腺病毒介导gax基因转染在培养的血管平滑肌细胞中的表达

目的以腺病毒介导gax基因转染血管平滑肌细胞(VSMC),增强VSMC中gax基因的表达.方法采用位置特异性重组方法构建携带大鼠gax基因表达序列的复制缺陷型5型腺病毒载体(AdCMV-gax),经293细胞扩增,纯化制备高滴度病毒转染液;以病毒转染液常规转染VSMC后,应用RT-PCR、流式细胞仪和免疫细胞化学等方法分别检测VSMC中gax mRNA和蛋白质的表达.结果流式细胞仪检测和免疫细胞化学染色均显示AdCMV-gax转染VSMC后,VSMC的Gax蛋白表达率明显增高,转染后24小时即可达80%左右,高水平的表达可维持5天以上; AdCMV-gax转染前,PDGF-BB对gax基因转录和翻译水平的表达均有下调作用,AdCMV-gax转染后,无论有无PDGF-BB刺激,VSMC中gax基因的表达均比转染前显著增高.结论腺病毒载体可有效地介导gax基因转染VSMC,并且表达为蛋白质.这有利于进一步研究gax基因对VSMC生物学行为的影响.     

gax基因转染对血管平滑肌细胞中基质金属蛋白酶2和骨桥蛋白转录的影响

目的:探讨腺病毒介导gax基因转染血管平滑肌细胞(VSMC)后,VSMC中基质金属蛋白酶2(MMP2)和骨桥蛋白基因转录的变化。方法:以携带大鼠gax基因表达序列的复制缺陷型5型腺病毒载体(AdCMV-gax)转染VSMC后,应用免疫细胞化学染色检NGax蛋白表达的变化,以RT-PCR技术检测VSMC中MMP2 mRNA和骨桥蛋白mRNA的变化。结果:AdCMV-gax转染后:(1)VSMC中Gax蛋白的表达比转染前显著增高(P<0.01); (2)VSMC中MMP2 mRNA和骨桥蛋白mRNA 比未转染组均显著降低(P<0.05)。结论:增强gax基因表达可抑制MMP2和骨桥蛋白的转录。     

腺病毒介导血管平滑肌细胞转染表达血管紧张素Ⅱ2型受体的研究

目的：体外培养大鼠血平滑肌细胞（VSMC）并以腺病毒介导转染表达血管紧张素Ⅱ（AngⅡ）2型受体（AT2R）。方法：取大鼠主动脉血管,以常规组织块贴壁法培养VSMC;用同源重组方法构建带AT2R基因的复制缺陷型腺病毒载体（AdCMV-AT2R),并加以鉴定,扩增,以制取高滴度AdCMV-AT2R转染液,体外转染,VSMC,;用RT－PCR方法检测AT2RmRNA表达,免疫组织化学法及蛋白免疫印迹法检测AT2R蛋白表达,流式细胞仪检测AT2R表达率,同时检测AT1R的表达变化。结果：构建的AdCMV-AT2R体外转染培养VSMC表达率为89．51％,以免疫组化、免疫印迹和RT－PCR检测AT2R表明,转染后AT2R表达明显增强,并随表达时间延长而增加,48小时达峰值,AT2R转染表达不影响AT1R表达。结论：腺病毒载体可较高效率介导AT2R在体外培养的VSMC转染表达,AT1R表达不受其影响,转表达AT2R的SMC可作研究AngⅡ对其增殖、迁移以及凋亡等生物学行为影响的良好细胞模型。     

腺病毒介导钙离子ATP酶基因在骨髓间充质干细胞的表达

目的：以腺病毒（Ad—CMV）介导钙离子ATP酶基因（sarcoplasmic reticulum Ca^2＋ ATPase，SERCa2a）转染骨髓间充质干细胞（Bone marrow msenchymal stem cells，BMSCs），测定表达产物，为心力衰竭的细胞移植和基因治疗提供实验基础。方法：体外培养大鼠骨髓间充质干细胞，用已构建好的Ad5-CMVSERCa2a真核载体进行转染。通过流式细胞仪检测转染效率。逆转录-聚合酶链反应（RT-PCR）、Westem杂交等方法检测SERCa2amRNA和蛋白质的表达。结果：Ad5-CMV—SERCa2a对BMC的转染率为80％左右，没有明显的细胞毒性反应。RT-PCR检测到SERCa2a，Western杂交确定SERCa2a蛋白表达。结论：腺病毒载体可有效地介导SERCa2a基因转染BMC，并表达SERCa2a蛋白，为基因治疗心力衰竭的研究奠定了基础。     

