抗Fas锤头状核酶阻抑小鼠急性粒-单核白血病细胞免疫逃逸的研究

为了研究抗Fas锤头状核酶对T细胞Fas表达及其凋亡的影响和探讨增强供者淋巴细胞输注时移植物抗白血病（GVL）效应的新策略，构建可有效切割Fas mRNA的锤头状核酶真核质粒，用电穿孔法将其导入小鼠CTL细胞株CTLL-2之后，借助RT—PCR和Western blot检测其Fas的表达，同时检测转染前后其胱冬酶-3（Caspase-3）活性和凋亡（Annexin V—FITC法）的改变，并用MTT法检测空白对照组、空载体转染组及pU6-RZ596转染组CTLL-2细胞的增殖情况和体外杀伤小鼠急性粒-单核白血病细胞（WEHI-3）的活性。结果表明：构建的U6嵌合型锤头状核酶RZ596在细胞内能有效切割Fas，明显降低小鼠活化CTLL-2的Fas水平，与高表达Fas配体的WEHI-3孵育后，其存活率和体外杀伤WEHI-3活性明显高于对照组。结论：抗Fas核酶能显著降低小鼠活化CTLL-2的Fas表达，使其免于WEHI-3的膜Fas配体经Fas途径所致的凋亡，并提高CTL对小鼠急性粒-单核白血病细胞的杀伤力，从而阻抑小鼠急性粒-单核白血病细胞的免疫逃逸。     

Fas和FasL在Na+/H+交换器-1抑制诱导缺氧大鼠肺动脉平滑肌细胞凋亡中的作用

目的探讨Fas、FasL在Na^＋／H^＋交换器-1（NHE-1）抑制所诱导的缺氧大鼠肺动脉平滑叽细胞（PASMCs）凋亡中的作用。方法将转染NHE-1特异性核酶基因的大鼠PASMCs置于缺氧条件下（O2的体积分数低下1％）培养。缺氧培养2、6、12、24和48h后用原位末端标记法（TUNEL）检测细胞凋亡情况；半定量逆转录-聚合酶链反应（sqRT—PCR）疗法检测细胞内fas和fasL mRNA表达变化；免疫细胞化学法检测细胞内Fas和FasL蛋白表达变化。结果转染NHE-1特异性核酶基因的大鼠PASMCs在缺氧培养时，其细胞凋亡率随缺氧时间的延长而逐渐升高，但细胞内fas、fasL mRNA及Fas、FasL蛋白表达与对照组细胞比较差异均无显著性。结论Fas／FasL死亡通路可能不参与NHE-1抑制而诱导缺氧大鼠PASMCs凋亡的调控。     

切割bcr/abl核酶的逆转录病毒载体构建及其对K562细胞的影响

目的：研究ｂｃｒ／ａｂｌ融合基因在慢性粒细胞白血病（ＣＭＬ）中的作用以及探索ＣＭＬ基因治疗的可能性。方法：合成针对ｂｃｒ／ａｂｌ融合基因转录本ｂ３ａ２断裂点“锤头状”核酶的ｃＤＮＡ序列，定向克隆于逆转录病毒载体内，通过脂质体介导的ＤＮＡ转染法，将核酶基因导入Ｋ５６２细胞，并通过克隆分析法、流式细胞术（ＦＣＭ）、逆转录聚合酶链反应（ＲＴＰＣＲ）、ＤＮＡ电泳及电镜观察等方法检测核酶对Ｋ５６２细胞的影响。结果：①核酶转染Ｋ５６２细胞后４８小时克隆形成抑制率达８５％；②细胞内Ｐ２１０蛋白合成受到明显抑制；③ＲＴＰＣＲ半定量检测ｂｃｒ／ａｂｌｍＲＮＡ表达水平明显下降；④核酶处理组Ｋ５６２细胞发生凋亡，表现为：在ＦＣＭ图谱上可见明显的凋亡峰；ＤＮＡ电泳分析出现典型的梯状ＤＮＡ带；电镜观察呈现凋亡早期形态学改变。结论：核酶基因通过逆转录病毒载体导入Ｋ５６２细胞后可成功表达，使Ｋ５６２细胞ｂｃｒ／ａｂｌｍＲＮＡ及Ｐ２１０蛋白表达水平下降，抑制Ｋ５６２细胞增殖，同时诱导Ｋ５６２细胞发生凋亡。     

