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Gene therapy in gastric cancer
1 IntroductionWe have reviewed the gene therapy in gastrointestinal diseases[1]. Gastric cancer is common in China[2~20] ,and its early diagnosis andtreatment are still difficult up to now[13~36]. The expression of anexogenous gene introduced by gene therapy into patients with gliomascan be monitored non- invasively by positron- emission tomography[4]. In recent years, gene study in cancer is a hotspot, and great progress hasbeen achieved[33~41].
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重组腺相关病毒介导CD151基因治疗心肌梗死后侧支循环形成
目的 探讨重组腺相关病毒(rAAV)介导的CD151基因转染对小型猪心肌梗死模型后侧支循环重建的影响.方法 中国实验用小型猪(清洁级,由华巾农业大学种猪场提供)26头,随机分为:(1)正常对照绀(n=4),健康小型猪,小进行任何手术处理;(2)rAAV-GFP组(n=7),结扎冠状动脉序前降支(left anterior descending,LAD)制备小型猪急性心肌梗死模型,心电监护显示I、aVL导联ST段持续抬高20 min为模型制备成功,同时,在左心室前壁梗死边缘区心肌分10点注射rAAV-GFP(1×10 11 pfu/点);(3)rAAV-CD151纽(n=7),结扎LAD,左窜前壁梗死边缘区心肌分10点注射rAAV-CD151;(4)rAAV-anti-CD151组(n=8),结扎LAD,左室前壁梗死边缘区心肌分10点注射rAAV-anti-CD151.8周后用Western Blot测定心肌组织CD151蛋白的表达,冠脉造影评价冠脉侧支循环形成情况.氯化二苯基四氮唑(TTC)染色显示心肌梗死面积.运用单因素方差分析进行统计学分析,以P<0.05为差异具有统汁学意义.结果 与正常对照组和rAAV-GFP组比较,rAAV-CD151组心肌组织局部CD151蛋白表达增加(P<0.05).冠状动脉造影证实CD151基因转染明显促进闭塞动脉的侧支循环建立.rAAV-CD151组心肌梗死面积百分比明显小丁rAAV-GFP组[(12.82±2.26)%vs.(23.14±2.83)%,P<0.05],rAAV-anti-CD151纽心肌梗死面积百分比(32.52±3.47)%明显大于rAAV-GFP组和rAAV-CD151组(P<0.05).结论 CD151基因转染可以明显促进小型猪急性心肌梗死后侧支循环的建立,减小心肌梗死面积.
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慢性乙型肝炎病毒清除自杀基因平衡制约载体系统的构建
目的:建立清除乙型肝炎病毒(HBV)自杀基因平衡制约载体系统,靶向清除HBV感染的肝细胞,抑制慢性乙型肝炎和肝硬化患者免疫再活动.方法:逆转录PCR(RT-PCR)扩增丙型肝炎病毒(HCV)基因组中核糖体插入位点(IRES),以pcDNA3载体为基础,构建双顺反子表达载体.同时在IRES位点的上游克隆HBV表面基因的部分反义序列和HCV全长核心基因,在IRES位点下游克隆胸腺苷激酶基因,PCR、酶切和测序鉴定.构建的清除乙型肝炎病毒(HBV)自杀基因平衡制约载体系统分别转染培养基含有更昔洛韦的HepG2细胞和2.2.15细胞.结果:构建的清除乙型肝炎病毒(HBV)自杀基因平衡制约载体系统可以选择性促使2.215细胞凋亡(实验组细胞凋亡率为15%,而对照组仅为6%);抑制2.2.15细胞在培养基中分泌乙型肝炎病毒表面抗原(HBsAg),统计学分析表明,HBsAg在培养基中检测滴度,在两组之间有显著性差异(P<0.05).结论:构建以HBV表面基因为靶基因,自杀基因为效应基因的自杀基因制约平衡系统,可能为下一步慢性乙型肝炎的基因治疗提供理想的基因治疗载体.
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重组复制缺陷型腺病毒基因治疗肝纤维化的研究与应用
肝纤维化的特征性变化是肝内细胞外基质的过多沉积.抑制肝星形细胞的激活和细胞外基质的合成,促进基质降解和肝细胞再生是肝纤维化治疗的重要方法.近年来,针对上述环节构建重组复制缺陷型腺病毒,表达不同外源基因产物基因治疗肝纤维化研究取得了较大的进展.本文对重组腺病毒的结构、构建以及抗肝纤维化作用和安全性研究作一综述.