慢病毒载体介导绿色荧光蛋白基因转染血管内皮祖细胞的实验研究

目的探讨HIV-1来源的慢病毒载体介导绿色荧光蛋白（GFP）基因转染血管内皮祖细胞（EPCs）的可行性和方法。方法用梯度密度离心法分离人脐带血内皮祖细胞,在EGM-2培养基中培养。用细胞免疫荧光染色和流式细胞仪检测其表达情况。以HIV-1来源的慢病毒为载体、以GFP基因为目的基因转染EPCs,MTT法检测不同病毒滴度（MOI）时细胞增殖情况并观察转染率。结果单个核细胞经EGM-2培养基培养1周后即分化成EPCs。GFP转染后48 h细胞即发出绿色荧光。MOI 1∶10转染组细胞转染率低于MOI 1∶50组（P〈0.05）。MOI 1∶50转染后的细胞与未转染GFP组比较,生长曲线无明显差异（P〉0.05）。MOI 1∶100组转染后细胞的增殖处于停滞状态。结论采用HIV-1来源的慢病毒载体介导GFP基因转染标记EPCs是可行的。以MOI 1∶50进行转染对细胞生长影响小,转染效率高。     

Ap-1基因RNA干扰对大鼠血管平滑肌细胞增殖的影响

目的探讨Ap-1基因RNA干扰抑制血管平滑肌细胞（VSMCs）基因对VSMCs增殖的影响。方法以Ap-1基因RNA干扰VSMCs基因,应用MTr比色试验检测VSMCs增殖情况,流式细胞仪检测VSMCs细胞周期,免疫细胞化学染色观察增殖细胞核抗原（PCNA）表达。结果siRNA转染后,3个阳性siRNA转染组Ap-1基因表达水平均降低;VSMCs的MTY吸光度值和PCNA表达量均降低;VSMCs出现明显的G0／G1期阻滞。结论RNA干扰介导的Ap-1基因沉寂可显著抑制VSMCs增殖。     

大鼠microRNA-145慢病毒表达载体的构建及其对血管平滑肌细胞表型转化的影响

目的构建针对Rno-miR-145的慢病毒表达载体并探讨其在血小板源生长因子(PDGF)诱导的血管平滑肌细胞(VSMC)表型转化中的作用。方法人工合成含有酶切位点粘端miR-145 shDNA双链模板序列,克隆于LV3 pGLV/H1/GFP+Puro-miRNA慢病毒穿梭载体中,转染293T细胞,收获并浓缩慢病毒颗粒,感染大鼠原代VSMC,倒置荧光显微镜下观察VSMC感染后的荧光表达情况,实时荧光定量PCR检测miR-145的表达情况;实验分为空白对照组、PDGF组、PDGF+miR-145组和细胞转染阴性慢病毒载体组(miR-NC组);采用实时荧光定量PCR测定miR-145对VSMC增殖相关基因PCNA、c-Jun及分化相关基因SM22αmRNA表达水平的影响。结果成功构建了microRNA-145慢病毒载体,测定病毒滴度为1×109TU/mL。倒置荧光显微镜下观察大鼠microRNA-145慢病毒表达载体感染成功,MOI值为50,感染72 h时感染率最高。实时荧光定量PCR结果显示PDGF可使PCNA、c-Jun表达增加,而使SM22α表达降低;miR-145可使PDGF诱导的去分化型VSMC增殖相关基因PCNA、c-Jun表达降低,分化相关基因SM22α表达增加。结论 miR-145慢病毒载体可高效感染大鼠原代VSMC。感染miR-145慢病毒后可抑制VSMC的表型转化。     