构建两种启动子调控的自杀基因介导的肝癌细胞治疗

目的将外源基因p Survivin-HSV-TK导入质粒PBI-CMV2中,将外源基因p AFP-HSV-TK导入质粒PBI-CMV2中,构建两种特异性表达的质粒,比较两种启动子调停的优越性。方法由阳离子脂质体lipofectamine2000TM,运载两种质粒转染肝癌细胞Hu H-7,流式细胞术测定转染效率,半定量PCR检测TK基因在肝癌细胞中的表达水平。转染肝癌细胞Hu H-7,联合更昔洛韦杀伤细胞,流式细胞术检测细胞凋亡率,CCK-8检测加药(GCV)后细胞的增殖情况。结果阳离子脂质体lipofectamine2000TM介导Survivin-TK-p BI-CMV2质粒转染的Hu H-7细胞的转染率与介导AFP-TK-p BI-CMV2质粒转染的Hu H-7细胞的转染率分别为(14.43±0.88)%和(12.51±1.74)%(P=0.38),无统计学差异。半定量PCR检测HSVtk在转染的Hu H-7细胞中有表达,且转染Survivin-TK-p BI-CMV2质粒的细胞中TK基因的表达更高(P=0.001),在未转染细胞内未检测到表达。细胞凋亡实验表明GCV对转染的Hu H-7细胞有显著的杀伤作用,且转染Survivin-TK-p BI-CMV2质粒的Hu H-7细胞凋亡作用更明显(P=0.001)。结论肝细胞靶向基因运输载体PBI-CMV2介导的由AFP和Survivin启动子调控的HSVtk/GCV自杀基因系统对Hu H-7肝癌细胞均具有显著杀伤作用,且Survivin启动子调控作用相对明显,这为肝癌的靶向基因治疗策略的发展提供了新思路。     

抗bcl—2核酶在HL—60细胞中的表达及促进其凋亡的研究

为消除白血病细胞中ｂｃｌ－２基因对细胞凋亡的抑制作用开辟白血病基因治疗的途径。将合成的针对ｂｃｌ－２ｍＲＮＡ的“锤头型”核酶基因定向克隆于真核表达本ＰＤＯＲ－ｎｅｏ，构ｐＤＯＲ－ＲＺ重组体。通过脂质体Ｌｉｐｏｆｅｃｔｉｎ介导的ＤＮＡ转染法，把ｐＤＯＲ－ＲＺ导入ＨＬ－细胞。     

苦参碱对人肺癌SPC-A-1细胞Bcl-2、Fas表达的影响

目的了解苦参碱（matrine）作用人肺癌细胞株SPC-A-1后其相关凋亡基因Fas、Bcl-2的变化。方法不同浓度苦参碱作用于体外培养人肺癌细胞株SPC-A-1,流式细胞术检测其凋亡相关基因Bcl-2、Fas蛋白表达的改变。结果不同浓度的苦参碱处理人肺癌SPC-A-1细胞后,流式细胞仪检测随着苦参碱浓度的升高,Bcl-2的表达量减少、Fas的表达量增加且呈剂量效应（P〈0．05）。结论苦参碱诱导人肺癌细胞株SPC-A-1细胞凋亡,其机制可能与降低Bcl-2、升高Fas等活性有关。     

HA-1树突状细胞核酸疫苗的构建及其特异性CTL的诱导

本研究以次要组织相容性抗原HA-1为靶点，构建HA-1树突状细胞核酸疫苗用于造血干细胞移植后抗白血病治疗。体外培养移植供者树突状细胞，用流式细胞术、混合淋巴细胞反应检测其免疫活性，通过电转法将HA-1基因转染树突状细胞，构建树突状细胞核酸疫苗。48小时后检测HA-1蛋白表达情况。将转染后的树突状细胞与同基因淋巴细胞共孵育诱导特异性CTL，应用LDH释放实验检测其体外杀伤活性。结果表明：经外周血单核细胞诱导的树突状细胞表达树突状细胞表型，能刺激同种淋巴细胞增殖。电转48小时后，Western blot可检测到HA-1蛋白表达。诱导的CTL体外杀伤活性高于对照组。结论：次要组织相容性抗原HA-1可作为造血干细胞移植后抗白血病治疗的靶点。     

