人Midkine启动子驱动的HSV-TK自杀基因体外抗肝癌作用

目的 观察以人Midkine(MK)启动子调控单纯疱疹病毒胸苷激酶基因(HSV-TK)/丙氧鸟苷(GCV)自杀基因系统在体外对人肝癌细胞的杀伤效应.方法 以含有人MK启动子调控的HSV-TK基因的重组腺病毒分别感染体外培养的甲胎蛋白(AFP)阳性人肝癌细胞BEL-7402和AFP阴性人肝癌细胞SMMC-7721,RT-PCR法检测HSV-TK基因在上述两种细胞中的转录表达,观察GCV对人肝癌细胞的杀伤作用.结果 GCV在体外对重组腺病毒感染的BEL-7402及SMMC-7721均有明显的杀伤作用,又以前者为著.相同的病毒滴度,其杀伤作用随着GCV浓度的增高而增强.均具有旁观者效应.结论 在体外表现HSV-TK基因的AFP阳性及阴性人肝癌细胞均可被人MK启动子调控的自杀基因HSV-TK杀伤.     

HSV-TK+GFP/GCV自杀基因系统对小鼠胰腺癌的治疗作用

目的: 探讨HSV-TK+GFP/GCV自杀基因系统对小鼠胰腺癌细胞系MPC体外及体内杀伤作用及其产生的旁观者效应.方法: 通过RT-PCR从基因组文库中扩增出HSV-TK基因全长CDS序列, 并将其与GFP基因定向克隆到质粒表达载体pcDNA3.1(+), 构建重组质粒pcDNA3.1+/HSV-TK+GFP. 脂质体法将重组质粒转染小鼠胰腺癌细胞株MPC细胞, 得到带有HSV-TK和GFP基因的MPC/HSV-TK+GFP细胞, 并将其分别用于体外和体结果: 重组质粒pcDNA3.1+/HSV-TK+GFP导入小鼠胰腺癌细胞株MPC细胞. 体外实验结果显示, 当MPC/HSV-TK+GFP细胞数占混合细胞10%时, 低浓度(20 mg/L)的GCV就可将50%左右的肿瘤细胞杀死. 体内实验结果显示GCV可明显抑制MPC /HSV-TK+GFP细胞在昆明小鼠体内的肿瘤形成.结论: HSV-TK和GFP基因转入小鼠胰腺癌细胞株MPC细胞并获得稳定表达, HSV-TK+GFP/GCV自杀基因系统在体内外对小鼠胰腺癌均有杀伤作用, 且存在明显的旁观者效应.     

HSV-TK/GCV系统杀伤高、低转移卵巢癌细胞株旁观者效应的观察

目的探讨单纯疱疹病毒胸苷激酶/丙氧鸟苷（HSV-TK/GCV）系统杀伤高、低转移卵巢癌细胞时的旁观者效应及其与间隙连接蛋白（Cx）43表达的关系。方法比较经HSV-TK/GCV作用的高、低转移性卵巢癌细胞株Ho-8910pm和Ho-8910旁观者效应;检测两种细胞Cx43的表达。结果HSV-TK/GCV系统对Ho- 8910细胞产生明显的旁观者效应,而对Ho-8910pm细胞旁观者效应较弱;Ho-8910细胞的Cx43表达为7.12（平均荧光强度）,而Ho-8910pm细胞的Cx43表达为1.34,两者相比,P〈0.05。结论HSV-TK/GCV系统杀伤低转移卵巢癌细胞时旁观者效应强,与Cx43的表达水平有关。     

全反式维甲酸对卵巢癌SKOV3细胞增殖、分化的影响

目的 探讨全反式维甲酸（ATRA）对卵巢癌SKOV3细胞增殖、分化的影响.方法 以1×10^-6 mol/L的ATRA处理人卵巢癌SKOV3细胞为实验组,细胞不加ATRA处理为对照组.应用MTT法测定细胞增殖抑制率,流式细胞仪测定细胞周期分布,免疫组化法检测鼠抗人分化相关基因1（NDRG1）表达,显微镜观察细胞形态变化.结果 与对照组比较,实验组细胞增殖抑制率、NDRG1表达升高（P均＜0.05）;随着作用时间延长,实验组G/G0期细胞增多、S期细胞减少,显微镜下可见细胞生长差,增殖缓慢,分裂像少见.结论 ATRA有较强的抑制卵巢癌SKOV3细胞增殖和诱导其分化作用.     