血管紧张素Ⅱ2型受体转染表达对血管平滑肌细胞增殖及迁移的影响

目的　探讨血管平滑肌细胞 (VSMC)转染表达血管紧张素Ⅱ (AngⅡ ) 2型受体 (AT2 R)对其增殖和迁移的影响。方法　构建带AT2 R基因的重组复制缺陷型腺病毒载体 (AdCMV AT2 R) ,体外转染大鼠主动脉VSMC ,用RT PCR方法检测AT2 RmRNA表达 ,流式细胞仪检测AT2 R表达率 ,用细胞周期、分裂指数、MTT比色法和 5 溴尿苷(BrdU)掺入法检测VSMC增殖。VSMC迁移用改良Boyden‘s趋化小室法检测 ,用激光共聚焦显微镜检测细胞骨架蛋白F actin的表达。结果　构建的AdCMV AT2 R体外转染培养VSMC表达率为 89.5 1%。AT2 R峰值表达时 ,其S期和G2 M期细胞比率从 31.7%降低到 13.9% (P <0 0 5 ) ,MTT吸光度和BrdU掺入量分别降低 6 1.4%和 5 1.6 %(P <0 0 (1)。VSMC的跨膜迁移数降低 6 2 .2 % ,F actin表达明显减少。结论　AT2 R转染表达可显著抑制体外培养VSMC的增殖和迁移 ,这一作用对再狭窄防治是有益的。     

转铁蛋白受体介导的Bcl-2shRNA转移对HL-60细胞生长的抑制作用

目的 探讨Bcl一2短发夹状RNA（shRNA）表达载体对白血病细胞HL-60生长的抑制作用。方法 采用转铁蛋白-多聚乙烯亚胺介导的转染方法将携带绿色荧光蛋白基因的重组Bcl-2 shRNA1、shRNA2表达载体转入HL-60细胞。通过荧光显微镜和流式细胞仪观察绿色荧光蛋白的表达;采用台盼蓝拒染法计数活细胞;用免疫细胞化学方法检测Bcl-2蛋白表达水平。结果 Bcl-2 shRNA1、shRNA2载体分别转入HL-60细胞后,细胞中Bcl-2蛋白表达水平均显著降低（P〈0．05）,转染后48、72、96h细胞生长明显受到抑制。结论 转铁蛋白受体介导的Bcl-2 shRNA可显著抑制HL-60细胞生长。     

Smac基因过表达对白血病耐药细胞株K562/AO2化疗敏感性的影响

目的 探讨Smac基因过表达对K562/A02细胞株化疗敏感性的影响.方法 构建重组腺病毒载体pAdeno-Smac,转染K562/A02细胞.实验分组:A组:pAdeno-Smac转染组;B组:空载体转染对照组;C组:正常对照组.Western blot检测转染前后细胞内Smac蛋白的表达水平.将终浓度分别为0、0.5、1.0、1.5 mg/L的柔红霉素处理各组后,Annexin V/PI双染法经流式细胞仪检测各组细胞早期凋亡率.结果 成功构建腺病毒载体pAdeno-Smac质粒.Western blot结果证实,经Smac基因全长cDNA转染后,K562/A02细胞中的Smac蛋白表达显著升高.经不同浓度柔红霉素处理后,A组的细胞早期凋亡率明显高于B组和C组,组间比较差异有统计学意义(P＜0.05),且随着柔红霉素浓度的升高,细胞凋亡率随之升高,B组和C组之间差异无统计学意义.结论 重组腺病毒载体pAdeno-Smac可通过使K562/A02细胞中的Smac蛋白表达增高,增强细胞对化疗药物的敏感性.     

外源性P~(53)和Rb基因对血管平滑肌细胞迁移的影响

为研究外源性Ｐ５３和Ｒｂ基因对血管平滑肌细胞迁移的影响,体外培养人脐动脉平滑肌细胞,将Ｐ５３基因和Ｒｂ基因重组腺病毒载体转染平滑肌细胞７２ｈ后,检测平滑肌细胞的迁移。结果显示,对照组平滑肌细胞的迁移距离为０．８４±０．３６ｃｍ（Ｘ±Ｓ）,而Ｐ５３基因转染组和Ｒｂ基因转染组的细胞迁移距离分别为０．５３±０．４５ｃｍ和０．３７±０．２９ｃｍ,显著低于对照组（Ｐ＜０．０５,Ｐ＜０．００１）。提示外源性Ｐ５３基因和Ｒｂ基因导入可显著抑制血管平滑肌细胞迁移     

