艾滋病的基因治疗策略及慢病毒载体

高活性的抗病毒治疗可以显著地降低艾滋病患者血浆中的HIV病毒载量,但对潜伏的病毒库无效。对HIV的基因治疗包括诱导HIV潜伏感染的休止的CD4 T记忆细胞增生,使潜伏的HIV激活进入复制循环,结合药物治疗和激活潜伏的HIV基因表达但并不诱导细胞增生,而是通过载体携带的基因使细胞凋亡,以清除HIV潜伏感染的细胞,利用载体携带目的基因治疗脑中的病毒。     

Detection of 95 novel mutations in coagulation factor VIII gene F8 responsible for hemophilia A: results from a single institution

Hemophilia A (HA) is an X‐linked hereditary bleeding disorder defined by a qualitative and/or quantitative factor VIII (FVIII) deficiency. The molecular diagnosis of HA is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. The putative role of the novel mutations, especially missense mutations, may be difficult to interpret as causing HA. We identified 95 novel mutations out of 180 different mutations responsible for HA in 515 patients from 406 unrelated families followed up at a single hemophilia treatment center of the Bicêtre university hospital (Assistance Publique‐Hôpitaux de Paris [AP‐HP], Le Kremlin‐Bicêtre). These 95 novel mutations comprised 55 missense mutations, 12 nonsense mutations, 11 splice site mutations, and 17 small insertions/deletions. We therefore developed a mutation analysis based on a body of proof that combines the familial segregation of the mutation, the resulting biological and clinical HA phenotype, and the molecular consequences of the amino acid (AA) substitution. For the latter, we studied the putative biochemical modifications: its conservation status with cross‐species FVIII and homologous proteins, its putative location in known FVIII functional regions, and its spatial position in the available FVIII 3D structures. The usefulness of such a strategy in interpreting the causality of novel F8 mutations is emphasized. Hum Mutat 27(7), 676–685, 2006. © 2006 Wiley‐Liss, Inc.     

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO₂ level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.     

Hsa-mir-196b慢病毒表达载体的构建及鉴定

目的构建Hsa-mir-196b慢病毒表达载体并对其进行鉴定。方法以正常人外周血DNA为模板,PCR扩增得到目的基因。通过HpaⅠ/XhoⅠ双酶切及其后的连接将其插入Lentilox3.7（pLL-3.7）质粒中。PCR筛选阳性克隆,测序鉴定。用pLL-3.7-mir-196b、pCMV-VSV-G和pCMV-dR8.91三质粒系统共感染HEK-293FT细胞,包装生产慢病毒。将所得病毒悬液梯度稀释后感染HEK293FT细胞,以检测病毒滴度,并用实时定量PCR检测慢病毒感染后Hsa-mir-196b的表达变化。结果 PCR及测序结果证明成功构建了pLL-3.7-mir-196b重组质粒。所得慢病毒上清滴度为（7.2±1.1）×107TU/ml。感染慢病毒的239FT细胞中,Hsa-mir-196b的表达量相对于未感染细胞提高了约24倍。结论成功构建Hsa-mir-196b慢病毒表达载体。     

凝血因子Ⅷ制剂及其临床应用

本文主要介绍了凝血因子Ⅷ制剂冷沉淀和浓缩物的制备方法和性质特点,临床应用适应症、输注剂量、使用方法、注意事项和不良反应及预防,仅供同道参考。     

慢病毒介导的红色荧光蛋白基因在大鼠坐骨神经细胞的表达

目的 研究慢病毒载体介导的红色荧光蛋白(RFP)基因在坐骨神经雪旺细胞体内转染情况.方法 将60只Wistar大鼠随机分成A组、B组、C组和D组四个组,每组15只.四组均行右坐骨神经损伤模型,术毕向B组、C组和D组坐骨神经内注入滴度分别为2×10-6 TU、2×10-7 TU、2×10-8 TU的Lenti-RFP悬液8μl,向A组注入8μl平衡盐液(BSS)作为对照组.术后第1、2、4周观察红色荧光蛋白的表达情况,计算转染效率;并进行神经组织学检查.结果 术后第1周B、C、D组均出现RFP表达,术后第4周D组的转染效率最高.同一时间不同滴度组以及同一滴度组不同时间比较差异有统计学意义(P＜0.05).组织学检查病毒载体对神经未见明显炎性反应及组织学损伤.结论 慢病毒载体能安全有效的在体内转染雪旺细胞,并有浓度依赖性和时间依赖性,对组织无明显毒性作用.     