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AIM To explore the therapeutic potential of antisense oligodeoxynucleotides on hepatcellular cacinoma(HCC).METHODS Four antisense phosphorothioated oligodeoxynucleotides (asON), complementary to differentsites of HBV, were synthesized and assayed for their anti-HBV activity in HepG22. 2.15 cells with ELISA.The most effective asON was chosen for the following study: FACSCAN, TRAP and immuno-staining wereused respectively for checking apoptosis, telomerase activity and expression of oncogene p21ras and p62C-myc inHepG2.2.15 cells after treated by asON.RESULTS The oligomer directed against the initiator of pre-S2 was the most effective one with aninhibitory rate of 66% on HBsAg and 91% on HBeAg (P<0.02). Two inhibitory peaks (bimodal)appeared. Telomerase activity as well as the expression of p21fas and p62C-myc decreased drastically 3 days afterasON-HBpreS2 treatment. Meanwhile, apoptosis appeared in the experiments.CONCLUSION The inhibitory effects of as-preS2 on the HBV gene expression and the reversion of somemalignant behaviour in HepG2.2.15 cells were the significant, effective therapy against HBV infection andhepatocellular carcinoma.
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AIM To summarize the experience of surgical treatment of hilar cholangiocarcinoma and the results of aseries of experiments.METHODS AND RESULTS Personal perspectives of surgical treatment of hilar cholangiocarcinoma werebased on the experience of a series of patients with hilar bile duct cancer treated in the General Hospital ofPLA, Beijing from 1986 to 1999. A total of 157 cases were treated surgically, with 106 (67.5%) resections ofthe tumor , 37.6% of the resections was proved to be radical. The 1-, 2-, 3-, and 5-year survival rate of theradical resection group was 96.7%, 40.0%, 23.3% and 13.3%, respectively. No patient of the palliativeresection group lived beyond 3 years postoperatively. The recent trends of surgical management of hilar bileduct cancer were discussed. Experiments were carried out for cooperative clinicopathological study toevaluate the perineural space involvement, the neural cell adhesion molecule expression, p16 geneexpression, and the 3-dimensional reconstruction of the bile duct cancer specimens. The pathogeneticrelationship of HBV and HCV with extrahepatic cholangiocarcinoma was evaluated by histochemical and IS-PCR methods. And an inquiry into the possibility of gene therapy was made.CONCLUSION Hilar bile duct cancer rarely runs a “benign” course. It is a regional disease rather than alocal affection and may be related to HBV and HCV infection in China. It possesses the metastasing abilityalong the perineural space by a “jumping” fashion, therefore, in most cases, conventional surgical excision isbound to be unradical in the region of the porta hepatis for anatomical reasons.
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Gene therapy for cardiovascular diseases has developed from preliminary animal experiments to clinical trials. However, vectors and target genes used currently in gene therapy are mainly focused on viral, nonviral vector and single target gene or monogene. Each vector system has a series of advantages and limitations. Chimeric vectors which combine the advantages of viral and nonviral vector,chimeric target genes which combine two or more target genes and novel gene delivery modes are being developed. In this article, we summarized the progress in chimeric vectors and chimeric genes based cardiovascular gene therapy, which including proliferative or occlusive vascular diseases such as atheroslerosis and restenosis, hypertonic vascular disease such as hypertension and cardiac diseases such as myocardium ischemia, dilated cardiomyopathy and heart failure, even heart transplantation. The development of chimeric vector, chimeric gene and their cardiovascular gene therapy is promising.
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心律失常的分子机制和基因治疗研究进展
心律失常是全世界非常关注的重大公共卫生问题,也是我国重大疾病防治领域亟待解决的课题.国内外学者在药物、射频消融术、心脏起搏器、植入型心律转复除颤器(ICD)等心律失常治疗领域不懈努力,取得瞩目的成就.而基因治疗是当今生物医学发展的一个重要里程碑.它不仅是早期干预心脏病的新手段,也是当今"再生医学"研究领域的重要组成部分.基会项目:国家自然科学基金(30760079);新疆维吾尔自治区自然科学基金(2009211B18)
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干细胞移植与转基因治疗建立生物起搏器的实验研究进展
近来的研究表明,基因治疗和干细胞移植修复可以治疗多种心脏疾患,其中包括重建心脏生物起搏器(biological pacemaker)[1].因此,基因与干细胞移植治疗技术可能为缓慢心律失常的治愈带来了希望.
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Objective:In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by 60 Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34+ cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results:The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34+ cells increased by 173. 09±11.58×103/ml, which was significantly higher than that of non-radiation group(68. 04± 13. 73 × 103/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34+cells from human umbilical cord blood were one after the other transplanted i. v. into sublethally irradiated severe combined immunodeficient (SCID) mice. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, while no significant differences were found for CD45+ 、CD34+ cells in bone marrow cells. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, without significant differences being found for CD45+、CD34+ 、CFU-GM and marrow nucleared cells in bone marrow cells. Conclusions:The results suggested both in vivo and in vitro use of the gene therapy of FL gene regulated by Egr-1 promoter could protect hematopoiesis from irradiation-induced damage.