腺病毒介导的HSV-tk/GCV系统在hTERT启动子调控下治疗人膀胱癌的实验研究

目的观察人端粒酶逆转录酶（hTERT）启动子调控的腺病毒介导单纯疱疹病毒胸苷激酶（HSV-tk）基因/丙氧鸟苷（GCV）系统在体外及荷瘤裸鼠体内对人膀胱癌的治疗作用。方法利用hTERT启动子及小鼠巨细胞病毒（CMV）启动子调控的携带HSV-tk基因的重组腺病毒（Ad-hTERT-HSV/tk与Ad-CMV-HSV/tk）分别感染人膀胱癌细胞253 J和人正常肺成纤维细胞MRC-5,加入GCV,MTT法观察受染细胞的存活率。建立人膀胱癌裸鼠移植瘤模型,随机分为4组,A组（Ad-hTERT-HSV/tk＋GCV组）、B组（Ad-hTERT-HSV/tk＋PBS组）、C组（PBS＋GCV组）和D组（PBS对照组）,观察各组肿瘤生长状况、重要脏器病理变化及裸鼠存活情况。结果体外实验表明,应用GCV处理后,Ad-CMV-HSV/tk对253 J和MRC-5细胞均有杀伤作用,而Ad-hTERT-HSV/tk只对膀胱癌细胞253 J具有杀伤作用。动物实验显示,A组移植瘤的体积和瘤重低于其他3组（P〈0.01）。A组裸鼠平均存活期长于其他3组（P〈0.01）。结论 hTERT启动子调控的重组腺病毒介导HSV-tk/GCV系统是一种安全、有效且靶向性高的基因疗法。     

lncRNA RRS1-AS1/miR-142-3p/TRIM24分子轴对脂多糖诱导的血管内皮细胞凋亡的影响

目的 探讨长链非编码（long non-coding RNA,lncRNA）RRS1-AS1对脂多糖诱导的血管内皮细胞凋亡的影响及可能的作用机制。方法 以脂多糖10 μg/mL刺激人脐静脉血管内皮细胞系HUVECs。实验设立两组：转染阴性对照质粒为对照组,转染载有RRS1-AS1序列的质粒为实验组。采用实时定量聚合酶链反应（qRT-PCR）检测转染效率。细胞增殖实验（CCK-8法）和流式细胞术分别检测两组细胞的增殖能力和凋亡情况。采用生物信息学技术预测RRS1-AS1可能的作用机制。qRT-PCR和Western blot法检测RRS1-AS1可能的靶基因表达。结果 与对照组比较,实验组HUVECs细胞中RRS1-AS1的表达明显增加（P＜0.01）,实验组细胞增殖能力明显增加（P＜0.05）,实验组细胞凋亡比例明显降低（P＜0.01）。RRS1-AS1的靶基因可能是miR-142-3p,miR-142-3p的靶基因可能是三基序蛋白24（tripartite motif containing 24,TRIM24）。与对照组比较,实验组HUVECs细胞中miR-142-3p的表达明显减少（P＜0.01）,TRIM24基因的表达明显增加（P＜0.01）。结论 RRS1-AS1介导的miR-142-3p/TRIM24分子轴可增强脂多糖诱导的血管内皮细胞的增殖能力并抑制其凋亡。     

