糖尿病基因治疗中胰岛素基因表达调控的研究进展

目前糖尿病基因治疗的方向之一是寻找理想的胰岛素表达调控方案 ,以达到胰岛素生理性合成和释放的目的。肝脏是目前胰岛素基因异位表达的最佳靶器官 ,而且肝细胞可转化成为合成并分泌胰岛素的细胞。另外 ,通过控制内质网的凝聚过程、与胰岛 β细胞同源的K细胞及四环素调控基因实现胰岛素的可控分泌也是很有前途的胰岛素基因治疗途径     

糖尿病基因治疗中胰岛素基因表达调控的研究进展

目前糖尿病基因治疗的方向之一是寻找理想的胰岛素表达调迭方案，以达到胰岛素生理性合成和释放的目的。肝脏是目前胰岛素基因异位表达的最佳靶器官，而且肝细胞可转化成为合成并分泌胰岛素的细胞。另外，通过控制内质网的凝聚过程，与胰岛β细胞同源的K细胞及四环素调控基因实现胰岛素的可控分泌也是很有前途的胰岛素基因治疗途径。     

体内胰岛素基因转移治疗1型糖尿病的研究现状

基因治疗是近年来随着基因重组和基因转移技术的迅速发展而出现的治疗 1型糖尿病的一条新途径 ,而胰岛素基因是糖尿病基因治疗的主要目的基因。文中分别就生殖细胞水平基因转移、体内间接基因转移以及体内直接基因转移 3个方面 ,从基因转移方法、途径等角度 ,就胰岛素基因转移治疗 1型糖尿病的研究现状作一简要综述。     

基因治疗与胰岛素依赖型糖尿病

近年来许多研究致力于探索胰岛素依赖型糖尿病基因治疗的途径和可行性。首先可利用各种现代分子生物学技术改造胰岛Ｂ细胞系或建立人工Ｂ细胞，以获得能够分泌胰岛素并可对引起胰岛素分泌的生理刺激作出正确反应的各种细胞，然后再进行细胞移植。另外还可通过体内胰岛素基因转移直接在体内表达胰岛素     

人胰岛素基因转染鼠成纤维细胞致糖尿病大鼠血糖下降效应的研究

目的研究人胰岛素基因表达质粒转染鼠成纤维细胞（Ｌｔｋ－）后对糖尿病大鼠血糖的影响。方法重组的人胰岛素基因表达质粒通过脂质体法转染鼠成纤维细胞（Ｌｔｋ－），经Ｇ４１８选择性筛选获得一株较高胰岛素表达水平的细胞株（Ｌｔｋ－／ＰＲＩ１２），大量扩增，直接注射至糖尿病大鼠腹腔内，并与转染空载体的对照Ｌｔｋ－细胞比较，观察在糖尿病大鼠体内的胰岛素表达及对血糖等变化的影响。结果空载体转染的对照细胞与未转染组相似，无胰岛素表达，植入糖尿病大鼠腹腔后，血糖仍持续稳定在较高水平，体重进行性下降；而接受胰岛素表达载体细胞的糖尿病大鼠，在观察的２１天内血Ｃ肽持续表达，血糖明显下降，体重逐渐恢复。结论人胰岛素基因能成功转染非胰岛β细胞并表达目的基因使血糖水平下降。本研究为进一步开展糖尿病基因治疗研究奠定了基础。     

基因治疗与胰岛素依赖型糖尿病

近年来许多研究致力于探索胰岛素依赖型糖尿病基因治疗的途径和可行性，首先可利用各种现代分子生物学技术改造胰岛Ｂ细胞系或建立人工Ｂ细胞，以获得能够分泌胰岛素并可引起胰岛素分泌的生理刺激作出正确反应的各种细胞，然后再进行细胞移植，另外还可通过体内胰岛素基因转移直接在体内表达胰岛素。     

基因治疗 任重道远

1型糖尿病依赖胰岛素皮下注射治疗，而2型糖尿病随着病程逐渐延长，绝大多数最终也需要胰岛素治疗才能实现血糖达标。自从基因重组技术发明开始，人们就憧憬有朝一日实现胰岛素基因治疗，摆脱注射胰岛素的痛苦。但目前尚无糖尿病血糖控制方面的基因治疗方案，被美国食品药品监督管理局批准进行临床试验。因此，糖尿病血糖控制相关的基因治疗，还需走很长的路才能见到曙光。     