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胰腺癌基因治疗研究进展
胰腺癌恶性程度高,症状隐匿,绝大多数患者在确诊时已是晚期,不能手术完整切除,病程较短,其生存期一般不到1年.据统计全球胰腺癌的发病率和死亡率几乎相同,对化疗和放疗均不敏感,仅占20%的可手术切除的胰腺癌中,有70%~80%在手术时已发生淋巴洁转移.
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Our previous studies have revealed that amyloidβ(Aβ)-binding alcohol dehydrogenase (ABAD) decoy peptide antagonizes Aβ42-induced neurotoxicity. However, whether it improves oxidative stress injury remains unclear. In this study, a recombinant adenovirus constitutively secreting and expressing Aβ-ABAD decoy peptide (rAAV/ABAD-DP-6His) was successfully constructed. Our results showed that rAAV/ABAD-DP-6His increased superoxide dismutase activity in hydro-gen peroxide-induced oxidative stress-mediated injury of PC12 cells. Moreover, rAAV/ABAD-DP-6His decreased malondialdehyde content, intracellular Ca2+concentration, and the level of reactive oxygen species. rAAV/ABAD-DP-6His maintained the stability of the mitochondrial membrane potential. In addition, the ATP level remained constant, and apoptosis was reduced. Overall, the results indicate that rAAV/ABAD-DP-6His generates the fusion peptide, Aβ-ABAD decoy peptide, which effectively protects PC12 cells from oxidative stress injury induced by hy-drogen peroxide, thus exerting neuroprotective effects.
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3β-Hydroxysteroid-Δ24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloidβdeposition. hTe present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human (h) and rat (r). Recombinant Ad-r(h)SYN1-DHCR24 was transfected into AD-293, N2A (mouse neuroblastoma), and MIN6 (mouse pancreatic carcinoma) cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no speciifc band was found in MIN6 cells. hTis demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunolfu-orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our ifndings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer’s disease.
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聚酰胺-胺型纳米载体在基因治疗中的应用
高效、安全和靶向的载体系统是基因治疗成功的关键.病毒载体系统因毒性、免疫原性等安全隐患,其临床应用受到很大限制;而非病毒载体系统转染效率一直不如前者.近年来出现的纳米微粒基因转移技术有可能突破这一窘境,使基因治疗成为临床常规治疗手段.
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纳米基因载体及其在内耳基因治疗中的研究进展
基因治疗是借助载体把目的基因高效且安全地输送到特定的病变细胞或组织中,从而进行疾病治疗的一种方法.内耳疾病在耳鼻喉科的发病率较高,严重影响人们的健康和生活质量,且目前尚无确切的治疗方法.随着分子生物学的发展,内耳基因治疗研究为攻克这一顽症带来希望.但目前阻碍内耳基因治疗的一大难题就是缺乏理想的基因载体[1].
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1 Gene therapyGene therapy includes the treatment of both genetically based and infectious diseases by introducing genetic materials which have therapeutic effects[1~3]. In its simplest terms, a wild type gene (which is non-functional in the cell leading to disease development) is introduced into the somatic cell lacking this gene to restore the normal gene function in this cell. Many gene therapy strategies, however, utilize genes to destroy specific cells.
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脑血管疾病的干细胞移植和基因治疗
脑血管疾病是神经科常见的疾病之一,我国每年新发病例在180万例以上,对其治疗药物和手段虽已进行过大量研究,但仍然存在许多尚未解决的问题.其中20%~30%留有不可逆的神经功能缺损,导致家庭和社会的严重负担.基于组织工程和基因工程技术发展起来的细胞(或组织)替代治疗和基因治疗是近年来医学领域乃至整个生命科学领域中的研究热点和前沿学科,为人类征服脑血管疾病提供了新的途径和希望.
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冠心病基因治疗研究现状与前景
冠心病(coronary heart disease)又称缺血性心脏病,是一种严重危害人类健康的疾病,是由于冠状动脉粥样硬化造成血管痉挛,从而引起血管腔狭窄或阻塞,发生冠脉循环障碍,造成心肌缺血、缺氧甚至坏死的一种常见心脏病.
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肝癌的分子生物学诊断与基因治疗的现状与存在的问题
全球范围内因患原发性肝细胞癌(HCC)年死亡人数约45万~100万,位居恶性肿瘤死因的第三或四位.