腺病毒、腺相关病毒载体转导心肌细胞的研究

目的和方法：构建含人β2肾上腺素能受体（β2－AR）基因的重组腺病毒(rAd) 和腺相关病毒（rAAV),并感染体外培养的心肌细胞,检测目的基因在心肌细胞内的表达。结果：RT－PCR检测结果显示感染的心肌细胞均表达人β2-ARmRNA,western印迹杂交显示感染的心肌细胞可表达人β2－AR蛋白;放射性配基检测表明两种重组病毒感染的心肌细胞的β－AR密度无明显差异（P＞0．05）,但均高于对照组（P＜0．01）。结论：腺相关病毒（AAV）载体与腺病毒载体均中有效的转染心肌细胞并使目的基因表达。     

神经生长因子家族及其受体研究进展

过去几年在神经营养因子、受体和神经元细胞程序性死亡的研究领域中取得了几项引人注目的进展：（１）神经生长因子（ＮＧＦ）基因家族的其他一些成员包括脑源性神经营养因子（ＢＤＮＦ）、神经营养素－３（ＮＴ－３）、神经营养素－４（ＮＴ－４）、神经营养素－５（ＮＴ－５）的发现;（２）神经生长因子三维结构及功能和进化之关系的阐明;（３）定性了两种神经生长因子受体Ｐ７５＾ＮＧＦＲ和原癌基因ｐ１４０＾ｔｒｋＡ以及相关     

PDX-1腺病毒载体构建及其在脂肪间充质干细胞中的表达

研究通过重组腺病毒表达系统包装重组腺病毒Ad—PDX-1,通过重组腺病毒将Pancreaticduodenalhomeoboxl（PDX-1）导入大鼠脂肪间充质干细胞（ratadiposemesenchymalstemcells,rASCs）中,评价PDX．1在rASCs中的表达情况,为进一步探讨ASCs．PDX-1在体内修复糖尿病的潜能提供了实验基础。研究通过构建pAdEasy-CMV—PDX-1腺病毒质粒,在重组腺病毒表达系统PadEasy-1中成功包装出重组腺病毒Ad-PDX-1。培养及鉴定大鼠脂肪间充质干细胞,将携带PDX-1的重组腺病毒感染rASCs,通过RT-PCR、免疫荧光及免疫印迹（Westernblotting）检测PDX-1在rASCs中的表达及产物定位。结果显示,构建的重组腺病毒包装成功,具有较高滴度,并能成功感染大鼠脂肪间充质干细胞。PDX．1蛋白定位于细胞核,并可稳定表达,为后续将大鼠脂肪间充质干细胞向胰岛B细胞诱导分化奠定了基础,为糖尿病的干细胞治疗提供依据。     

腺病毒载体介导的肝癌细胞专一性自杀基因表达

构建由肝癌细胞专一的ａｆｐ基因表达调节元件控制自杀基因ＨＳＶ－ｔｋ的穿梭质粒,将它与缺陷型腺病毒载体重组,得到ＡｄｒＡＦＰＴＫ病毒。经ＰＣＲ及Ｓｏｕｔｈｅｒｎ杂交等证实它们含ａｆｐ元件和ｔｋ基因。空斑形成试验表明病毒效价达１×１０１５ｐｆｕ／Ｌ。同时构建由ＣＭＶ启动子控制ｔｋ基因的类似载体作为对照。将这两个重组腺病毒分别感染ＡＦＰ阳性（ＨｅｐＧ２）或阴性（ＨｅＬａ,ＢＲＬ－３Ａ）细胞株（ｍ．ｏ．ｉ．＝１００）,以丙氧鸟苷（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）处理后,用ＭＴＴ法测定杀伤细胞的效应。结果,ＡｄＣＭＶＴＫ感染这三种细胞后,ＧＣＶ半杀伤浓度分别为１．３、２、＜１μｍｏｌ／Ｌ;但是,ＡｄｒＡＦＰＴＫ感染的ＨｅＬａ和ＢＲＬ－３Ａ细胞的ＧＣＶ半杀伤浓度都＞１０００μｍｏｌ／Ｌ,而对ＨｅｐＧ２细胞只有＜１μｍｏｌ／Ｌ,表现出极高的细胞专一性。重组腺病毒ＡｄｒＡＦＰＴＫ可望用于肝癌的专一性基因治疗     