逆转录病毒与慢病毒介导HSV-TK/GCV防治移植物抗宿主病的比较研究

移植物抗宿主病（GVHD）是限制异基因造血干细胞移植（allo-HSCT）临床应用的最主要并发症，现有的防治方法均不理想，应用自杀基因系统防治GVHD是一个新的尝试。本实验将携带单纯疱疹病毒胸苷激酶（HSV-TK）基因的逆转录病毒和慢病毒分别感染供鼠（C57BL／6小鼠）脾细胞，感染后细胞与供鼠骨髓细胞混合移植到经Co^60γ射线照射后的受鼠（BABL/C小鼠），移植后分别给受鼠腹腔注射更昔洛韦（GCV），初步观察HSV-TK／GCV系统控制GVHD的效果，并在体内外实验中对两种载体进行比较研究。     

腺病毒介导CD/UPRT自杀基因系统治疗胰腺癌的实验研究

目的 探讨CD/UPRT自杀基因系统在胰腺癌基因治疗中的作用.方法 采用同源重组技术构建重组腺病毒Ad-CD、Ad-CD/UPRT和Ad-GFP;体外感染人胰腺癌SW1990细胞,RT-PCR检测UPRT mRNA表达;MTT法检测细胞对5-FC的敏感性及旁观者效应;流式细胞仪分析细胞凋亡率;建立胰腺癌裸鼠皮下移植瘤模型,瘤体内直接注射腺病毒,观察CD/UPRT自杀基因的原位治疗作用.结果 转染CD/UPRT的胰腺癌SW1990细胞对5-FC的敏感性明显提高,Ad-CD/UPRT组的IC50为25 μmol/L,而Ad-CD组和Ad-GFP组分别为100 μmol/L和＞1 000 μmol/L.Ad-CD组、Ad-CD/UPRT组和Ad-GFP组的细胞凋亡率分别为61.3%、40.5%和3.0%,3组比较有显著差异(P＜0.05).CD/UPRT基因转染存在旁观者效应.Ad-CD/UPRT瘤内注射治疗后种植癌体积缩小81%,Ad-GFP治疗肿瘤缩小63%.结论 CD/UPRT自杀基因系统能提高对胰腺癌细胞的杀伤作用.     

siRNA干扰GADD45联合顺铂对人卵巢癌细胞增殖及凋亡的影响

目的研究siRNA沉默GADD45联合顺铂对人卵巢癌细胞SKOV-3增殖和凋亡的影响及相关作用机制。方法将化学合成的GADD45基因siRNA序列转染至SKOV-3细胞中,将SKOV-3细胞分为5组:空白对照组、阴性对照组、干扰组、顺铂组、联合组(干扰+顺铂)。通过MTT比色法、流式细胞术和分光光度法分别检测SKOV-3细胞增殖率、细胞凋亡率、细胞周期各时相分布、Caspase-3和Caspase-9的活力变化以及胞质中Cytochrome C水平变化。结果 GADD45 siRNA转染后,相对于顺铂处理组,联合组细胞凋亡率明显增加(P<0.01);细胞周期各时相重新分布,G0/G1期细胞显著减少(P<0.01),S期和G2/M期细胞则显著增多(P<0.01);Caspase-3和Caspase-9活力显著升高,胞质中细胞Cytochrome C水平明显增加(P<0.01)。结论 GADD45靶向siRNA沉默联合顺铂处理通过调节细胞周期进程阻碍DNA损伤修复进而促进了线粒体依赖性的细胞凋亡,GADD45可作为人卵巢癌基因治疗的后选靶点。     