黏着斑激酶对血小板源性生长因子刺激的血管平滑肌细胞迁移和黏附的调控

目的探讨黏着斑激酶(FAK)信号分子在雷帕霉素抑制血小板源性生长因子(PDGF)诱导血管平滑肌细胞(VSMC)迁移、黏附中的调控作用。方法将培养的大鼠VSMC分为对照组、PDGF组、雷帕霉素+PDGF组(雷帕霉素组)和FAK反义寡核苷酸+PDGF组(FAK组)。用PDGF诱导VSMC的迁移和黏附,计数贴壁细胞;采用Boyden检测细胞迁移;采用RT-PCR、Western blot、免疫沉淀方法分别检测FAK基因、蛋白及蛋白磷酸化表达量。将FAK反义寡核苷酸经脂质体转染VSMC,观察FAK mRNA及蛋白磷酸化、细胞迁移和黏附的变化。结果与对照组比较,PDGF组明显诱导细胞迁移和黏附,上调FAK mRNA的表达,提高FAK蛋白和磷酸化FAK表达,差异有统计学意义(P0.05),与PDGF组比较,FAK组和雷帕霉素组细胞迁移、黏附能力减弱,FAK mRNA、FAK蛋白和磷酸化FAK表达水平明显降低,差异有统计学意义(P0.05)。结论 PDGF诱导细胞迁移和黏附可能是FAK介导的,雷帕霉素可能是通过抑制FAK蛋白和磷酸化FAK来抑制VSMC的迁移和黏附。     

Kallistatin stimulates vascular smooth muscle cell proliferation and migration in vitro and neointima formation in balloon-injured rat artery

Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [(3)H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.     

JE mRNA accumulates rapidly in aortic injury and in platelet-derived growth factor-stimulated vascular smooth muscle cells.

The early response to vascular injury is characterized by migration of inflammatory cells, including monocytes, and platelets to the damaged vessel wall. These inflammatory cells may serve as a source of growth factors and cytokines that stimulate vascular smooth muscle cell (VSMC) migration and proliferation associated with intimal hyperplasia. JE is a platelet-derived growth factor (PDGF)-inducible "early" gene that encodes a monocyte chemoattractant and, as such, could play an important role in inflammation. We now report that JE mRNA levels are increased in intact aorta after balloon injury. The time course of this increase, with maximal levels at 4 hours, is similar to that seen in PDGF-treated cultured rat aortic VSMCs. The accumulation of JE mRNA in cultured VSMCs is accompanied by a marked increase in the secretion of JE protein. The elevation of JE mRNA levels in VSMCs shows specificity for PDGF, because angiotensin II, alpha-thrombin, and epidermal growth factor fail to increase JE mRNA levels. In contrast to 3T3 fibroblasts, the accumulation of JE mRNA in VSMCs in response to PDGF is predominantly due to an increase in JE mRNA stability. The accumulation of JE mRNA in VSMCs stimulated by PDGF appears to occur via a novel pathway(s) independent of Ca2+ mobilization, Na(+)-H+ exchange, protein kinase C activation, or elevation in cAMP levels. These findings suggest that VSMCs may take part in the early inflammatory response after injury through the production of JE, a potent monocyte chemoattractant. Finally, our data suggest that JE may be a marker for PDGF-specific effects on VSMCs, both in vitro and in vivo. Thus, in addition to direct effects on VSMC growth and migration, PDGF may play a role in the early inflammatory response after vascular injury by inducing chemoattractants, such as that encoded by JE.     

水蛭素对凝血酶诱导的血管平滑肌细胞增殖的影响

为研究重组水蛭素对凝血酶诱导的兔胸主动脉血管平滑肌细胞增殖的影响及其作用机制.选用第2～5代培养的血管平滑肌细胞,在与凝血酶(4.0 ku/L)共同孵育的条件下,分别给予水蛭素(6.0 ku/L)和肝素(6.O ku/L)进行干预.通过四唑盐比色实验检测细胞生长活性,流式细胞术检测细胞周期,免疫组织化学检测血小板生长因子和增殖细胞核抗原在血管平滑肌细胞中的表达.结果发现,重组水蛭素能明显抑制凝血酶诱导的平滑肌细胞增殖,其抑制作用可能与减低了血管平滑肌细胞对血小板生长因子和增殖细胞核抗原的表达有关.     

