基因治疗及基研究进展

基因治疗作为一种新型的疾病治疗手段，正在广泛用于血友病、糖尿病、癌症、心血管病、爱滋病等多种疾病的治疗领域。基因治疗的一般步骤包括：目的基因的转移和目的基因的表达两个方面。其中目的基因的导入和目的基因表达的精确调控是两个关键步骤。新发展的还有反义疗法以及应用核酶进行基因治疗等。本文就基因治疗的基本步骤、基因治疗的对象、方式及基因治疗的现状和前景作一个简单介绍。     

肿瘤基因治疗的临床应用

基因治疗方法是通过基因转导技术赋于靶细胞一种新的功能或改变靶细胞某些基因的表达状况。目前基因治疗的疾病主要包括遗传性疾病、免疫缺陷病、肿瘤等。1991年12月在复旦大学遗传学研究所进行了国际上首例血友病的基因治疗。两位由于凝血因子Ⅸ缺乏而患有血友病B的患者被重新植入了大量转移有正常Ⅸ因子基因的自身皮肤成纤维细胞,结果病人体内Ⅸ因子含量都成倍上升,取得较好疗效。1990年9月,美国国立卫生研究院(NIH)用基因疗法治疗了一位由于体内腺苷脱氨酶(ADA)缺乏而患者严重联合性免疫缺陷的病人,他们将转移有正常ADA基因的患者自身淋巴细胞体外培养后重新大量输回病人体内,实验结果令人鼓舞。目前基因治疗已应用于很多种疾病。 肿瘤的基因治疗始于九十年代。1990年美国NIH首次对一位晚期恶性黑色素瘤病人进行了基因转导的肿瘤浸润淋巴细胞(TIL)     

基因治疗及其研究进展

基因治疗作为一种新型的疾病治疗手段 ,正在广泛用于血友病、糖尿病、癌症、心血管病、爱滋病等多种疾病的治疗领域 .基因治疗的一般步骤包括 :目的基因的转移和目的基因的表达两个方面 .其中目的基因的导入和目的基因表达的精确调控是两个关键步骤 .新发展的还有反义疗法以及应用核酶进行基因治疗等 .本文就基因治疗的基本步骤、基因治疗的对象、方式及基因治疗的现状和前景作一个简单介绍     

四例血友病B患者基因治疗的临床试验

报道了对四例血友病Ｂ患者基因治疗的临床试验，取自四例血友病Ｂ患者的自体皮肤成纤维细胞，通过反转录病毒介导的基因转移，能高效地分泌人凝血因子Ⅸ。用胶原包埋经遗传修饰的自体皮肤成纤维细胞，再移植到血友病Ｂ患者皮下。四例患者经治疗后，自发出血症状得到了减轻，血浆中凝血因子Ⅸ蛋白浓度和凝血活性明显地增加，其中两例患者增加较大，达到正常值的４％￣５％。另外两例患者体内的凝血因子Ⅸ和凝血活性也有不同程度的提高     

血友病B基因治疗研究

血友病Ｂ是一种由于人凝血因子Ⅸ (ｈＦＩＸ)缺陷导致的严重出血性遗传疾病 ,临床治疗主要依靠输血或凝血酶原复合物 ,但这些常规的替代治疗不仅费用昂贵 ,易引起输血反应 ,更可能感染肝炎病毒或爱滋病病毒 .理论上 ,只有基因治疗可望从根本上治疗血友病Ｂ .本研究先后建立反转录病毒基因转移系统 ,高效转染小鼠、大鼠、兔和血友病Ｂ患者皮肤成纤维细胞等不同细胞 ,尤其是人ＦＩＸ在骨髓基质细胞的表达在国际同类研究中是首次报道 .人ＦＩＸ在兔体内表达水平和持续时间在同类研究中居世界领先水平 ;在国内首先建立了有关基因治疗的安全性检…     

编码下的容颜

人的容貌在基因操作的层面上面临怎样的命运？选择姣好相貌和永葆青春的面容能否真正实现？当复旦大学遗传学研究所的薛京伦教授第一次听到＂基因美容＂这个词的时候,他显得有些茫然。毕竟,薛京伦教授已经从事遗传学方面的研究40余年,在基因治疗领域具有卓越的贡献,是学界公认的权威,曾经在国际上首次成功地实现了血友病B患者 的基因临床治疗。     