过氧化氢酶过度表达对血管平滑肌细胞凋亡的影响

目的 探讨腺病毒戢体介导的过氧化氢酶基因转染对体外培养的人血管平滑肌细胞凋亡的影响。方法 用含过氧化氢酶基因的重组腺病毒转染人血管平滑肌细胞，采用Western blot方法检测血管平滑肌细胞过氧化氢酶的表达。应用流式细胞术、TUNEL法等方法检测血管平滑肌细胞的凋亡。结果 含过氧化氢酶基因的重组腺病毒转染后血管平滑肌细胞过氧化氢酶表达明显增多；流式细胞术显示含过氧化氢酶基因的重组腺病毒组与对照组比较凋亡率明显增加（P〈0．01）。经TUNEL分析显示，含过氧化氢酶基因的重组腺病毒组凋亡细胞明显多于对照组。两者之间有显著性差异（P〈0．001）。结论 腺病毒载体介导的过氧化氢酶基因转染导致过氧化氢酶过度表达。促进人血管平滑肌细胞的凋亡，这可能是防治经皮腔内冠状动脉血管成形术后再狭窄的一种新方法。     

Herpes simplex virus thymidine kinase and ganciclovir suicide gene therapy for human pancreatic cancer

AIM: To investigate the in vitro effects of suicide gene therapy system of herpes simplex virus thymidine kinase gene (HSV-TK) in combination with the treatment of nucleotide analog-ganciclovir (GCV) on human pancreatic cancer, and to provide a novel clinical therapeutic method for human pancreatic cancer. METHODS: We used a replication defective recombinant retrovirus vector GINaTK (bearing HSV-TK gene) to make packaging cell PA317 produce progeny virions. We then transferred the HSV-TK gene to target cells SW1990 using these progeny virions, and treated these gene-modified tumor cells with GCV to study the sensitivity of the cells to GCV and their bystander effects by routine MTT-method. RESULTS: Packaging cell PA317/TK was successfully constructed, and we acquired SW1990/TK through virus progeny infection. These gene-modified pancreatic cancer cells were sensitive to the treatment of GCV compared with unmodified tumor cells (t=4.15, n=10, P<0.0025). We also observed a remarkable bystander effect by mixing two kinds of cells at different ratio. CONCLUSION: Our data demonstrate that HSV-TK/GCV suicide gene therapy system is effective for treating experimental human pancreatic cancer, which is largely resistant to the common therapies, so the suicide gene therapy system may be a potential treatment approach for pancreatic cancer.     

Comparison of gene therapy with the herpes simplex virus thymidine kinase gene and the bacterial cytosine deaminase gene for the treatment of hepatocellular carcinoma

BACKGROUND: Bystander effects induced by suicide gene/prodrug systems play an essential role in achieving successful antitumor effects. Although it has been shown in several in vitro studies that the bacterial cytosine deaminase (CD) gene/5-fluorocytosine (5-FC) system is superior to the herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system, we examined here which suicide gene system was more promising in vivo for the treatment of hepatocellular carcinoma (HCC). METHODS: BNL1ME A.7R.1 murine HCC cells were retrovirally transduced with the HSV-TK or CD gene, and bystander effects caused by the appropriate prodrug treatment were examined not only in vitro but also in vivo. RESULTS: The CD/5-FC system was superior to the HSV-TK/GCV system in HCC cell elimination in vitro. The bystander effect of the HSV-TK/GCV was shown to be substantially dependent on cell-to-cell contact, whereas that of the CD/5-FC was not. However, antitumor effects on HCC and tumor immunity to parental HCC induced by the HSV-TK/GCV system were not inferior and even superior to those induced by the CD/5-FC system. Bystander effects induced by the suicide gene/prodrug systems in immunocompetent syngeneic mice were much more profound than those induced in vitro. However, significant bystander effects were not observed in athymic nude mice. CONCLUSIONS: These results suggest that both HSV-TK/GCV and CD/5-FC systems are useful for the treatment of HCC. The results also suggest that T-cell-mediated immune responses elicited by the suicide gene/prodrug systems play a substantial role in antitumor effects in vivo.     