重组腺病毒介导的白细胞介素—12（IL—12）在肝癌细胞中的表达

利用腺病毒表达系统在肝癌细胞中成功地表达了有生物活性的白细胞介素－１２（ＩＬ－１２）。ＩＬ－１２的Ｐ３５和Ｐ４０ ｃＤＮＡ分别克隆到腺病毒载体ｐＡＣＣＭＶ．ｐＬｐＡ,构建ｐＡＣ／Ｐ３５和ｐＡＣ／Ｐ４０表面质粒。与腺病毒重组质粒ｐＪＭ１７共转染２９３细胞,通过基因重组产生ＩＬ－１２Ｐ３５和Ｐ４０重组腺病毒。用重组腺病毒感染肝癌细胞株ＨｅｐＧ２和ＳＭＭＣ７７２１,经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ检测证明,     

含HIV-1 gag、gagV3基因的重组腺病毒伴随病毒的构建及其免疫原性的研究

将HIV－1中国株42（B亚型）gag基因及gag与gp120 V3区的嵌合基因gag V3插入腺病毒伴随病毒（AAV）表达载体（pSNAV）质粒中,构建重组质粒pSNAV-gag及pSNAV-gagV3;采用脂质体转染的方法分别将重组质粒转入BHK细胞,G418筛选得到转入重组质粒并能表达外源基因的细胞系,命名为BHK－gag及BHK－gagV3。用具有重组腺病毒伴随病毒（rAAV）包装功能的一种重组单纯疱疹病毒（rHSV）分析感染这两株细胞系,纯化后得到rAAV,电镜观察可见到大量实心病毒颗粒,核酸杂交检测重组病毒滴度达到10^12病毒颗粒／ml,重组病毒感染293细胞,ELISA检测有gag及gagV3基因的表达。用重组病毒免疫Balb/C小鼠,检测抗体及细胞免疫水平,证明重组病毒可以在小鼠体内诱导产生细胞及体液免疫。     

睫状神经营养因子对体外培养骨骼肌细胞的伸增殖效应

目的：探讨睫状神经营养因子（ＣＮＴＦ）对骨骼肌细胞的直接营养作用,从而为神经肌肉系统损伤和进行性病变的治疗提供新的思路。结果：ＣＮＴＦ可以促进体外培养的Ｌ６－ＴＧ肌母细胞和新生ＳＤ大鼠原代骨骼肌细胞增殖。结论：ＣＮＴＦ对体外骨骼肌细胞具有营养作用。ＣＮＴＦ的神经和肌肉双重营养性能在神经肌肉损伤和退行性为的治疗上发挥重要作用。     

表达神经营养因子Neurturin的骨髓间充质干细胞的构建及体外活性检测

研究神经营养因子Neurturin(NTN)在由于神经元损伤而造成的神经退行性疾病中对神经元的保护和修复作用。利用重组腺病毒载体将NTN基因转入恒河猴骨髓间充质干细胞(rMSC),通过RT-PCR、IF及Western blot方法检测NTN的转录和表达,并采用鸡胚背根神经节体外培养实验和胚胎大鼠中脑多巴胺能神经元存活实验对NTN进行体外活性检测。结果表明NTN在rMSC中稳定表达和分泌,并具有体外生物学活性,为由于神经元损伤造成的神经退行性疾病的干细胞移植治疗奠定了一定的基础。     