体内胰岛素基因转移治疗1型糖尿病的研究现状

基因治疗是近年来随着基因重组和基因转移技术的迅速发展而出现的治疗1型糖尿产现的一条新途径，而胰岛素基因是糖尿病基因治疗的主要目的基因。文中分别就生殖细胞水平基因转移、体内间接基因转移以及体内直接基因转移3个方面，从基因转移方法、途径等角度，就胰岛素基因转移治疗1型糖尿病的研究现状作一简要综述。     

肝型丙酮酸激酶启动子与人胰岛素基因逆转录病毒介导感染pT67、HepG2细胞的蛋白质表达

目的检测新构建肝型丙酮酸激酶启动子（LPKp）与人胰岛素基因（hInsg）逆转录病毒表达载体（pM54,LPKp-hInsg）在pT67、HepG2细胞的表达情况,为基因治疗或基因结合干细胞治疗糖尿病寻找新的优化生物载体。方法（1）限制酶切父、母本质粒p54、pMDN-SIN,取相关片段构建含人Ins基因一逆转录病毒载体pM54（pMDN-SIN＋p54;LPKp-hInsg）;pM54扩增、纯化、酶切鉴定;（2）脂质体FuGENE6转染pM54质粒进入pT67、PhoenixE和3T3细胞系,同时转染pCMVβGal和父本质粒p54做对照;（3）制备转染后细胞培养拟逆转录病毒上清液;（4）用转染后细胞培养上清液旋转感染pT67、HepG2细胞;（5）ELISA法检测转染、感染后细胞培养上清液Ins含量。结果酶切鉴定质粒与构建预期相符;ELISA法检测出转染、感染上清液中均含较高水平Ins。对照结果均为阴性。结论LPKp-hlnsg基因逆转录病毒表达载体pM54构建成功。人胰岛素基因逆转录病毒介导旋转感染pT67等细胞,目的蛋白Ins获得了预期的表达。实验为基因治疗或基因结合于细胞治疗糖尿病的进一步相关研究奠定了基础。     

利用非胰岛细胞进行1型糖尿病基因治疗的研究进展

1型糖尿病是由于胰腺 β 细胞特异的自身免疫破坏 ,使胰岛素的产生减少所造成的。目的在于进行胰岛素替代的基因治疗战略目前主要有 :①刺激 β 细胞生长 ;②诱导 β 细胞分化和再生 ;③移植基因工程改造的胰岛或 β 细胞 ;④利用基因工程技术使非 β 细胞产生胰岛素。对于 1型糖尿病 ,由于 β 细胞的自身免疫破坏作用持续存在 ,依赖于 β 细胞的增殖和再生的治疗方法可能效果有限。而利用基因工程技术将胰岛素基因转入非 β 细胞 ,发展 β 细胞替代物 ,可避免 β 细胞特异的自免疫攻击。以非 β 细胞为靶细胞的胰岛素基因治疗应很好的实现…     

Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.     

Adenoviral gene therapy in gastric cancer： A review

Gastric cancer is one of the most common malignancies worldwide. With current therapeutic approaches the prognosis of gastric cancer is very poor, as gastric cancer accounts for the second most common cause of death in cancer related deaths. Gastric cancer like almost all other cancers has a molecular genetic basis which relies on disruption in normal cellular regulatory mechanisms regarding cell growth, apoptosis and cell division. Thus novel therapeutic approaches such as gene therapy promise to become the alternative choice of treatment in gastric cancer. In gene therapy, suicide genes, tumor suppressor genes and anti-angiogenesis genes among many others are introduced to cancer cells via vectors. Some of the vectors widely used in gene therapy are Adenoviral vectors. This review provides an update of the new developments in adenoviral cancer gene therapy including strategies for inducing apoptosis, inhibiting metastasis and targeting the cancer cells.     

Human gene therapy

Recent advances in molecular genetics have made possible the use of retroviral "vectors" to transfer cloned human genes into somatic cells. With this new technology, the genetic defect underlying many recessive inherited disorders can probably be corrected by inserting a normal gene into the patient's hematopoietic stem cells. This article reviews the design and safety of the viral vectors and the results of in vivo studies in mice and large animals that have led to the first human trials. Other target cells for gene transfer, such as endothelial cells, fibroblasts, keratinocytes, and hepatocytes, are also discussed. The use of recombinant retroviruses for gene transfer in vivo is still a new area of research, but the feasibility of "gene therapy" for genetic disorders is rapidly gaining medical and scientific acceptance.     