凝血因子IX的体外检测方法研究

凝血因子ＩＸ的检测是血友病Ｂ基因治疗研究中的重要工作，在实验室原有基础上，发现和完善了凝血因子ＩＸ在蛋白质水平上的检测系统，为基因治疗血友病Ｂ提供了更为直观可靠的依据。首先，建立了以鼠抗人ＦＩＸ单克隆抗体Ａ－７为一抗的检测活性ＦＩＸ蛋白量的ＥＬＩＳＡ法，为检测活性ＦＩＸ提供了快速简便的方法。其次，实现了用Ｗｅｓｔｅｒｎｂｌｏｔ法检测转染细胞培养液上清中ＦＩＸ，确证了体外培养的转有人ＦＩＸｃＤＮＡ细     

不同重组慢病毒介导人凝血因子Ⅸ cDNA在血友病B小鼠中表达的影响

血友病B是凝血因子Ⅸ(hFⅨ)缺乏所导致的严重凝血功能障碍、X-连锁隐性遗传性疾病,在男性中的发病率为三万分之一．目前没有理想的治疗措施,而基因治疗可能是根治该病的安全、有效方法．该实验构建了ubiquitin—c和ABP(肝特异性)启动子指导hFⅨ表达的载体FUXW、FAXW,利用磷酸钙法共转染三质粒制备高滴度的重组慢病毒．将不同剂量的重组FUXW、FAXW病毒,分别用尾静脉液压法和静脉缓注法注射入血友病B小鼠体内,各治疗组小鼠血浆均可持续检测到hFⅨ抗原,最高峰值为45ng／mL,表达持续超过60d．结果表明：hFⅨ蛋白的表达与病毒的剂量成正相关,重组FAXW病毒的hFⅨ表达量高于重组FUXW病毒,而尾静脉液压组和静脉缓注组在表达量、表达时间上没有显著差异．     

凝血因子Ⅸ基因剔除小鼠遗传稳定性及其临床表型研究

通过对凝血因子Ⅸ基因剔除小鼠遗传稳定性及其相应临床表型的研究,为以该动物模型为研究对象的血友病Ｂ基因治疗提供相应背景资料,遗传分析表明,该小鼠繁殖过程中无回复突变出现,亦未发现Ⅸ因子基因未敲除部分被转录的证据;血浆ＰＴ,ＫＰＴＴ和Ⅸ因子活性检定结果提示该小鼠符合人血友病Ｂ相应临床表征。     

电击法转移含人凝血因子IX基因重组质粒的影响因素

反转录病毒载体在基因治疗中可能会产生野生型病毒颗粒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者合ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸｃＤＮＡ，后者仅含ｈＣＭＶ启动于控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动于驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１０８０细胞中的ＦＩＸ表达量分别为２２０、２１２ｎｇ／（１０６细胞·２４ｈ）通过与ｐＣＭＶＩＸ共转染靶细胞，可以增加人ＩＸ因子表达１．５～３．５倍，显示了用质粒载体转染靶细胞在血友病Ｂ基因治疗中是一条潜在可行的途径．本项研究用自制的电击仪转移ＰＡ３１７和ＨＴ１０８０等细胞，最高的转移效率达１０－３，并探讨了细胞种类，ＤＮＡ量，ＤＮＡ结构，电压，脉冲时间与转移效率及表达量的关系．     

Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells

Haemophilia B, or Christmas disease, is an inherited X-chromosome-linked bleeding disorder caused by a defect in clotting factor IX and occurs in about 1 in 30,000 males in the United Kingdom. Injection of factor IX concentrate obtained from blood donors allows most patients to be successfully managed. However, because of impurities in the factor IX concentrate presently in use, this treatment involves some risk of infection by blood-borne viruses such as non-A, non-B hepatitis and the virus causing acquired immune deficiency syndrome (AIDS). Because of the recent concern about the increasing incidence of AIDS amongst haemophiliacs, a factor IX preparation derived from a source other than blood is desirable. Here, we report that after introduction of human factor IX DNA clones into a rat hepatoma cell line using recombinant DNA methods, we were able to isolate small amounts of biologically active human factor IX.     

Haemophilia B caused by a point mutation in a donor splice junction of the human factor IX gene

Haemophilia B (Christmas disease) is an inherited, recessive, sex-linked, haemorrhagic condition caused by a defect in the intrinsic clotting factor IX. This disease occurs in males at a frequency of approximately 1 in 30,000. Patients differ in the severity of their clinical symptoms, and variation in the clotting activity and in the concentration of factor IX antigen in their plasma has been demonstrated. There is probably heterogeneity in the molecular defects of the factor IX gene causing the disease. Here we study a severely affected, antigen-negative patient, and show that the only significant sequence difference from the normal factor IX gene is a point mutation changing the obligatory GT to a TT within the donor splice junction of exon f. We infer that this change is the cause of the disease in this individual. In addition, we have used oligodeoxynucleotide probes specific for this mutation to demonstrate the feasibility of carrier detection and prenatal diagnosis for relatives of the patient.     

Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots

Haemophilia A is a common disorder of blood coagulation caused by a deficiency of factor VIII. It is inherited as an X-linked recessive trait, and one-third of all cases are thought to result from de novo mutations. The clinical severity of haemophilia A varies markedly among different families and a subset of the patients with severe disease develop antibodies against factor VIII, called inhibitors. Because of this heterogeneity, it is likely that many different molecular lesions result in haemophilia A. Indeed, of the nine mutations described to date, all appear to be unique changes. However in this study of 83 patients with haemophilia A we have identified two different point mutations, one in exon 18 and one in exon 22, that have recurred independently in unrelated families. Each mutation produces a nonsense codon by a change of CG to TG, and each occurred de novo on the X-chromosome donated by the maternal grandfather. These observations strongly support the view that CpG dinucleotides are mutation hotspots.     

Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene

Haemophilia A is the most common inherited bleeding disorder in man, affecting approximately 1 male in 10,000. The disease is caused by a deficiency in the gene for factor VIII, a component of the intrinsic coagulation pathway. Due to the broad range of clotting activity in normal and heterozygous females, it is often difficult to confirm the status of women at risk for carrying the disease. A genetic marker in the form of a restriction fragment length polymorphism (RFLP) within or tightly linked to the factor VIII gene would serve as a tag for the haemophilia gene, thus allowing both accurate carrier detection and improved, earlier prenatal diagnosis by chorionic villi sampling. The recent isolation of the factor VIII gene has allowed a search for RFLPs within the gene, and we report here the identification of a common polymorphism within the factor VIII gene, revealed by the restriction enzyme BclI, which can be used diagnostically in about 42% of all families. Although the disease haemophilia A has been mapped to the distal portion of Xq, the BclI RFLP makes possible higher-resolution genetic linkage mapping with respect to other polymorphic markers on this portion of the X chromosome. We have established close linkage of the factor VIII gene to several useful RFLP markers, including the highly informative marker St14. These markers should also be useful for prenatal diagnosis of haemophilia A and for detection of its carriers.     

无约束优化问题新的谱共轭梯度法(英文)

通过结合牛顿法与PRP谱共轭梯度法提出一新的谱共轭梯度法.该方法为下降方法且为Birgin谱共轭梯度法与PRP共轭梯度法的线性组合.在适当的假设下算法全局收敛.     

共轭梯度法和最速下降法的混合算法

将共轭梯度法与最速下降法有机地结合起来,构造了一种共轭梯度法和最速下降法的混合算法,并证明了该算法的全局收敛．混合算法既提高了共轭梯度算法的收敛速度,又解决了目标函数“性态不优”时,最速下降法难以求解的问题．同时也可以看到共轭梯度法与最速下降法仅仅是混合算法的特例．     

在乙醇—水介质中采用硫酸钡沉淀滴定法测定氯化钡中钡的含量

提出了在乙醇—水介质中采用硫酸钡沉淀滴定法测定氯化钡中钡的含量.试样溶解并消除干扰后,在pH2.0~3.0的乙醇—水介质中,以茜素S为指示剂,用Na2SO4标准溶液进行滴定.结果表明,改进后的方法简单、快捷、准确、省时、干扰小;与重量法相比,相对误差小于0.5%,说明该方法的准确度与重量法相当;其相对标准偏差仅为0.20%,说明其重现性好.因此,该方法是一种既便于教学实验,又可满足现场生产要求的快速分析方法.     

一个求解非线性最小二乘问题的新方法

在Gauss-Newton(G-N)方法和Levenbery-Marquardt(L-M)方法(阻尼最小二乘法)的基础上给出了一种新的求解非线性最小二乘问题的方法，它是通过寻求新的非线性方程组的数值方法来实现的，首先给出了不用计算导数的求解非线性方程组的收敛迭代方法，该方法是建立在求解动力系统的稳定点的基础上，采用了较稳定的常微分方程初值问题的数值方法进行迭代求解，并采用Steffensen加速技术以提高收敛速度，最后，给出了用Matlab试算的数值例子、试验结果表明了该方法的有效性。     

等波纹线性相位FIR滤波器的1种设计方法

介绍了一种用Remez函数设计等波纹线性相位FIR滤波器的设计方法,并利用MATLAB强大的功能进行仿真。仿真结果表明,设计结果的各项性能指标均达到较高的要求,并且证明该方法是一种优化的设计方法。在同样的技术指标下,用这种方法设计得到的滤波器要比窗函数法和频率采样法得到的滤波器的长度均要小,而且设计过程简单易行。