Ly49A基因转染的淋巴细胞对H-2~d小鼠正常和肿瘤细胞杀伤活性的实验研究

目的　观察Ly49A基因转染的C5 7BL/ 6小鼠的淋巴细胞对BALB/c小鼠正常成纤维细胞和 4T1乳腺癌细胞杀伤能力的变化与差异。方法　构建逆转录病毒表达载体pLXSN Ly49A ,经由PA317包装细胞包装后转染C5 7BL/ 6小鼠淋巴细胞。流式细胞仪检测Ly49A受体在转染后淋巴细胞上的表达率。MTT法检测转染后淋巴细胞对BALB/c小鼠正常成纤维细胞和 4T1乳腺癌细胞的杀伤活性 ,以空载体转染和未转染的淋巴细胞作对照。结果　Ly49A基因转染的C5 7BL/ 6小鼠的淋巴细胞 2 4h后Ly49A受体表达率为 (4 6 .6 7± 0 .35 ) % ,空载体转染组为 (18.73± 0 .85 ) % ,未转染对照组为 (19.6 0± 0 .2 7) % ,其对BALB/c小鼠正常成纤维细胞的杀伤活性明显降低 (抑制率 2 2 %～ 2 5 % ) ,对 4T1乳腺癌细胞的杀伤活性无明显改变 (P >0 .0 5 )。结论　转染Ly49A的C5 7BL/ 6小鼠淋巴细胞对BALB/c小鼠正常细胞的杀伤作用明显降低 ,但仍保留了对肿瘤细胞的杀伤活性 ,为解决异基因骨髓移植后移植物抗宿主病提供了实验依据     

抗bcl－2核酶在HL－60细胞中的表达及促进其凋亡的研究

为消除白血病细胞中ｂｃｌ－２基因对细胞凋亡的抑制作用，开辟白血病基因治疗的途径。将合成的针对ｂｃｌ－２ｍＲＮＡ的“锤头型”核酶（Ｒｉｂｏｚｙｍｅ，ＲＺ）基因定向克隆于真核表达载体ｐＤＯＲ－ｎｅｏ，构成ｐＤＯＲ－ＲＺ重组体。通过脂质体Ｌｉｐｏｆｅｃｔｉｎ介导的ＤＮＡ转染法，把ｐＤＯＲ－ＲＺ导入ＨＬ－６０细胞。用Ｓｏｕｔｈ－ｅｒｎ印迹，ＲＮＡ斑点杂交法观察ＲＺ基因在ＨＬ－６０细胞内表达，并通过电镜、流式细胞仪（ＦＣＭ），光镜和ＤＮＡ电泳观察ＲＺ对ＨＬ－６０细胞的影响。结果：①ＲＺ在转染ＨＬ－６０后７２小时得以表达，细胞内ｂｃｌ－２蛋白合成受到抑制。②在ＦＣＭ图谱上可见到明显的凋亡峰，形态观察ＲＺ处理的ＨＬ－６０细胞呈典型的凋亡形态。③足叶乙甙（促凋亡）敏感性试验表明，与未转染的细胞和转染空载体ｐＤＯＲ－ｎｅｏ的细胞相比，转染ｐＤＯＲ－ＲＺ的细胞ＤＮＡ电泳易出现梯状条带。结果说明ＲＺ基因通过逆转录病毒表达载体导入ＨＬ－６０细胞后可成功地表达并抑制ＨＬ－６０细胞ｂｃｌ－２蛋白的合成，并促进ＨＬ－６０细胞凋亡     