Do there exist synergistic antitumor effects by coexpression of herpes simplex virus thymidine kinase with cytokine genes on human gastric cancer cell line SGC7901？

AIM: To evaluate the synergistic antitumor effects of herpes simplex virus thymidine kinase (HSV-TK) together with tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2) gene expression on gastric cancer cell line SGC7901. METHODS: Recombinant vectors pL(TT)SN and pL(TI)SN,which express TK-IRES-TNF-α and TK-IRES-IL-2 genes separately, as well as the control plasmids pL(TK)SN and pLXSN were employed to transfect PA317 cells respectively to generate the viruses that can stably express the objective genes through G418 selection. The gastric cancer cells were then transfected by the retroviral serum from the package cells and maintained in culture to determine the cell growth and apoptosis. The cytotoxic effects of HSV-TK together with TNF-α or IL-2 gene expression on the transfected cancer cells were evaluated by the cell viability and bystander effects in the presence of GCV supplemented in the cultural medium. RESULTS: Expression of recombinant proteins including TNF-α and IL-2 by stable transfectants was confirmed by Western blotting. The percentage of cell apoptosis in the SGC/0, SGC/TK-TNF-α SGC/TK-IL-2 and SGC/TK done was 2.3%, 12.3%, 11.1% and 10.9% respectively at 24 h posttransfection. Cell growth status among all the experimental groups as judged by cell absorbance (A) at 570nm did not exhibit any significant difference (P&gt;0.05); although it was noted to be slightly lower in the SGC/TT group. Cell survival rate in SGC/TI, SGC/TT and SGC/TK group was significantly decreased in a dose-dependent manner of GCV compared with that of the SGC/0 group (P&lt;0.05-0.01). Among all studied cells, the SGC/TT was shown most sensitive to GCV with a half lethal dose of 0.5 mg.L^-1. In contrast, the survival rate of SGC/0 cells was not affected by the presence of GCV with the doses less than 10 mg-L^-1 The half lethal dose of GCV for SGC/0 cells was more than 100 mg-L^-1. Marked bystander effect induced by SGC/TI, SGC/TT and SGC/TK cells was confirmed by the fact that 20% of these stable transfectants could kill 50% of the co-cultured cells, in which the most prominent bystander effect was found in the circumstance of SGC/TT presence. However, no significant difference of these variables was found among SGC/TI,SGC/TT and SGC/TK cells (P&gt;0.05). CONCLUSION: The synergistic antitumor effects produced by the co-expression of HSV-TK with TNF-α or IL-2 geneswere not present in the transfected SGC7901 cells. The mechanism underlying these phenomena was not known.     

肺癌的自杀基因放射导向治疗实验研究

目的：利用放射敏感性调控序列诱导单纯疱疹病毒胸苷激酶（HSV－TK）基因的肿瘤靶向表达，以提高肺癌自杀基因治疗的选择性和有效性。方法：利用基因重组构建放射可调控的HSV－TK表达载体；转染肺癌A549细胞系，观察γ线照射后细胞HSV－TK表达，以及丙氧鸟苷（GCV）作用下细胞相对存活率的变化。利用裸鼠肺癌模型观察不同处理后的抑瘤效应。结果：γ线可诱导HSV－TK在被转染肺癌细胞表达显增强，呈剂量依赖性；经放射诱导后，转染细胞对GCV的敏感性明显升高（P＜0．05），细胞生工活性显受抑；放射联合GCV可明显抑制感染tgEgr-HyTK的裸鼠移植瘤，并可使50％的肿瘤完全消退。结论：由辐射敏感性启动子诱导的自杀基因肿瘤靶向性表达，是一种高产、安全的肺癌基因治疗新策略。     