Local drug and gene delivery through microbubbles

Ultrasound contrast agents (microbubbles) lower the threshold for cavitation by ultrasound energy. Ultrasound microbubbles may be used as cavitation nuclei for drug and gene delivery. By tailoring the physical properties of microbubbles and coating materials, drugs and genetic drugs can be incorporated into ultrasound contrast agents. As the microbubbles enter the region of insonation, the microbubbles cavitate, locally releasing the therapeutic agents. Cavitation also causes a local shockwave that improves cellular uptake of the therapeutic agent. As a result of the human genome project and continuing advances in molecular biology, many therapeutic genes have been discovered. In the cardiovascular system, gene therapy has the potential to improve myocardial vascularization and ameliorate congestive heart failure. For successful development of clinical gene therapy, however, effective gene delivery vectors are needed. Ultrasound contrast agents can be used to develop new, more effective vectors for gene delivery. Transthoracic ultrasound can be focused on the heart so that an intravenous injection of gene-bearing microbubbles will deliver genes relatively selectively to the myocardium. Using this technique, we have produced high levels of transgene expression in the insonated region of the myocardium. This new technology, using microbubbles and ultrasound for drug and gene delivery, merits further study and development.     

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.     

Systemic gene therapy for cardiovascular disease

Systemic gene therapy involves the transfer into the body of a gene whose protein product reaches the blood and has a beneficial effect on a patient. Both retroviral and adenovirus-associated viral vectors have resulted in stable but only moderate systemic levels of blood proteins. Adenoviral vectors have resulted in very high levels of expression that diminishes over days or weeks. Hepatic gene therapy has achieved levels of the anticoagulant protein C in blood that would protect against spontaneous thromboses in homozygous protein-C deficiency, and levels of tissue plasminogen activator that can lyse pulmonary emboli. Hypercholesterolemia has been ameliorated transiently by transfer of the low-density lipoprotein receptor gene into the livers of animals with familial hypercholesterolemia or by promoting lipid transfer via a variety of alternative mechanisms. Hypertension has been reduced by the transfer of genes for kallikrein or atrial natriuretic peptide into the liver, or by expressing antisense for the angiotensin II type I receptor after intravenous injection in neonates. Finally, fasting but not fed hyperglycemia has been ameliorated in animal models of diabetes by transfer of an insulin gene into the liver or by expression of insulin from implanted fibroblasts. Gene therapy has the potential to treat these cardiovascular diseases. However, improvements in levels of long-term expression and the ability to regulate expression in response to physiologic changes will be required before this approach will be implemented for most of these disorders in humans.     

Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus

Delivering transgenes to human cells through transduction with viral vectors constitutes one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the most promising vectors for these approaches. When the genetic modification of the cell must be performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined by the characteristics of the surface proteins carried by the vector. In this regard, an important property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses, widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved at the molecular level and what the properties and the potentialities of the different envelope proteins that can be used for pseudotyping these vectors are.     

Gene therapy of hepatocellular carcinoma

The extraordinary versatility of gene therapy opens new possibilities for the treatment of incurable diseases, including hepatocellular carcinoma. Gene therapy strategies against tumors include prodrug activation therapy by the transfer of suicide genes, immunogene therapy, tumoral cell phenotype correction by the inhibition of oncogenes or the transfer of tumor suppressor genes, antiangiogenesis and transfer of oncolytic viruses. The experience accumulated during the last decade of clinical gene therapy indicates that genes can be expressed inside the tumor tissue, but the overall results of the studies conducted so far are still disappointing, mainly due to the poor performance of the currently available gene therapy vectors. This review covers the general aspects of gene therapy vectors, preclinical data available in animal models of hepatocellular carcinoma, and finally a brief summary of the gene therapy clinical trials aimed at the treatment of liver cancer.     

Virus infection recognition and early innate responses to non-enveloped viral vectors

Numerous human genetic and acquired diseases could be corrected or ameliorated if viruses are harnessed to safely and effectively deliver therapeutic genes to diseased cells and tissues in vivo. Innate immune and inflammatory response represents one of the key stumbling blocks during the development of viral-based therapies. In this review, current data on the early innate immune responses to viruses and to the most commonly used gene therapy vectors (using adenovirus and adeno-associated virus) will be discussed. Recent findings in the field may help develop new approaches to moderate these innate immune anti-viral responses and thus improve the safety of viral vectors for human gene therapy applications.