腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

目的　探讨腺病毒载体介导的内皮抑素基因（Ad-mES）在体外和体内的生物学活性。方法　不同感染复度（MOI）的腺病毒体外感染靶细胞;RT-PCR法检测目的基因的表达;MTT法检测Ad-mES对靶细胞生物活性的影响。观察各组小鼠黑素瘤的生长、转移和生存率;免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达。电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况。结果　腺病毒体外能够有效感染靶细胞,MOI为10,20,50,100,200,500时,B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%。靶细胞明确表达内皮抑素基因;Ad-mES对B16F10细胞的增殖没有影响;而Ad-mES能抑制ECV304细胞的增殖,且随MOI增大,抑制内皮细胞增殖效果越强。瘤细胞接种后第8天,各组成瘤率100%。开始出现小鼠死亡的最早日：PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天。结论 Ad-mES体外和体内均影响靶细胞的生物学活性;Ad-mES治疗组小鼠平均生存时间延长（P < 0.05）,肿瘤体积增长减慢（P < 0.05）。     

喜树碱抗血管生成作用的研究

目的 探讨喜树碱在体外对人脐静脉血管内皮细胞ECV304增殖抑制和凋亡诱导作用。方法 将不同浓度的喜树碱作用于ECV304细胞株,用MTT法、光镜、电镜、DNA梯状带检测药物对细胞的增殖抑制和凋亡诱导作用。结果 喜树碱能抑制ECV304细胞的增殖。24h,36h,48h最大抑制率分别为67.9%,96.7%,97.2%。形态学观察细胞生长受抑制,可见凋亡小体。凝胶电泳显示典型的DNA梯度带。结论 喜树碱体外能抑制人脐静脉血管内皮细胞增殖并诱导其凋亡,证明喜树碱可能具有抗血管生成作用。     

沙利度胺等3种药物对内皮细胞细胞间黏附分子-1表达的影响

目的：探索沙利度胺、氨苯砜、雷公藤内酯醇等药物对内皮细胞诱导性细胞间黏附分子—1(ICAM—1)表达的影响。方法：用逆转录—聚合酶链反应(RT—PCR)方法检测肿瘤坏死因子—α(TNF—α)、佛波酯(PMA)或脂多糖(LPS)对人内皮细胞株ECV304细胞上ICAM—1表达的诱导，以及沙利度胺、氨苯砜、雷公藤内酯醇、吲哚美辛、丙酸氯倍他索等药物对ECV304细胞上诱导性ICAM—1表达的影响。结果：TNF—α和PMA可显著上调ECV304细胞上ICAM—1的表达；沙利度胺、氨苯砜可分别抑制TNF—α或PMA诱导的ICAM—1表达；吲哚美辛可显著上调ECV304细胞上ICAM—1的诱导表达。结论：沙利度胺和氨苯砜的抗炎作用可能与其对内皮细胞上ICAM—1表达的抑制有关。     

血管内皮细胞和角质形成细胞对血卟啉单甲醚吸收特性的比较

目的 探讨血管内皮细胞(ECV304)和角质形成细胞(HaCaT)对光敏剂血卟啉单甲醚(hematoporphyrin monomethyl ether,HMME)的吸收特性.方法 取对数生长期的ECV304和HaCaT细胞分别与50、100、150、200、250 mg/L HMME孵育16 h,或与150 mg/L HMME孵育15 min、30 min、1h、3h、8h、12h、24 h.激光扫描共聚焦显微镜检测孵育浓度及孵育时间对两种细胞吸收HMME的影响.结果 与上述5种浓度HMME孵育后,ECV304细胞的平均荧光强度分别为74.00、125.57、135.24、141.99、132.09,HaCaT细胞的平均荧光强度分别为93.88、102.45、112.59、108.23、104.70.与150 mg/LHMME孵育上述时间后,ECV304细胞的平均荧光强度分别为95.07、103.97、105.96、108.99、112.93、115.36、122.91,HaCaT细胞的平均荧光强度分别为104.25、106.60、108.72、113.75、117.66、114.90、118.14.结论 ECV304和HaCaT细胞对HMME的吸收在一定浓度范围和时间范围内均呈浓度和时间依赖性.     