藤梨根乙酸乙酯提取物对肺癌A549细胞凋亡的诱导作用

目的观察藤梨根乙酸乙酯提取物对肺癌A549细胞凋亡的诱导作用。方法体外培养肺癌A549细胞,分别给予40、80、160μg/ml的藤梨根乙酸乙酯提取物（实验组）,同时设对照组（0.04%DMSO＋肺癌A549细胞）。采用DNA琼脂糖凝胶电泳检测用药后DNA梯状条带、TUNEL检测用药后肺癌A549细胞凋亡率的变化、免疫组化SP法检测用药后肺癌A549细胞Survivin蛋白表达变化、RT-PCR法检测用药后A549细胞Survivin mRNA的表达。结果实验组细胞DNA凝胶电泳均出现凋亡细胞所特有的DNA梯状条带图谱,而对照组细胞未出现。各实验组细胞随着藤梨根乙酸乙酯提取物浓度的增加,细胞凋亡率明显增加,并存在时相性和量—效依赖性;其Survivin蛋白和mRNA表达均低于对照组（P均〈0.05）,亦存在时相性和量—效依赖性。结论藤梨根乙酸乙酯提取物可明显诱导肺癌A549细胞凋亡,其机制可能与降低Survivin蛋白和mRNA表达有关。     

TL1A在高糖诱导的人脐静脉内皮细胞凋亡中的作用及机制探讨

目的 研究肿瘤坏死因子配体相关分子1A（TL1A）在高糖诱导的人脐静脉内皮细胞凋亡中的作用,并探讨机制.方法 采用RT-PCR、Western blot法检测不同浓度（5.6、11.2、22.4、33.6 mmol/L）葡萄糖与人脐静脉内皮细胞共同孵育48 h以及22.4 mmol/L葡萄糖作用于内皮细胞12、24、48、96 h后,TL1A mRNA及蛋白质在人脐静脉内皮细胞中的表达情况.在22.4 mmol/L葡萄糖培养的人脐静脉内皮细胞中加入不同浓度的TL1A单克隆抗体（1、3、9 μg/mL）,48 h后进行Annexin V/PI双染色流式细胞凋亡计数,以纯化重组人TL1A作为内源性TL1A的对照.结果 在人脐静脉内皮细胞中,随着葡萄糖浓度升高及作用时间延长,TL1A mRNA和蛋白水增高（P＜0.05）.流式细胞计数显示,高糖培养人脐静脉内皮细胞经TL1A单克降抗体处理后凋亡明显减少（P＜0.05）.结论 高浓度葡萄糖诱导体外培养的人脐静脉内皮细胞TL1A的表达呈浓度、时间依赖性增高;TL1A表达增多可能是高糖诱导人脐静脉内皮细胞凋亡的重要因素.     

Effect of blocking IGF-Ⅰ receptor on growth of human hepatocellular carcinoma cells

AIM: To study the expression level and localization of insulin-like growth factor -Ⅰ receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the growth of HepG2 cells.METHODS: The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry.The influences of αIR3 on proliferation and apoptosis were examined by the 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and electron microscopy, respectively. Flow cytometry (FCM) was applied for the analysis of cell cycle and apoptosis was observed under electron microscope.RESULTS: IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1 μg/mL αIR3 for 48 h in vitro, the cell growth index (GI) of HepG2 cells was significantly higher than that of control (103.41% vs 100%, P ＜ 0.01).However, the αIR3 for 24 h at final concentration of 4.0 μg/mL made the GI of HepG2 cells lower than that of control (93.37% vs 100%, P ＜ 0.01). Compared with control, treated with αIR3 for 48 h at final concentrations ranging from 1.0 μg/mL to 4.0 μg/mL markedly reduced the GIs of HepG2 cells (97.63%, 97.16%, 95.13%,92.53% vs 100%, P ＜ 0.05 or P ＜ 0.01), treated with αIR3 for 72 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL decreased the GIs of HepG2 cells obviously (95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P ＜ 0.01), and treated with αIR3 for 96 h at final concentrations ranging from 0.5 μg/mL to 4.0 μg/mL made GIs of HepG2 cells lower significantly (88.86%,83.97%, 79.81%, 77.24%, 70.51% vs 100%, P ＜ 0.05 or ,P ＜ 0.01). Moreover, treated with αIR3 from 24 h to 96 h at final concentrations ranging from 0.2 μg/mL to 4.0 μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also,αIR3 treatment for 72 h at final concentration from 0.5 μg/mL to 2.0 μg/mL increased the proportion of G0/G1 phase cells(61.73%, 67.1%, 83.7%,76.87% vs 44.47%,P ＜ 0.01) and significantly decreased that of S phase cells(28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P ＜ 0.01), in contrast to the proportion of G2/M phase cells.The apoptotic rates of HepG2 cells were increased more than that of control (7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P ＜ 0.01).CONCLUSION: The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGFIR. The blockage of IGF-IR with αIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.     

