毒介导的脑源性神经生长因子（AxCA-BDNF）体外转基因成肌细胞移植和静脉内注射大剂量甲基强的松龙（MP）对大鼠脊髓损伤后细胞凋亡的影响。方法：120只Wistar大鼠分为：脊髓挫伤组（A组），脊髓挫伤后AxCA-BDNF基因转染的成肌细胞移植组（B组），脊髓挫伤后静脉内注射大剂量MP治疗组（C组），脊髓挫伤后同时应用AxCA-BDNF和MP组（D组）。手术后1、3、7、14、28d用行为学和电生理检查观察大鼠功能恢复情况，并用计算机图像分析技术对脊髓损伤区细胞凋亡（TUNEL法）和Bcl-2蛋白表达（免疫组化法）进行定量分析。结果：四组中均发现凋亡细胞及Bcl-2蛋白阳性表达细胞．图像分析发现四组凋亡细胞核数为A组〉B组〉C组〉D组；Bcl-2免疫反应阳性细胞表达顺序为D组〉C组〉B组〉A组。大鼠后肢功能恢复和电生理检查也有类似的变化趋势。结论：体外转基因成肌细胞移植和大剂量MP都能抑制大鼠脊髓损伤后的细胞凋亡，促进大鼠后肢功能恢复，两者联合应用具有协同作用。     

体外转基因成肌细胞移植对大鼠损伤脊髓细胞凋亡的影响

目的：探讨大鼠脊髓损伤后胚胎脊髓和腺病毒介导的脑源性神经生长因子（AxCA-BDNF)体外转基因成肌细胞移植对大鼠脊髓细胞凋亡的影响。方法：将动物分为：大鼠脊髓半切洞损伤明胶海绵填充组（A组）,大鼠脊髓半切洞损伤应用胚胎脊髓移植组（B组）,脊髓半切洞损伤损伤AxCA-BDNF基因转染的成肌细胞移植组（C组）大鼠脊髓半切洞损伤后应用胚胎脊髓和AxCA-BDNF基因转染的成肌细胞移植组（D组）。手术后1、3、7、14、28d应用行为学和电生理检查观察大鼠功能恢复情况,对脊髓损伤区进行细胞凋亡的检测（TUNEL）以及Bcl-2蛋白表达的测定（免疫组化法）。采用计算机图像分析技术,进行定量分析。结果：A、B、C、D四组中均发现凋亡细胞及Bcl-2蛋白阳性表达细胞,图像分析发现,各组凋亡细胞核为A＞B＞C＞D;Bcl-2免疫反应阳性细胞表达顺序为D＞C＞B＞A,Bcl-2免疫反应阳性细胞的表达与大鼠后肢功能恢复有同样的变化趋势。结论：大鼠胚胎脊髓和体外转基因成肌细胞移植能抑制脊髓损伤后的细胞凋亡。     

硫酸软骨素酶ABC对大鼠脊髓损伤后轴突髓鞘化和胶质瘢痕的影响

目的探讨硫酸软骨素酶ABC(chondroitinase ABC,ChABC)对大鼠脊髓损伤后轴突再生、髓鞘化和胶质瘢痕形成的影响。方法将72只雄性SD大鼠随机分为ChABC治疗组(A组)、生理盐水治疗组(B组)和假手术组(C组),每组24只。A、B组采用改良Allen撞击法制作大鼠T9中度脊髓损伤模型,分别在伤后1 h和之后连续7 d每天1次经蛛网膜下腔注射6μL浓度为1 U/mL ChABC和生理盐水;C组仅打开椎管,不损伤脊髓。术后1、7、14、28 d,采用BBB评分评定大鼠后肢运动功能恢复情况;取出损伤段脊髓组织,行HE染色和Nissl染色,并采用免疫组织化学染色检测髓鞘碱性蛋白(myelin basic protein,MBP)、生长相关蛋白43(growth associated protein 43,GAP-43)和胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的变化情况。结果术后各时间点C组BBB评分均明显高于A、B组(P<0.05);术后随时间延长A、B组BBB评分逐渐增加,14、28 d时A组BBB评分明显优于B组(P<0.05)。HE和Nissl染色显示术后各时间点A组脊髓组织形态和神经元数量均优于B组。术后各时间点A、B组MBP和GAP-43的积分吸光度(IA)值及GFAP染色阳性面积均高于C组(P<0.05),术后7、14、28 d时A组MBP和GAP-43的IA值明显高于B组(P<0.05),GFAP染色阳性面积明显小于B组(P<0.05)。结论 ChABC能有效改善大鼠脊髓损伤区域微环境,提高MBP和GAP-43表达并抑制GFAP表达,促进轴突再生与髓鞘化,抑制胶质瘢痕形成,促进神经功能恢复。     