热休克蛋白110对人乳头瘤病毒16型E711-20配体的佐剂效应研究

目的 探讨热休克蛋白（HSP）110对人乳头瘤病毒（HPV）16型E711-20配体的免疫佐剂效应。方法 体外构建HSP 110与HPV 16型E711-20配体的复合物。将C57BL/6雌性6周龄小鼠15只随机分为3组：复合物组、配体组和磷酸盐缓冲液（PBS）组,每组5只,腹腔注射免疫复合物100 μg,肽10 μg,PBS 100 μl,2周后重复1次,第2次免疫2周后,分离脾细胞作为效应细胞。免疫小鼠后采用噻唑蓝法（MTT）判断脾淋巴细胞增殖活性,干扰素γ（IFN-γ）胞内染色法检测小鼠脾细胞中特异性细胞毒T淋巴细胞,并以标准51Cr释放实验检测特异性细胞毒T淋巴细胞对靶细胞的杀伤效应。采用SPSS10.0软件进行t检验。结果 HSP110与HPV16型E711-20配体的复合物免疫小鼠后脾淋巴细胞增殖活性（增殖指数为1.87 ± 0.12）和CD8+ IFN-γ+ T细胞的频率（3.9%）显著高于配体组（分别为0.32 ± 0.071和0.4%）,差异均有统计学意义（P < 0.01）。配体的复合物对靶细胞的杀伤效应（效靶比为100 ∶ 1、50 ∶ 1、25 ∶ 1、12.5 ∶ 1时,杀伤率分别为54.7%、72.2%、61.5%、39.8%）显著高于配体组（分别为35.2%、49.3%、28.1%、17.4%）。结论 HSP110能增强HPV16型711-20配体的免疫效应,具有较好的免疫佐剂作用。     

HPV16E7_(11-20)配体抗肿瘤免疫的作用

目的比较HPV16型E7抗原CTL表位E711-20(YMLDLQPETT)与其修饰多肽配体APL(YLLDLQPE-VT)诱发特异CTL免疫应答的能力和抗肿瘤免疫治疗的作用。观察E711-20及其APL的小鼠TC-1细胞肿瘤免除率、存活时间与荷瘤小鼠的肿瘤体积变化,了解其预防和治疗效果。方法以E711-20及其修饰多肽配体(APL)免疫C57BL/6小鼠,测定免疫后小鼠脾淋巴细胞的增殖指数;用LDH释放试验检测免疫后小鼠脾淋巴细胞的CTL杀伤活性。结果脾细胞增殖实验中,3组增殖指数如下:IFA+APL组2.42±0.38,IFA+E711-20组1.68±0.32,IFA组1.07±0.22;CTL杀伤效应实验中,3组靶细胞溶破率(%)如下:IFA+APL组54.21±0.12,IFA+E711-20组62.33±0.46,IFA组0.88±0.07;IFA组肿瘤免除率为0,IFA+E711-20组肿瘤免除率为40%,IFA+APL组肿瘤免除率为80%;荷瘤小鼠中,对照组IFA肿瘤体积迅速增大,E711-20和APL组的肿瘤生长速度较慢。APL免疫诱导的小鼠脾淋巴细胞,其CTL杀伤活性和增殖能力强于E711-20;APL对TC-1肿瘤细胞的预防和治疗效果优于E711-20。结论 APL诱导特异性抗肿瘤免疫应答的能力和抗肿瘤效果优于E711-20,可能为针对HPV16E7抗原的肽疫苗分子设计提供理想的CTL表位。     

凉血活血复方对体外培养HaCaT、ECV304细胞端粒酶活性的影响

目的观察凉血活血复方水醇粗提液对人永生化表皮细胞株(HaCaT)和人脐静脉血管内皮细胞株(ECV304)端粒酶活性的影响,探讨该复方治疗银屑病可能的作用机制。方法将不同浓度的凉血活血复方水醇粗提液分别地作用于体外培养的HaCaT和ECV304细胞株,应用端粒重复序列扩增-酶联吸附(TRAP-ELAISA)试剂盒检测两株细胞的端粒酶活性。结果凉血活血复方分别作用两株细胞48h,细胞端粒酶活性在所测定实验浓度范围内,随浓度的增加对细胞的端粒酶活性的下调作用逐渐增强。各加药组与对照组在统计学上有显著性差异,且各加药组之间差异亦有统计学意义(P0.01)。结论凉血活血复方以剂量依赖性抑制端粒酶活性。     