TNF-α联合阿霉素对人胃癌细胞株SGC-7901的作用及其机制研究

背景:肿瘤坏死因子-α(TNF-α)是抗肿瘤活性很强的细胞因子,但较多的全身毒副作用限制了其应用。目前发现小剂量TNF-α联合化疗药物可提高化疗效果。目的:探讨TNF-α联合阿霉素(ADM)对人胃癌细胞株SGC-7901的治疗作用及其机制。方法:体外常规培养人胃癌细胞株SGC-7901,并分为ADM组(64、16、4、1、0.25μg/mL)、TNF-α组(16 000、4000、1000、250、62.5 IU/mL)、联合组(ADM 5μg/mL+TNF-α4000 IU/mL)、阴性对照组和空白对照组。以MTT法观察细胞生长,流式细胞术检测细胞周期和凋亡,蛋白质印迹法检测caspase-8蛋白表达。构建裸鼠胃癌移植瘤模型,分为阴性对照组、ADM组(2 mg/kg)和联合组(ADM 2 mg/kg+TNF-α5×104IU/kg),观察肿瘤生长情况。结果:与ADM组相比,联合组SGC-7901细胞存活率明显降低,S期和G2期细胞比例、细胞凋亡率、caspase-8蛋白表达均明显升高;体内实验亦显示联合组对裸鼠移植瘤的抑瘤率明显高于ADM组。结论:TNF-α与ADM联合应用可增加化疗药物对胃癌细胞的杀伤作用,其机制部分与促进细胞凋亡和改变细胞周期有关。     

红景天苷对人恶性黑色素瘤细胞A375增殖和凋亡的影响

目的：观察红景天苷对人恶性黑色素瘤细胞A375增殖及凋亡的影响。方法分别用0、0．01、0．1、1、10、100μg/mL红景天苷处理人恶性黑色素瘤细胞A375后,MTT法检测各组细胞增殖率,Annexin V-FITC/PI双染色流式细胞术检测细胞凋亡率,RT-PCR检测各组细胞周期依赖性激酶CDk4和CDk2 mRNA、细胞周期素CyclinE和CyclinD1 mRNA的影响。结果与阴性对照组比较,各给药组细胞增殖抑制率明显增多（P＜0．05）。给药组细胞以G0/G1期为主,S期细胞增加,G2/M期细胞减少（P＜0．05）。给药组中的CDk2、 CDk4、CyclinD1和CyclinE mRNA表达较阴性对照组明显降低（P＜0．05）。结论红景天苷能够抑制人恶性黑色素瘤细胞A375增殖和促进其凋亡。     