特异性eIF-4EsiRNA重组腺病毒构建及其对人卵巢癌细胞SKOV-3转移相关能力的影响

目的：构建表达真核细胞起始因子-4E（eukaryoticinitiationfactor4E,eIF-4E）特异性siRNA的重组腺病毒载体,观察其对人卵巢癌细胞SKOV-3体外转移能力的影响。方法：应用基因重组技术将eIF．4EsiRNA序列构建于腺病毒载体pLP－Ade－X,经包装细胞包装后得到高滴度重组腺病毒pLP—Ade-4EsiRNA（psiE）。将腺病毒psiE感染SKOV．3细胞,用定量PCR进行elf．4E基因表达检测,然后应用transwell小室法观察对细胞侵袭和运动能力的影响,同时检测感染后细胞内VEGF、MMP－2、MMP－9蛋白表达。结果：病毒检测结果与预期相符,real—timePCR可检测到感染重组腺病毒psiE后SKOV．3细胞没有eIF-4E基因表达;病毒感染后transwell小室法检测到SKOV-3细胞的侵袭和运动能力均受到显著的抑制（均为P〈0．01）;此外病毒感染的SKOV．3细胞中VEGF、MMP-2、MMP-9蛋白表达降低。结论：封闭eIF-4E基因表达对人卵巢癌细胞SKOV．3的侵袭和运动都有抑制作用,其作用机制可能与VEGF、MMP-2、MMP-9表达相关。     

视黄酸核受体γ重组腺病毒构建及其鉴定

目的构建携带大鼠视黄酸核受体γ（retinoic acid receptor γ,RARγ）的重组腺病毒,为研究RAR3,在骨髓间充质干细胞（mesenchymal stem cells,MSCs）成神经分化中的作用奠定基础。方法体外扩增大鼠础研基因,将其定向克隆至腺病毒穿梭质粒pAd Trace—TOX构建重组质粒pAdTrace—RARγ,并在BJ5183菌中与骨架质粒pAd Easy-1重组获得腺病毒载体pad—RARγ,Pac I酶切后转染HEK293细胞包装腺病毒。腺病毒Ad—RARγ感染大鼠MSCs,Real—time PCR和Western印迹检测RARγ的表达。结果PCR,酶切及测序均证实础研正确克隆至腺病毒质粒载体中,Ad—RARγ对MSCs感染率达60％～70％,并明显增强础研基因和蛋白的表达。结论成功构建携带RARγ的重组腺病毒,并具有上调大鼠MSCsRA脚基因和蛋白表达的功能。     

腺病毒介导BDNF基因对无血清培养SH-SY5Y细胞的生物学作用研究

目的 :为了有效的将腺病毒介导的脑源性神经营养因子 (BDNF)用于神经损伤的保护治疗。方法 :在体外用BDNF重组腺病毒 (Ad BDNF)对SH SY5Y细胞进行感染 ,在无血清培养条件下对细胞的生长分化进行了形态学的观察 ,利用MTT法检测不同浓度的Ad BDNF对SH SY5Y细胞的促存活作用 ,并对细胞凋亡作用进行了检测。结果和结论 :腺病毒介导的BDNF可有效的促进感染后的SH SY5Y细胞的存活 ,生长和分化 ,并可有效的抑制无血清状态下细胞凋亡的发生     

含人纤溶酶原K5基因的腺病毒载体制备和功能研究

应用PCR将人纤溶酶原信号肽序列引入K5cDNA基因 ,与真核表达载体pcDNA3重组 ,形成重组质粒pcDNA3K5 ,与穿梭质粒pShuttle重组得pShuttleK5 ,经与腺病毒DNA重组 ,PCR鉴定正确 ,即为pAd K5。脂质体法将其转染 2 93细胞后 ,制备细胞裂解液 ;噬斑分析法测定病毒滴度为 5× 10 8pfu mL。将病毒以不同的感染系数 (MOI)感染人脐静脉内皮细胞株ECV30 4和人乳腺癌细胞株MDA MB 2 31,MTT法检测两者的增殖情况 :ECV30 4细胞增殖受抑制 ,而MDA MB 2 31细胞增殖未受明显影响。将感染病毒的ECV30 4细胞接种于ECMatrixTM胶 ,显示内皮细胞分化和毛细血管管腔形成受抑制。表明所构建的含人纤溶酶原K5基因的重组复制缺陷型腺病毒具有抑制ECV30 4细胞增殖、分化和管腔形成的作用而对MDA MB 2 31细胞的生长则无影响。     