T-钙黏蛋白对小鼠皮下氮烯咪胺耐药黑素瘤B16F10细胞株的影响

目的观察T-钙黏蛋白(cadherin)对小鼠皮下氮烯咪胺(dacarbazine,DTIC)耐药黑素瘤B16F10细胞株的影响。方法通过体外分步诱导法诱导B16F10氮烯咪胺耐药细胞株(DTIC-R B16F10)。采用CCK-8法检测DTIC-R B16F10细胞的增殖情况。将T-cadherin基因转染至DTIC-R B16F10细胞。免疫组织化学检测转染后T-cadherin的表达情况。培养DTIC-R B16F10细胞,分为6组:对照组、pEGFP-N1组、T-cadherin组、DTIC组、pEGFP-N1联合DTIC组和T-cadherin联合DTIC组。通过CCK-8法和Transwell小室实验检测T-cadherin与氮烯咪胺联合对DTIC-R B16F10细胞活性和迁移的影响。建立荷瘤小鼠模型,观察T-cadherin对小鼠皮下注射DTIC-R B16F10细胞后肿瘤生长的影响。采用析因分析法评价T-cadherin联合氮烯咪胺对细胞活性、迁移和小鼠皮下瘤生长的影响。结果DTIC-R B16F10组、B16F10组与DTIC-R B16F10+DTIC组A值差异无统计学意义(P>0.05)。免疫组织化学检测表明细胞表达T-cadherin。T-cadherin联合DTIC组细胞活性、迁移细胞数及肿瘤重量均显著低于其他组(P<0.05)。析因分析显示,T-cadherin联合氮烯咪胺对DTIC-R B16F10的细胞活性、迁移和小鼠皮下瘤生长的抑制有协同作用。结论T-cadherin可抑制黑素瘤小鼠皮下瘤的生长,增加黑素瘤对氮烯咪胺的敏感性。     

2-甲氧基雌二醇对恶性黑素瘤B16细胞株增殖与凋亡的影响北大核心CSCD

目的 探讨2-甲氧基雌二醇（2-ME）对小鼠恶性黑素瘤B16细胞增殖与凋亡的影响,并初步探讨其机制.方法 选用小鼠B16细胞进行体外培养,实验分为阴性对照组和药物处理组,药物处理组加入2-ME,使其终浓度分别为5、10、20、40 μmol/L,阴性对照组不加2-ME.作用不同时间后,倒置相差显微镜下观察细胞形态变化;根据磺酰罗丹明B方法测得的各组A490值绘制生长曲线;采用磺酰罗丹明B法检测黑素瘤细胞活性;流式细胞仪检测细胞周期及凋亡;逆转录PCR和实时PCR法分析凋亡诱导基因（gadd45b）及原癌基因（c-myc）的表达情况.结果 重复测量的方差分析显示,5、10、20、40 μmol/L的2-ME作用于黑素瘤细胞不同时间后对其增殖的抑制作用差异有统计学意义（F=1170.94,P＜ 0.01）;上述浓度的2-ME作用24、48、72 h对黑素瘤细胞增殖的抑制作用差异亦有统计学意义（F=1843.04,P＜ 0.01）;药物浓度和培养时间存在交互作用（F=272.79,P＜ 0.01）.10、20、40μmol/L 2-ME作用48 h后,B16细胞的凋亡率分别上升至（4.13±1.12）％、（11.25±2.38）％、（19.46±2.9）％,与阴性对照组[（0.23±0.5）％]相比,差异均有统计学意义（P＜0.01）;细胞出现G0/G1期阻滞,对照组细胞、10、20、40 μmol/L 2-ME处理组细胞G0/G1期比例分别为（44.1±3.4）％、（59.5±5.6）％、（63.4±8.2）％、（70.8±4.4）％,且随着浓度的增加,G0/G1期细胞明显增加,各处理组及对照组间比较,G0/G1期细胞比例差异有统计学意义（F=13.56,P＜ 0.05）.与阴性对照组相比,20 μmol/L和40 μmol/L 2-ME作用24 h可升高gadd45b的表达（均P＜ 0.01）,10、20、40μmol/L 2-ME均可降低c-myc基因的表达,差异均有统计学意义（均P＜0.05）.结论 2-ME在体外能够抑制小鼠恶性黑素瘤B16细胞的增殖,能够使c-myc基因的表达降低,使gadd45b基因的表达升高.     