不同剂量结核病DNA疫苗的电转染免疫原性研究

目的 研究不同剂量结核病DNA疫苗电转染后的免疫原性.方法 40只BALB/c小鼠通过随机数字表法分为8组,每组5只.其中4组分别用100 μl生理盐水和10 μg、50 μg、100 μg结核分枝杆菌Ag85A DNA肌内注射免疫小鼠;另4组用100 μl生理盐水和10 μg、50 μg、100 μgAg85ADNA肌内注射加电转染免疫小鼠.每2周免疫1次,共3次.最后1次免疫后2周杀死小鼠.用ELISA方法检测小鼠脾细胞培养上清液中γ干扰素（IFN-γ）和白细胞介素4（interleukin 4,IL-4）水平;用流式细胞术检测小鼠外周血单个核细胞（PBMC）分泌IFN-γ的辅助性T细胞（helper T cell,Th）1细胞百分比、分泌IL-4的Th2细胞百分比,以及Th1与Th2的细胞比值.用SAS 9.2软件处理数据,实验数据以“-x±s”表示,对有关数据进行两因素析因设计的方差分析,两两比较采用t检验,P＜0.05为差异有统计学意义.结果 免疫结束后2周,小鼠脾淋巴细胞分泌IFN-γ水平,50 μg[（646.05±342.53） pg/ml]和100 μgAg85ADNA肌内注射组[（738.61±372.68） pg/ml]显著高于生理盐水组[（1.73±3.88）pg/ml]（f值分别为4.065、4.647,P值均＜0.05）和10 μg Ag85A DNA组[（87.83±120.82）pg/ml]（t值分别为3.513、4.094,P值均＜0.05）;10 μg Ag85A DNA电转染组[（357.06±105.18） pg/ml]显著高于生理盐水组（t=2.247,P＜0.05）,高于100 μg Ag85ADNA电转染组[（86.08±135.73） pg/ml],但差异无统计学意义（t=1.706,P＞0.05）;50 μg Ag85ADNA电转染组[（648.60±439.41）pg/ml]显著高于生理盐水组（t=4.081,P＜0.05）和100 μg Ag85A DNA电转染组（t=3.539,P＜0.05）.与直接肌内注射组IFN-γ水平[（87.83±120.82） pg/ml]比较,10 μg Ag85A DNA电转染组增高3倍[（357.06 pg/ml）/（87.83 pg/ml）]; 50 μg Ag85A DNA肌内注射组[（646.05±342.53） pg/ml]与电转染组[（648.60±439.41）pg/ml]比较,差异无统计学意义（t=-0.016,P＞0.05）;100 μg Ag85A DNA电转染组[（86.08±135.73）pg/ml]较肌内注射组[（738.61±372.68） pg/ml]降低88.35％（t=4.105,P＜0.05）.小鼠PBMC分泌IFN-γ的Th1细胞百分比,不同剂量Ag85A DNA肌内注射组[10 μg Ag85A DNA：（1.39±0.84）％;50 μg Ag85A DNA：（1.55±0.33）％;100 μg Ag85A DNA：（2.13±0.47）％]和DNA电转染组[10 μg Ag85A DNA：（1.42±0.47）％; 50 μg Ag85A DNA：（1.88±0.51）％; 100 μg Ag85ADNA：（1.43±0.68）％]均高于生理盐水组[（0.65±0.31）％]（t值分别为2.002、2.431、4.015、2.084、3.332和2.105,P值均＜0.05）,但不同剂量Ag85A DNA电转染组与肌内注射组之间差异均无统计学意义（t值分别为0.081、0.901和-1.91,P值均＞0.05）.小鼠PBMC分泌IL-4的Th2细胞百分比,不同剂量Ag85A DNA肌内注射组[10 μg Ag85A DNA：（1.42±1.18）％; 50 μg Ag85A DNA：（1.14±0.78）％; 100 μg Ag85A DNA：（1.24±0.76）％]和DNA电转染组[10 μg Ag85A DNA：（1.19±1.09）％; 50 μg Ag85A DNA：（2.06±0.96）％;100 μgAg85A DNA：（1.47±0.65）％]均显著低于生理盐水电转染组[（4.14±2.55）％]（t值分别为-3.392、-3.738、-3.616、-3.676、-2.599和-3.325,P值均＜0.05）,但不同剂量DNA肌内注射组与DNA电转染组之间差异无统计学意义（t值分别为0.284、-1.139和-0.292,P值均＞0.05）.结论 DNA电转染免疫可以增强低剂量DNA疫苗的免疫应答,使用少量的DNA疫苗就可以产生较好的免疫效果.