造血干细胞作为基因治疗靶细胞的研究进展

基因治疗是指将人的正常基因或有治疗作用的基因通过一定方式导入人体靶细胞,以纠正基因的缺陷或发挥治疗作用,从而达到治疗疾病的目的。造血干细胞具有自我更新能力和可移植性,可在体内长期存活,可使治疗基因在患者体内长期稳定地表达,因而具有广泛的应用前景。来源于HIV的慢性病毒载体因为可有效转染处于静止状态的造血干细胞而成为研究热点。造血干细胞的基因治疗技术逐渐成熟且已应用于一些疾病的临床治疗。     

The identification and classification of 41 novel mutations in the factor VIII gene (F8C)

Hemophilia A is a bleeding disorder caused by a quantitative or qualitative deficiency in the coagulation factor VIII. Causative mutations are heterogeneous in nature and are distributed throughout the FVIII gene. With the exception of mutations that result in prematurely truncated protein, it has proved difficult to correlate mutation type/amino acid substitution with severity of disease. We have identified 81 mutations in 96 unrelated patients, all of whom have typed negative for the common IVS-22 inversion mutation. Forty-one of these mutations are not recorded on F8C gene mutation databases. We have analyzed these 41 mutations with regard to location, whether or not each is a cross-species conserved region, and type of substitution and correlated this information with the clinical severity of the disease. Our findings support the view that the phenotypic result of a mutation in the FVIII gene correlates more with the position of the amino acid change within the 3D structure of the protein than with the actual nature of the alteration.     

鼠内皮抑素基因腺病毒载体的构建及对血管内皮细胞的生长抑制

构建含鼠内皮抑素基因的腺病毒载体（pAd／mEnd），并观察内皮抑素蛋白对人脐静脉血管内皮细胞（HUVEC）的生长抑制、细胞周期和凋亡的影响。结果发现pAd／mEnd感染BEL7402细胞后，能有效表达内皮抑素蛋白，显著抑制HUVEC增殖，阻滞HUVEC细胞S期，凋亡指数显著增加。     

Mutational‐screening in the factor VIII gene resulting in the identification of three novel mutations,one of which is a donor splice mutation

Hemophilia A is an X‐linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1‐26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the a ®c transversion at position 3 of the donor‐splice‐site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A. Hum Mutat 13:504, 1999. © 1999 Wiley‐Liss, Inc.     

中国人动脉血栓性疾病与活化蛋白C裂解FV和FVIII基因位点突变的相关性研究

目的 :检测脑血栓 (CT)和急性心肌梗死 (AMI)患者活化蛋白C(APC)抵抗 (APCR)及APC裂解凝血因子V (FV )和凝血因子VIII(FVIII)肽链的基因位点突变。方法 :用单链构象多态性分析 (SSCP)方法 ,检测 10 2例CT、46例AMI和 10 5例健康人的APC裂解FV肽链 (Arg3 0 6、Arg 5 0 6、Arg 679和结合部位 1865～ 1875 )、裂解FVIII肽链 (Arg3 3 6、Arg 5 62和结合部位 2 0 0 5～ 2 0 18)的基因位点。结果 :对照组和患者组均未检出FV的突变基因。结论 :中国人动脉血栓性疾病患者无APC裂解FV和FVIII肽链的基因突变 ,患者的APC敏感性的降低可能由其他因素所致     

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication. Hum Mutat 12:393–402, 1998. © 1998 Wiley-Liss, Inc.     

Common intragenic and extragenic polymorphisms of blood coagulation factors VIII and IX are different in Chinese and Caucasian populations

In order to examine the possibilities of carrier detection and prenatal diagnosis in hemophilia A and B in the Chinese region of Suzhou, we analyzed four different RFLPs within the factor IX gene and two intragenic RFLPs and one extragenic RFLP for the factor VIII gene. The results obtained show important differences between the Chinese and Caucasian populations. No polymorphism was found within the factor IX gene in the Chinese population and the informativity obtained for the factor VIII gene was quite different between the two populations for each RFLP studied.     

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

Hemophilia A is a common X-linked bleeding disorder caused by various types of mutations in the factor VIII gene F8C. The most common intron 22-inversion is responsible for about 40% of the severe hemophilia A cases while large deletions, point mutations and small (less than 100 bp) deletions or insertions are responsible for the disease in the rest of patients. We report on nine novel (6 deletions, two indels and one partial duplication) and five recurrent small rearrangements identified in 15 German patients with severe hemophilia A, negative for the intron 22-inversion. c.2208-2214delTTATTAC/c.2207-2215insCTCTT and c.4665-4678del/c.4664-4678insAAGGAA identified in the present study are the first small indels described in the factor VIII gene. Our analyses suggest that the prevalence of this type of mutations (predominantly located in exon 14) among patients with severe phenotype and negative for the common intron 22-inversion, is about 30%. The correlation between these molecular defects and formation of factor VIII inhibitors as well as the parental origin of the de novo mutations are evaluated. Finally we show that denaturing HPLC (DHPLC) and classic heteroduplex analysis (HA) are able to detect these sequence alterations on 100% and could be preferred as a screening approach when analysing for mutations in factor VIII in severely affected patients.     