Wnt10b诱导再生毛囊的表达特性研究

目的 研究Wnt10b诱导再生毛囊的表达特性及诱导作用机制。 方法 HEK-293细胞内扩增并用氯化铯梯度离心纯化Wnt10b过表达腺病毒及对照腺病毒,皮内注射至C57BL/6J小鼠背部皮肤,在处理后2.5、5、7、9、14、28 d时取材,HE染色及免疫组化染色观察毛囊结构特征、信号通路表达特征及增殖特性。 结果 HE染色发现,AdWnt10b处理组从第5天开始出现新生毛囊结构,正常生长,第28天左右进入退化期。免疫组化染色发现,AdWnt10b处理组从处理后5 d开始新生毛囊具有AE15表达,随着毛囊生长而增加,至处理后28 d开始减少。在AdWnt10b处理后5 d,观察到β连环素的核表达,Lef1特异性表达于毛芽和毛母质部位,且全为核表达。在AdWnt10b处理后28 d,Lef1表达减弱。AdWnt10b处理后2.5 d即可见Ki67表达于表皮和毛囊外根鞘。处理后2.5、7、9、14 d均在隆突区见到Ki67的表达;从处理后7 d开始,Ki67表达于毛母质细胞。 结论 Wnt10b诱导的再生毛囊具有正常的毛囊结构,Wnt10b激活了经典Wnt信号通路,其作用的靶细胞是毛囊干细胞及其子代细胞。     

Gene transfer of secreted-type modified interleukin-18 gene to B16F10 melanoma cells suppresses in vivo tumor growth through inhibition of tumor vessel formation

Interleukin-18 is a novel cytokine identified as a strong inducer of interferon-gamma. Interleukin-18 has been shown to have similar bioactivities to interleukin-12 and to have antitumor efficacy in experimental models. In this study, we investigated whether the introduction of the interleukin-18 gene to B16F10 melanoma cells can induce antitumor response or not. Before the transfection, we modified the interleukin-18 gene to enable transfected tumor cells to secrete bioactive interleukin-18, because interleukin-18 does not have a signal sequence and requires processing by the interleukin-1 converting enzyme to attain the mature form. We found that B16 melanoma cells transduced with hybrid cDNA consisting of the interferon-beta signal sequence and mature interleukin-18 sequence, but not native interleukin-18, secreted a large amount of interleukin-18 and exhibited retarded tumor growth when injected in syngeneic mice. The antitumor effect was mostly abrogated by administration of anti-interferon-gamma antibody, but was not affected by in vivo depletion of CD8+ T cells or natural killer cells. Histologic analysis revealed that vascularization was markedly reduced and that necrosis was extensively induced in interleukin-18-secreting B16F10 melanoma (B16/IL18) tissues, whereas abundant tumor vessel formation was observed in B16/IL18 tissues of interferon-gamma-neutralized mice. We also found that chemokines, interferon-inducible protein-10 and monokine induced by interferon-gamma, were produced in B16/IL18 tissues and that the expression of both chemokines was dependent on that of interferon-gamma in the tumor tissues. Further, we showed that B16 melanoma cells secreted both chemokines in response to interferon-gamma. In addition, the expression of angiogenin, an angiogenic factor of melanoma, in B16 melanoma cells was reduced by interferon-gamma treatment. These results indicate that gene transfer of secreted-type interleukin-18 to B16F10 melanoma cells is a useful method of triggering an antitumor response without any systemic adverse effects and that the antitumor efficacy is mainly mediated by antiangiogenic activity, which is possibly involved in at least two dynamic changes induced by interferon-gamma inside B16 melanoma cells: the upregulation of antiangiogenic chemokines, interferon-inducible protein-10 and monokine induced by interferon-gamma, and the downregulation of angiogenic factor, angiogenin.     