RPB5介导蛋白与HBx蛋白共表达对肝癌HepG2细胞凋亡的影响

目的通过乙型肝炎病毒X蛋白(HBx)与RPB5介导蛋白(RMP)在肝癌Hep G2细胞中的共表达,探索RMP与HBx对肝癌细胞凋亡的影响。方法脂质体介导瞬时转染细胞;实时荧光定量RT-PCR和western blot分别检测RMP与HBx在肝癌Hep G2细胞中的表达量;流式细胞仪检测细胞凋亡率;逆转录PCR检测细胞凋亡相关基因Bax、Caspase3、P53、Bcl-2 mRNA相对表达水平,western blot检测凋亡相关蛋白Bax、Bcl-2的表达。结果与未经任何处理的Hep G2细胞比较,转染RMP质粒的稳定表达HBx的Hep G2细胞中RMP、HBx在基因及蛋白质水平表达均显著升高(P0.01);细胞凋亡率显著降低;促凋亡基因Bax、Caspase3和P53 mRNA相对表达水平均显著下调(P0.01),凋亡抑制基因Bcl-2 mRNA相对表达水平显著上调(P0.01);促凋亡蛋白Bax表达量明显降低,抗凋亡蛋白Bcl-2表达量明显升高。结论 RMP与HBx共表达可降低肝癌Hep G2细胞凋亡率,提示RMP和HBx可能在肝癌发生和发展过程中起着重要的作用。     

Dual Signaling of the Fas Receptor: Initiation of Both Apoptotic and Necrotic Cell Death Pathways

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, tetrapeptide inhibitors of caspase-1– and caspase-3–like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas–induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor κB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.     

Tumor necrosis factor-alpha and interleukin-1beta increase the Fas-mediated apoptosis of human osteoblasts.

Our recent work demonstrated functional Fas expression on human osteoblasts, and the histologic examination of the periarticular osteoporosis region in patients with rheumatoid arthritis (RA) showed apoptosis in osteoblasts. High concentrations of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IL-6--which are thought to increase bone resorption--have been determined in RA synovium. We investigated the effect of these cytokines on the Fas-mediated apoptosis of human osteoblasts. The human osteoblastic cell line MG63 and human primary osteoblast-like cells from bone biopsy specimens were used as human osteoblasts. Fas expression on these cells was examined by flow cytometry, and Fas-mediated apoptosis induced by anti-Fas immunoglobulin M (IgM) was determined by a chromium 51 release assay, the presence of cells with hypodiploid DNA, staining with Hoechst 33258 dye, and the detection of DNA fragmentation on agarose gel electrophoresis. The proliferation of osteoblasts was analyzed by a tritiated thymidine incorporation assay. Spontaneous apoptosis was not found on cultured osteoblasts. The apoptosis of human osteoblasts was not induced by TNF-alpha, IL-1beta, or IL-6 alone in the absence of anti-Fas IgM. In addition, proliferation of the cells was not affected by these cytokines. Fas was constitutively expressed on unstimulated osteoblasts, and treatment of these cells with IL-1beta or TNF-alpha significantly augmented Fas expression. Human osteoblasts were committed to apoptosis with anti-Fas IgM, and the treatment of both IL-1beta and TNF-alpha markedly increased Fas-mediated apoptosis. TNF-alpha augmented both Fas expression and Fas-mediated apoptosis more efficiently than did IL-1beta. In addition, an additive effect on both Fas expression and Fas-mediated apoptosis was demonstrated when TNF-alpha and IL-1beta were added to osteoblasts. IL-6 influenced neither Fas expression nor the Fas-mediated apoptosis of osteoblasts. Furthermore, no synergistic effect of IL-6 with IL-1beta or TNF-alpha was observed. IL-1beta, TNF-alpha, or IL-6 did not change Bcl-2 expression. Our results suggest that IL-1beta and TNF-alpha regulate osteoblast cell number by up-regulating the Fas-mediated apoptosis of osteoblasts, one of the putative mechanisms inducing periarticular osteoporosis in patients with RA.     

Lack of cell surface Fas/APO-1 expression in pulmonary adenocarcinomas.