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

Objective To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on vascular smooth muscle cells (VSMCs). Methods Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression of cytochrome c, procaspase-3, cleaved caspase-3, cleaved caspase-9 and inhibitor of apoptosis protein-1(IAP-1) were examined by western blot and RT-PCR. Results TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th day after gene transfer, the apoptotic rates in the TFPI group were 11.95%, 71.96% and 37.83%, respectively, whereas those in the LacZ group were 1.34%, 1.83% and 6.37%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. Cytochrome c, cleaved caspase-3 and cleaved caspase-9, which are all involved in mitochondrial pathway, were detected in the cytoplasm on the 3rd, 5th and 7th day after TFPI gene transfer. Procaspase-3 expression was significantly decreased over time in the TFPI group (each P < 0.05), which were not seen in the Ad-LacZ and DMEM groups. The expression of IAP-1 mRNA in the TFPI group was also decreased compared with the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd and 7th day after gene transfer. Conclusion The results demonstrated that overexpression of TFPI gene might induce VSMC apoptosis in vitro through the mitochondrial pathway; meanwhile, IAP-1 expression is decreased. These findings indicated that TFPI might inhibit restenosis by inducing apoptosis in VSMCs.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target-derived trophic factor for basilar pontine mossy fibers.     

Distinct effect of neurotrophins delivered simultaneously by an adenoviral vector on neurite outgrowth of neural precursor cells from different regions of the brain

For many years, it has been demonstrated that neurotrophins regulate the adult nervous system, implicating their potential as therapeutic agents for the treatment of neurodegenerative diseases. We generated adenoviral vectors encoding brain-derived neutotrophin factor (BDNF) and neurotrophin-3 (NT3) and tested either separately or together for the ability to induce differentiation of neuronal precursor cells with two different origins. Separate transduction of adenovirus delivering BDNF (BDNF-Ad) or NT3 (NT3-Ad) induced the neuronal differentiation in hippocampal and cortical precursor cells. NT3-Ad infected cells extended short neurites, whereas BDNFAd infected cells had longer neurites. In the early differentiation of hippocampal precursor cells, simultaneous infection of BDNFAd and NT3-Ad promoted further differentiation and neurite elongation compared with the separate infection of each virus. In contrast, simultaneous infection did not show the synergistic effect in the cortical precursor cells, suggesting that the neurotrophins play distinct roles in different regions of the brain. However, the numbers of neurites and spines per differentiated cells were markedly increased in cortical as well as hippocampal precursor cells, indicating the promotion of efficient neurite elongation and formation of dendritic spine, when BDNF-Ad and NT3-Ad were co-infected. These results suggest more studies in the effect of a combinatorial use of neurotrophins on different sites of brain need to be carried out to develop gene therapy protocols for neurodegenerative diseases.     

Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu significantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor specific immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3⁺CD8⁺ T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy.     

Adenovirus mediated overexpression of CYP2E1 increases sensitivity of HepG2 cells to acetaminophen induced cytotoxicity

To study the biochemical and toxicological properties of cytochrome P450 2E1 (CYP2E1), an adenovirus containing human CYP2E1 cDNA (Ad-CYP2E1) was constructed and was shown to successfully mediate the overexpression of CYP2E1 in HepG2 cells. Acetaminophen (APAP) toxicity to HepG2 cells infected with Ad-CYP2E1 was characterized as a preliminary proof of principle experiment to validate the functionality of the CYP2E1 adenovirus. Compared with cells infected with Ad-LacZ, HepG2 cells infected with Ad-CYP2E1 were more sensitive to APAP induced necrosis and apoptosis when the cells were depleted of intracellular reduced glutathione (GSH). The APAP cytotoxicity was dependent on both the concentration of APAP and the multiplicity of infection of the Ad-CYP2E1 virus. Apoptosis induced by APAP in HepG2 cells overexpressing CYP2E1 was caspase dependent and could be inhibited by the pan-caspase inhibitor Z-VAD-fmk. After treatment with APAP, mitochondrial membrane potential was dramatically decreased in the CYP2E1-expressing cells. APAP protein adducts were elevated in HepG2 cells infected with Ad-CYP2E1 compared with that in cells infected with Ad-LacZ; two bands around 90 KD were found only in the CYP2E1-expressing cells. These results demonstrate that adenovirus-mediated overexpression of human CYP2E1 activates APAP to reactive metabolites which damage mitochondria, form protein adducts, and result in toxicity to HepG2 cells. The Ad-CYP2E1 may be useful for studies designed to investigate the role of CYP2E1 in APAP and alcoholic liver injury and to further characterize the actions and effects of CYP2E1.     