Cbl-b基因沉默对小鼠原代淋巴细胞免疫活性影响的研究

目的 体外初步研究Cbl-b基因经特异性siRNA沉默后对小鼠淋巴细胞免疫活性的影响。 方法 摘取C57BL/6小鼠的脾脏,体外无菌分离小鼠脾脏淋巴细胞后进行培养。通过EntransterTM-R4000试剂将Cbl-b siRNA转染入小鼠原代淋巴细胞以沉默细胞内Cbl-b的表达。转染72 h后通过酶联免疫吸附实验（ELISA）检测淋巴细胞培养上清中干扰素γ（INF-γ）及肿瘤坏死因子α（TNF-α）的表达量。通过与B16F10黑素瘤细胞共培养,研究Cbl-b基因沉默后的淋巴细胞对B16F10黑素瘤细胞免疫杀伤活性的影响。 结果 Cbl-b siRNA成功转染进小鼠原代淋巴细胞并能有效沉默细胞内Cbl-b的表达。与阴性对照转染组及空白组相比,转染Cbl-b siRNA的淋巴细胞IFN-γ、TNF-α分泌量增加（P < 0.05）。共培养检测结果显示,Cbl-b siRNA转染组比转染阴性对照组能更高效地杀伤小鼠B16F10细胞。 结论 Cbl-b基因沉默能够促进小鼠淋巴细胞INF-γ、TNF-α分泌能力,并能增强淋巴细胞对B16F10黑素瘤细胞的体外免疫杀伤作用。     

Tumor apoptosis by indole-3-acetic acid/light in B16F10 melanoma-implanted nude mice

Recently, we reported that UVB-activated indole-3-acetic acid (IAA) induces the apoptosis of G361 human melanoma cells. In the present study, we used IAA and visible light combinations to treat B16F10 melanoma-implanted nude mice using an experimental intense pulsed light (IPL) therapy model. We first investigated whether activated IAA by horseradish peroxidase (HRP) or UVB causes apoptosis of B16F10 melanoma cells. IAA/HRP or IAA/UVB combination lead to apoptosis of B16F10 cells, as reported in other cell lines. Interestingly, IAA alone was not cytotoxic. These findings suggested the potential use of IAA in the treatment of melanoma. For the future clinical use, we also tested whether visible light has the same effects like UVB and found that visible light also activates IAA to produce free radicals and that IAA/visible light decreased cell viability significantly. Based on these results, IAA/IPL combination was tried whether it can induce apoptosis in vivo status. TUNEL staining showed that IAA/IPL treatment induced apoptosis of tumor cells. In addition, the expressions of p53, Fas, and PARP were upregulated in the IAA/IPL-treated group than in untreated control, demonstrating that IAA/IPL treatment caused apoptosis in melanoma-implanted nude mice. In conclusion, we showed that IAA/IPL induces melanoma regression in B16F10 melanoma-implanted nude mice. These results suggest the potential use of IAA/IPL in the treatment of malignant melanoma.     