High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation

Hemophilia A (HEMA) is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular genetic testing for the factor VIII gene is challenging due to its large size. Here we present results of high throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon-intron boundaries of the factor VIII gene. The results of mutation analysis on 89 hemophiliac males showed presence of a disease-causing mutation in 80 individuals (90%, 95% CI of 82%-95%). Seven out of nine mutation-negative individuals were severe cases of hemophilia A with < 1% factor VIII protein in the blood. The correlation of phenotype with genotype as observed in this study was not absolute. This finding is supported by similar observations in the international database for hemophilia A mutations (HAMSTeRS). This issue raises the importance of genotypes at other loci that can act as modifiers for the phenotype. Thirty-four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations. The mutations cover the full spectrum including rearrangements, deletions, frameshift, and point mutations. The novel missense mutations require careful evaluation. Prediction of a mutation as the disease-causing allele was made from the nature of the substitution and the degree of conservation of the mutated amino acid among species that have diverged in evolution. In some cases segregation analysis of the mutation with disease condition was performed when other family members were available.     

环孢素A延长腺病毒基因治疗载体体内存在时间的研究

目的研究重组腺病毒载体在免疫抑制疗法应用中的安全性并探讨环孢素A对该载体在体内清除的影响。方法采取腹膜后注射纯化重组腺病毒建立动物模型,连续给予环孢素A 14 d,取不同时间点大鼠样本,HE染色观察各主要脏器病理反应,免疫组化方法检测肝、脾、肺、肾、肌肉及淋巴结腺病毒的表达情况。结果①HE染色切片,各脏器呈现一过性可逆性轻微炎性反应。②免疫组化检测,对照组中重组腺病毒逐渐减弱至阴性,实验组中重组腺病毒存在时相性明显延长(P<0.05)。③腺病毒分布呈现肝脾肺高表达而其它组织表达相对较低。结论重组腺病毒作为基因治疗载体在免疫抑制疗法应用中具有一定的安全性,环孢素A能够部分抑制实验动物体内的免疫反应从而延长重组腺病毒在体内存在的时间。     

Hepatitis B Virus Vector Carries a Foreign Gene into Liver Cells In Vitro

Hepatitis B viruses (HBV) specifically target the liver, where they efficiently infect quiescent hepatocytes. Thus, HBV virus has potential to be used as vectors for liver-directed gene transfer. We constructed a new HBV-based vector system. It is composed of transfer vector for transferring a foreign gene, green fluorescence protein (GFP) gene, and a helper vector. When the transfer vector and the helper vector were cotransfected into HepG2 cells, the recombinant HBV (rHBV) particles were generated by trans-complementation between two vectors. The rHBV particles carrying the foreign gene were identified by the Southern blot assay. To test gene delivery and the transduction of the rHBV, we infected primary human hepatocytes and immortalized, HepG2 cells with rHBV in vitro. The results using fluorescence microscopy confirmed that the inserted GFP gene was successfully transferred and expressed both in primary human hepatocytes and HepG2 cells.     

Removal/neutralization of hepatitis A virus during manufacture of high purity,solvent/detergent factor VIII concentrate

Recent reports have suggested an increased risk of type A viral hepatitis in hemophilic patients treated with high purity factor VIII concentrates prepared using ion exchange chromatography coupled with solvent/detergent treatment for in-activation of viruses. To determine the capacity for removal or inactivation of hepatitis A virus during the factor VIM manufacturing process, human plasma and various factor VIM production intermediates were spiked with cell culture-propagated virus and subjected to scaled down conditions mimicking the manufacture of solvent/ detergent factor VIII. The combination of antibody-mediated neutralization, cryoprecipitation, anion exchange chromatography, and lyophilization in the absence of sucrose resulted in a minimal reduction of 5.5 to 8.55 Iog₁₀ in the infectivity of hepatitis A virus. © 1994 Wiley-Liss, Inc.     

海南地区人群中凝血因子XIIIA链567位点多态性的研究

目的研究海南地区汉族人群、黎族人群凝血因子XIIIA链567位点（GAA→GAG）分布频率。方法用聚合酶链反应、限制性片段长度多态性分析检测205名汉族正常人、236名黎族正常人凝血因子XIIIA链567位点多态性。结果汉族人群和黎族人群均未发现567位点GAA→GAG突变。结论凝血因子XIIIA链567位点变异在中国海南人群中很少见。