野生型P53基因诱导血管平滑肌细胞P21基因表达

为研究野生型P53基因导入诱导血管平滑肌细胞P21基因的表达，探讨P53基因调节细胞周期进程的作用机理，体外培养了人脐动脉平滑肌细胞。将野生型P53基因导入细胞后，应用逆转录－聚合酶链反应半定量测定P21mRNA水平，以免疫组织化学法观察P21蛋白表达的变化，并用流式细胞术分析细胞周期。结果发现，正常生长的血管平滑肌细胞中P21mRNA水平较低，用免疫组织化学法检测不到P21蛋白。野生型P53基因导入并在平滑肌细胞中表达后，显著增加了P21mRNA水平，在免疫组织化学检测中呈现很强的阳性显色反应、引起平滑肌细胞停滞在G0／G1期。以上结果提示，野生型P53基因通过诱导P21基因表达调控血管平滑肌细胞周期。     

Phosphoinositide-dependent kinase 1 and p21-activated protein kinase mediate reactive oxygen species-dependent regulation of platelet-derived growth factor-induced smooth muscle cell migration

Smooth muscle cell migration in response to platelet-derived growth factor (PDGF) is a key event in several vascular pathologies, including atherosclerosis and restenosis. PDGF increases intracellular levels of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs), but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. In VSMCs, PDGF dose-dependently increased migration compared with nonstimulated cells, with a maximum increase at 10 ng/mL. Pretreatment with the antioxidant N-acetyl-cysteine, the flavin-containing enzyme inhibitor diphenylene iodonium, or the glutathione peroxidase mimetic ebselen significantly attenuated migration (PDGF alone, 5.0+/-1.1-fold; NAC, 1.8+/-0.2-fold; diphenylene iodonium, 1.4+/-0.3-fold migration; and ebselen, 2.0+/-0.5-fold migration), as did overexpression of catalase. Pretreatment of VSMCs with the Src inhibitor PP1 or dominant-negative Rac adenovirus significantly inhibited migration, but only Src activation was attenuated by ROS inhibitors. Phosphorylation of the Src- and Rac-effector p21-activated protein kinase (PAK) 1 on Thr423 (the phosphoinositide-dependent kinase-1 [PDK1] site) was attenuated by ROS inhibition, and infection of VSMCs with dominant-negative PAK1 adenovirus attenuated migration. Moreover, kinase-inactive K111N-PDK1 inhibited PAK1 phosphorylation on Thr423, and both K111N-PDK1 and Y9F-PDK1 significantly inhibited VSMC migration. PDK1 tyrosine phosphorylation was also ROS dependent. These data indicate that PDGF-induced VSMC migration is ROS dependent and identify the Src/PDK1/PAK1 signaling pathway as an important ROS-sensitive mediator of migration. Such information is critical to understanding the role of ROS in vascular diseases in which migration of VSMCs is an important component.     

Hammerhead ribozyme targeting human platelet-derived growth factor A-chain mRNA inhibited the proliferation of human vascular smooth muscle cells

Platelet-derived growth factor (PDGF) A-chain contributes to the pathogenesis of cardiovascular proliferative diseases, such as hypertensive vascular disease, atherosclerosis, and re-stenosis of an artery after angioplasty. To develop a ribozyme against human PDGF A-chain mRNA as a gene therapy for human arterial proliferative diseases, we designed and synthesized a 38-base hammerhead ribozyme to cleave human PDGF A-chain mRNA at the GUC sequence at nucleotide 591. In the presence of MgCl(2), synthetic hammerhead ribozyme to human PDGF A-chain mRNA cleaved the synthetic target RNA to two RNA fragments at a predicted size. Doses of 0.01-1.0 microM hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited angiotensin II (Ang II) and transforming growth factor (TGF)-beta(1)-induced DNA synthesis in vascular smooth muscle cells (VSMC) from human in a dose-dependent manner. One micromolor of hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited Ang II-induced PDGF A-chain mRNA and PDGF-AA protein expressions in VSMC from humans. These results indicate that the designed hammerhead ribozyme to human PDGF A-chain mRNA effectively inhibited growth of human VSMC by cleaving the PDGF A-chain mRNA and inhibiting the PDGF-AA protein expression in human VSMC. This suggests that the designed hammerhead ribozyme to PDGF A-chain mRNA is a feasible gene therapy for treating arterial proliferative diseases.