功能训练促进颈脊髓损伤大鼠功能恢复的机制研究

目的：探讨大鼠颈脊髓不完全性损伤后前肢功能训练促进大鼠前肢功能恢复的机制.方法：在立体定位仪的引导下,致伤大鼠双侧红核和皮质脊髓背侧束后,对大鼠行前肢功能训练6周.免疫组化检测损伤脊髓节段脑源性神经生长因子（brain-derived neurotrophic factor,BDNF）的表达,皮质脊髓束投射神经元（corticospinal neurons,CSNs）中生长相关蛋白43（growth-associated protein 43,CAP43）和神经营养素共同受体P75（P75NTR）的表达,荧光金逆行示踪CSNs存活情况.结果：大鼠不完全性颈脊髓损伤后,前肢功能训练可上调脊髓前角神经元BDNF与CSNs中GAP43和P75NTR的表达,减少CSNs死亡.结论：大鼠颈脊髓不完全性损伤后,前肢功能训练通过上调脊髓前角神经元BDNF与CSNs中GAP43和P75NTR的表达以及减少CSNs的死亡等机制增加未损伤皮质脊髓腹侧束（vCST）的出芽,进而促进大鼠前肢功能恢复.     

一个与小鼠精子发生相关的基因—rjs 基因

应用改良的mRNA差异显示技术,对不同发育期小鼠（分别为1周龄至4周龄）睾丸曲细精管进行了差异表达的研究,并对其中一个在3周龄小鼠中具有高表达的差异表达片段进行了克隆和测序分析,其序列与Genebank中rjs基因具有高度同源性,表明rjs基因可能与小鼠精子发生相关。     

一个与小鼠精子发生相关的基因——ris基因

应用改良的mRNA差异显示技术,对不同发育期小鼠(分别为1周龄至4周龄)睾丸曲细精管进行了差异表达的研究,并对其中一个在3周龄小鼠中具有高表达的差异表达片段进行了克隆和测序分析,其序列与Genebank中ris基因具有高度同源性,表明ris基因可能与小鼠精子发生相关。     

基因敲除技术在精子发生相关基因功能研究中的应用

从基因水平研究男性不育症发病机理将有助于发现男性不育治疗的新途径。基因敲除技术是目前研究基因功能的主流方法。筛选鉴定精子发生过程中相关基因,探索其特性和功能,对了解睾丸功能、探索男科疾病新的治疗靶点具有重要意义。本文概述基因敲除技术在精子发生相关基因功能研究中的应用。     

基因芯片在膀胱癌基因研究中的应用

膀胱癌是泌尿系统最常见的恶性肿瘤,以空间上的多中心与时间上的反复发作为其生物学特点,其发生由多个基因控制、多步骤进行.基因芯片技术是一种高通量的基因分析平台,现已广泛用于疾病机制的研究、疾病的分类和诊断、疾病的预测和治疗,并用于膀胱癌发生、发展相关基因的筛选、分子治疗靶点的筛选、寻找膀胱癌亚型的分子标记以及肿瘤预后分类的研究.     