The Fas receptor and ligand initiate an apoptotic pathway. Alterations in this pathway within tumor cells can result in escape from apoptosis and immune surveillance. We evaluated Fas protein expression in 42 primary pulmonary adenocarcinomas, and Fas expression and function in the lung adenocarcinoma cell lines A549 and A427. Immunohistochemical analysis demonstrated Fas protein expression in 47.6% of the tumors; however, Fas-positive tumors demonstrated cytoplasmic staining without cell surface expression. Northern blot analysis indicated that levels of Fas mRNA were similar in Fas protein-positive tumors to levels in normal lung tissue, but were reduced in Fas protein-negative tumors. Soluble form Fas was not detected in the majority of these tumors either by RT-PCR or Western blot analysis. Cell surface Fas protein expression was minimal in A549 and A427 cell lines as determined by flow cytometry. Both cell lines demonstrated Fas mRNA expression by Northern blot analysis and abundant protein expression by Western blot analysis. Transfection of the Fas cDNA derived from A549 cells induced surface Fas protein in COS cells; however, stable transfection of a native Fas cDNA into A549 cells failed to induce surface Fas protein expression. Parental A549 cells and A549 cells transfected with a Fas expression vector were resistant to Fas-mediated apoptosis. Transgenic expression of a FLAG-tagged Fas cDNA in A549 cells, with visualization of the Fas-FLAG protein using confocal microscopy, demonstrated that the Fas-FLAG protein was retained within cytoplasmic portions of the cell and was not translocated to the cell surface. These findings suggest that the Fas protein is reduced or not present on the cell surface in the primary lung tumors and is sequestered within A549 tumorigenic lung cells, and these alterations directly affect the cells resistance to Fas-mediated apoptosis.     

bcl-2基因转染阻断Fas介导的Jurkat细胞凋亡

目的：通过ｂｃｌ２阻断Ｆａｓ介导的细胞凋亡了解ｂｃｌ２基因在淋巴瘤癌变过程中逃避机体免疫监控的作用机制。方法：应用基因重组技术重组ｐＬＸＳＮｂｃｌ２，采用电穿孔法将其与空载体分别转染包装细胞系ＰＡ３１７，将阳性克隆病毒上清感染人Ｔ淋巴瘤细胞株Ｊｕｒｋａｔ，用Ｇ４１８筛选，其阳性克隆进行免疫组化检测，应用抗Ｆａｓ抗体诱导细胞凋亡来模拟细胞毒Ｔ细胞的杀伤机制，观察ｂｃｌ２在淋巴瘤逃避免疫机制中的作用。结果：转染人ｂｃｌ２基因的Ｊｕｒｋａｔ（Ｊｕｒｋａｔｂｃｌ２）细胞较对照Ｊｕｒｋａｔ及转染空载体Ｊｕｒｋａｔ（Ｊｕｒｋａｔｎｅｏ）细胞，ｂｃｌ２表达量明显增加，而Ｆａｓ基因表达量无明显变化。其Ｊｕｒｋａｔｂｃｌ２能明显耐受抗Ｆａｓ抗体诱导的细胞凋亡。结论：人ｂｃｌ２基因在淋巴瘤中过量表达，能部分阻断抗Ｆａｓ抗体诱导的细胞凋亡，ｂｃｌ２基因过量表达可能是淋巴瘤癌变过程中逃脱机体免疫监控的机制之一。     

MK基因特异性siRNA对乳腺癌细胞凋亡的影响

目的探讨RNA干扰技术抑制Midkine对人乳腺癌癌细胞株MCF-7细胞凋亡的影响。方法人工合成抑制Midkine基因的siRNA片段,通过脂质体转染到MCF-7细胞内,应用RT-PCR检测bcl-2mRNA表达,流式细胞术检测细胞线粒体膜电位变化,通过测定光密度值检测Caspase-3活性。结果转染siRNA后的MCF-7细胞,bcl-2mRNA表达显著下降、细胞线粒体膜电位明显下降和Caspase-3酶活性升高。结论MK-siRNA通过下调hcl-2基因,而改变细胞线粒体膜电位使Caspase-3酶活性升高诱导细胞的凋亡。