携带MART-1基因的减毒单核细胞增多性李斯特菌抑制小鼠恶性黑素瘤的研究

目的 探讨携带黑素瘤分化抗原MART-1基因的减毒单核细胞增多性李斯特菌（LM）对恶性黑素瘤的抑制作用及其机制。方法 构建pERL3-MART-1载体,通过电转化建立携带MART-1基因的△inlB LM-MART-1和△actA/△inlB LM-MART-1重组李斯特菌。浓度梯度稀释法测定各减毒菌株半数致死量（LD50）。C57BL/6小鼠随机分为磷酸盐缓冲液（PBS）组,△inlB LM- MART-1组和△actA/△inlB LM- MART-1组。首次免疫后第7天于小鼠腹部皮下接种B16F10细胞,第14天、21天重复免疫小鼠,观察并记录肿瘤大小及小鼠生存状态。应用实时定量PCR检测肿瘤组织中MART-1表达量。流式细胞术检测小鼠脾细胞中CD4+CD25+ T细胞阳性率。结果 经鉴定成功构建△inlB LM-MART-1和△actA/△inlB LM-MART-1。减毒株△inlB LM 和△actA/△inlB LM的LD50比LM-EGDe标准株毒力分别下降100倍和10 000倍。体内抑瘤试验显示,与PBS组比较,△inlB LM- MART-1组抑瘤率为46.95%,△actA/△inlB LM-MART-1抑瘤率为83.96%,差异均有统计学意义（P < 0.05和0.01）。PBS组、△inlB LM-MART-1组和△actA/△inlB LM-MART-1组肿瘤组织中MART-1表达量递增,以ＰＢＳ组表达量为１,后两组为8.988 ± 0.207和11.315 ± 0.445,各组间差异均有统计学意义（P值均 < 0.05）,且三组小鼠脾细胞中CD4+CD25+ T细胞阳性率依次为（2.52 ± 0.20）%、（1.14 ± 0.13）%和（0.44 ± 0.15）%,组间差异均有统计学意义（P值 < 0.01）。结论 携带MART-1基因的减毒LM毒性明显低于LM标准株,而且可以有效抑制黑素瘤生长,延长小鼠生存期。     

In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer

CD4(+)CD25(+) T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25(+) regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8(+) T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8(+) T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4(+) T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25(+) T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25(+) T cells did not induce vitiligo-like coat color changes, though that of CD4(+) T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25(+) T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4(+)T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25(+) regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25(+) T-cell depletion is mediated through CD8(+) T cells and may differ from the enhancing mechanism caused by CD4(+) T-cell depletion.     

干扰BAG-1表达对黑素瘤B16F10细胞放射敏感性的影响

目的: 明确BAG-1基因表达对黑素瘤B16F10细胞放射敏感性的影响及机制。方法: B16-F10细胞株分为未转染组(Control组)、阴性对照质粒转染组(NC-shRNA组) 和转染BAG-1-shRNA 组(BAG-1-shRNA组)。 克隆计数法计算克隆形成率和存活分数,描绘细胞存活曲线。流式细胞仪检测细胞凋亡率,Western blot检测BAG-1沉默对黑色素瘤细胞凋亡相关蛋白Caspase3、8、9、PARP表达。结果: X线8Gy 剂量照射后,Control组、NC-shRNA组和BAG-1-shRNA组细胞的存活率分别为1.9%,1.75%和0.32%,细胞凋亡率分别为7.1%,9.2% 和25.3%,差异具有统计学意义（均P＜0.05）。BAG-1-shRNA组调亡蛋白c-PARP及c-caspase 3、caspase 8、caspase 9水平分别为0.36, 0.37,0.35和0.30高于control组（0.10,0.04,0.11, 0.2）和NC-shRNA组（0.13, 0.12, 0.20, 0.26）。讨论: 干扰BAG-1基因表达可显著增高黑素瘤B16F10细胞对X放射线的敏感性,促进细胞凋亡可能是放射增敏的机制之一。     

Responses of B16 melanoma cell lines, F1 and F10, to hyperthermia, lymphokine-activated killer cells and a combination of both in vitro

The cytolytic and/or cytostatic effects of hyperthermia, lymphokine-activated killer cells (LAK cells) and the combination of both were assayed using F1 and F10 B16 melanoma cell lines. F10 cells with a high metastatic potential showed a greater sensitivity to hyperthermia than F1 cells which have low metastatic potential. The F10 cells were lysed to a lesser extent by LAK cells than the F1-B16 cells. When the cell lines were subjected to hyperthermia at 43 degrees C for 3 h and then interacted with LAK cells, the maximum cytolysis reached almost 100%. When the interaction with LAK cells was followed by hyperthermia at 43 degrees C, the total release of 51Cr from the cell lines was 75-85%. The extent of 51Cr release from the B16 melanoma cell lines was inversely correlated with the survival rate as calculated by the plating efficiency of the incubated cells. The survival rate of mice intravenously injected with B16-F10 cells and subjected to hyperthermia at 41 degrees C for 3 h in vitro increased compared to that of controls. This was further increased by the simultaneous administration of LAK cells.