Nonviral gene transfer for cancer gene therapy

Although the pre‐clinical and clinical results of gene therapy have shown promise for some cancers, cancer gene therapy is still at an early stage of clinical development. Due to the complexity of targeted vector delivery to the tumor, our strategy for gene therapy is focussed on the development of local non‐viral gene transfer to treat tumors. The local application of non‐viral gene therapy is of particular value in the context of pre‐ or intraoperative application of therapeutic genes. This ensures accessibility of targeted tumor areas and will contribute to better local control of the disease. In this regard, applicable transfer technologies are needed in gene therapy. Different physical procedures, such as in vivo electroporation, sonoporation, ballistic transfer etc. are employed to deliver naked DNA into the target cells or tissues in vitro and in vivo. Among the various non‐viral gene delivery technologies jet‐injection is gaining increasing acceptance, since this technique allows gene transfer into different tissues with deep penetration of naked DNA. The jet‐injection technology is based on low‐volume jets of high‐velocity to penetrate skin and deeper tissues associated with efficient transfection of the affected area. For non‐viral in vivo gene transfer a jet‐injector prototype was created and tested. The beta‐galactosidase (LacZ), green fluorescence protein reporter gene constructs were successfully jet‐injected into different syngeneic mouse and patient‐derived xenotransplanted human tumor models of colon‐ or mammary carcinoma and malignant melanoma. Qualitative and quantitative expression analysis of jet‐injected tumor tissues revealed the efficient expression of these genes. Therapeutic in vivo experiments using the jet‐injection transfer of the cytosine deaminase suicide gene in tumors demonstrated antitumor effects with significant growth inhibition of the jet‐injected xenotransplanted colon carcinomas. Furthermore, jet‐injection was also successfully used for the application of a heat‐inducible TNF‐α expressing vector system leading to efficient in vivo tumor growth inhibition in the combined non‐viral TNF‐α gene transfer and hyperthermia approach. Based on our pre‐clinical experiments for non‐viral gene transfer, a phase I clinical trial has been conducted at the Clinic for Surgery and Surgical Oncology, Charité, Berlin to evaluate the feasibility, efficiency, and safety of jet‐injection aided LacZ‐reporter gene transfer in patients with cutaneous metastases from breast cancer and malignant melanoma. In this study naked GMP‐plasmid DNA was applied intratumorally by jet‐injection. The jet‐injection was well tolerated by all patients and no side effects have been experienced. The study clearly demonstrated that the single application of plasmid‐DNA is safe and leads to the expression of the LacZ‐reporter gene in the tumor tissue, as shown at mRNA‐ and at protein level.     

Evaluation of gene promoters for liver expression by hydrodynamic gene transfer

OBJECTIVE: Gene therapy represents a promising treatment for hepatic disease. Most approaches today use viral methods to target tissues. While nonviral gene therapy is less prominent, hydrodynamic gene delivery represents a promising approach to direct gene expression to the liver. The purpose of the present study was to evaluate promoters for efficient gene expression in hepatocytes in vivo by hydrodynamic delivery and to test the findings in a model of hemophilia A. MATERIALS AND METHODS: Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor-1 alpha (EF1alpha), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2 x 10(5) HEK 293 cells was followed by in vivo whole animal bioluminescence and luminometry after hydrodynamic tail vein injection of plasmid DNA. Six-month-old FVB factor VIII (FVIII)-deficient mice were similarly injected with CBA- or EF1alpha-promoted constructs containing the FVIII heavy and light chains and expression was examined. RESULTS: In vitro transfection demonstrated a hierarchy of expression: hCMV-intron>CAG>EF1alpha>hCMV>PGK. In vivo luminometry demonstrated that the CAG construct produced 2.6x, 3.0x, 3.4x, and >1000x the expression of the hCMV-intron, EF1alpha, hCMV, and PGK constructs respectively. FVIII plasmid injected hemophilic mice demonstrated higher levels of FVIII expression with CAG versus EF1alpha, confirming the reporter gene studies. All FVIII-deficient mice injected with EF1alpha-FVIII or CAG-FVIII plasmids survived after tail clipping. CONCLUSIONS: The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver.     

Multispecies comparison of the casein gene loci and evolution of casein gene family

Caseins, the major milk proteins, are present in a genomic cluster spanning 250–350 kb. The divergence at the coding level between human, rodent, and cattle sequences is rather extensive for most of the genes in this region. Nevertheless, comparative analysis of genomic sequences harboring the casein gene cluster region of these species (with equal evolutionary distances 79–88 Myr) shows that the organization and orientation of the genes is highly conserved. The conserved gene structure indicates that the molecular diversity of the casein genes is achieved through variable use of exons in different species and high evolutionary divergence. Comparative analysis also revealed the presence within two species of uncharacterized casein family members and ruled out the previously held notion that another gene family, located in this region, is primate-specific. Several other new genes as well as conserved noncoding sequences with potential regulatory functions were identified. All genes identified in this region are, or are predicted to be, secreted proteins involved in mineral homeostasis, nutrition, and/or host defense, and are mostly expressed in the mammary and/or salivary glands. These observations suggest a possible common ancestry for the genes in this region.