腺病毒介导的NT3基因转染对噪音损伤的耳蜗螺旋神经节细胞的保护作用

神经营养素 3(neurotrophin 3,NT3)作为螺旋神经节细胞特异的营养因子 ,可有效地支持内耳传入神经元的存活 ,因此有望成为治疗因其退变而引起的感音性神经性耳聋的有效因子。实验采用腺病毒介导lacZ基因 ,检测了外源基因在豚鼠内耳中的长期表达。用噪音制备了豚鼠耳聋模型 ,在噪音损伤后第 7天 ,通过圆窗膜注入 1× 10 8重组腺病毒。注入神经营养素 3重组腺 (Ad NT3)的组为实验组 ,注入Ad lacZ的为对照组。 4周后 ,经NT3抗体免疫细胞化学染色可见 ,在注入Ad NT3病毒的实验组中 ,在内耳多种细胞中有明显的NT3蛋白的表达。HE染色显示 ,注射Ad lacZ组的豚鼠耳蜗螺旋神经节细胞明显退变 ,螺旋神经节内细胞间隙拉大 ,细胞密度明显低于注射Ad NT3实验组动物 (P <0 .0 1)。这一结果说明 ,腺病毒介导的NT3基因可长期表达于内耳中 ,并且可在噪音引起毛细胞死亡后有效地抑制螺旋神经节细胞的退变。     

Developmental changes in the responsiveness of rat spiral ganglion neurons to neurotrophic factors in dissociated culture: differential responses for survival, neuritogenesis and neuronal morphology

The way that the development of the inner ear innervation is regulated by various neurotrophic factors and/or their combinations at different postnatal developmental stages remains largely unclear. Moreover, survival and neuritogenesis in deafferented adult neurons is important for cochlear implant function. To address these issues, developmental changes in the responsiveness of postnatal rat spiral ganglion neurons (SGNs) to neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were examined by using a dissociated cell culture system. SGNs at postnatal day (P) 0, P5 and P20 (young adult) were cultured with the addition of NT-3, BDNF, or LIF or of a combination of NT-3 and BDNF (N + B) or of NT-3, BDNF and LIF (ALL factors). SGNs were analyzed for three parameters: survival, longest neurite length (LNL) and neuronal morphology. At P0, SGNs required exposure to N + B or ALL factors for enhanced survival and the ALL factors combination showed a synergistic effect much greater than the sum of the individual factors. At P5, SGNs responded to a wider range of treatment conditions for enhanced survival and combinations showed only an additive improvement over individual factors. The survival percentage of untreated SGNs was highest at P20 but combinations of neurotrophic factors were no more effective than individual factors. LNL of each SGN was enhanced by LIF alone or ALL factors at P0 and P5 but was suppressed by NT-3, BDNF and N + B at P5 in a dose-dependent manner. The LNL at P20 was enhanced by ALL factors and suppressed by N + B. Treatment with ALL factors increased the proportion of SGNs that had two or more primary neurites in all age groups. These findings suggest that NT-3, BDNF, LIF and their combinations predominantly support different ontogenetic events at different developmental stages in the innervation of the inner ear.     

Neurotrophic factor regulation of developing avian oculomotor neurons: Differential effects of BDNF and GDNF

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target‐derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain‐derived neurotrophic factor (BDNF), neurotrophin 3 (NT‐3), neurotrophin 4 (NT‐4), and glial cell line–derived neurotrophic factor (GDNF) may be target‐derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio‐iodinated BDNF, NT‐3, NT‐4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT‐3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT‐3, and NT‐4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75^NTR and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 295–315, 1999