食管癌抗原和超抗原构建肿瘤疫苗的抗癌作用研究

目的利用食管癌肿瘤可溶性抗原(TSA)和超抗原(SEC)构建肿瘤疫苗,刺激外周血淋巴细胞,诱导产生细胞毒性T细胞(CTLs),对肿瘤细胞进行体内外杀伤作用研究,以探讨其抗肿瘤作用。方法外周血淋巴细胞经肿瘤疫苗作用,进行体外培养,诱导产生细胞毒性T细胞;细胞毒实验测定效应细胞杀伤活性;建立小鼠移植瘤模型,用肿瘤疫苗进行干预治疗,观察其治疗效果。结果经肿瘤疫苗刺激的淋巴细胞组诱导的CTLs对靶细胞杀伤活性显著高于单纯淋巴细胞组(P<0.05),对TSA来源的食管癌细胞具有选择性杀伤作用;体内研究发现肿瘤疫苗能显著减轻小鼠荷瘤负担,延长生存期。结论肿瘤可溶性抗原与超抗原SEC构建的肿瘤疫苗能产生高效特异性的抗肿瘤效果,显示出良好的抗肿瘤免疫治疗作用。     

乳腺癌干细胞RNA-DCs疫苗诱导特异性细胞毒性T淋巴细胞抗肿瘤免疫应答

目的制备负载乳腺癌干细胞RNA的树突状细胞(DCs)疫苗,研究其诱导的特异性细胞毒性T淋巴细胞(CTLs)的抗肿瘤免疫反应。方法采用细胞毒性试剂盒检测2种乳腺癌干细胞疫苗[(A组,CD44+CD24-+MCF-7-CTLs)、(B组,MCF-7-CTLs)]和DC-CTLs(C组)的体外细胞杀伤能力。取24只小鼠均分为四组:A1组皮下接种活化的A组疫苗+MCF-7乳腺癌细胞;B1组接种活化的B组疫苗+MCF-7乳腺癌细胞;C1组皮下注射活化的DCs-CTLs+MCF-7乳腺癌细胞;D1组单用MCF-7乳腺癌细胞作为对照。分析裸鼠接种后肿瘤在体内的生长情况。结果体外对CD44+CD24-+MCF-7乳腺癌细胞杀伤能力强度:A组>B组>C组(P<0.05)。体外对MCF-7乳腺癌细胞杀伤能力强度:B组>A组>C组(P<0.05)。在体成瘤实验显示,B1组和A1组的成瘤时间分别为第7周和第6周,明显长于D1组(第1周)和C1组(第2周)(P<0.05)。结论 CD44+CD24-乳腺癌干细胞RNA-DCs疫苗诱导的CTLs能够产生特异性免疫应答,从而抑制乳腺癌细胞成瘤。     

基于戊肝病毒的嵌合病毒样颗粒对人乳头瘤病毒16型肿瘤免疫治疗作用的研究

目的 研究基于戊肝病毒(hepatitis E virus,HEV)的嵌合病毒样颗粒(virus-like particles,VLPs)对人乳头瘤病毒16型(human papillomavirus type 16,HPV 16)肿瘤免疫治疗作用。方法 将HPV 16 E7插入HEV的p239蛋白形成重组嵌合蛋白p239-HPV16 E7。所构建的重组蛋白经大肠杆菌表达、纯化、复性后,通过电镜和动态光散射对所得蛋白颗粒大小形态进行表征。将蛋白颗粒免疫C57B/L小鼠,通过流式细胞技术与酶联免疫斑点免疫试验检测脾淋巴细胞特异性免疫细胞分化情况;并且利用TC-1肿瘤细胞在C57B/L小鼠中构建肿瘤模型,以此评价蛋白颗粒在小鼠体内的抗肿瘤免疫效果。结果 体外复性后的嵌合蛋白在电镜下观察到颗粒结构,粒径大小为22.80 nm。所得蛋白颗粒在C57B/L小鼠体内诱导产生良好的特异性细胞免疫反应。与对照组相比,试验组脾淋巴细胞的CD3⁺/CD4⁺、CD3⁺/CD8⁺比例均有明显差异(P＜0.05),且分泌IFN-γ干扰素的效应T细胞显著增加。同时,所得蛋白颗粒能有效抑制TC-1荷瘤小鼠体内肿瘤细胞的生长,在实验周期内小鼠未出现死亡,而对照组小鼠体内肿瘤快速生长,且6周后全部死亡。结论 原核表达的嵌合蛋白p239-HPV16 E7形成病毒样颗粒并有效诱导针对HPV 16的抗肿瘤免疫。     

HMGB1对HPV16型重组腺病毒载体疫苗免疫效果的影响及机制研究

目的 探讨高迁移率族蛋白B1(HMGB1)分子与人乳头瘤病毒(HPV)16型E6E7重组腺病毒载体疫苗免疫效果的关系及机制。方法 通过TC-1细胞移植诱导建立小鼠肿瘤模型,在接种HPV重组腺病毒载体疫苗的同时,给与HMGB1特异性拮抗剂HMGB1 A box注射,开展肿瘤抑制试验、小鼠免疫状态观察、肿瘤相关信号检测等研究。结果 A box蛋白与疫苗联用,即可显著降低模型小鼠HMGB1血清水平,抑制肿瘤组织中NF-kB的mRNA高表达,提高疫苗的抗肿瘤活性。同时,它还可减少IL-4、促进IFN-g产生,使体内免疫平衡由Th2向Th1偏移。虽然A box对于Bax的影响并不显著,但在接种疫苗并注射A box蛋白后,由肿瘤引起的NF-kB、Bcl-2、c-IAP2、VEGF、MMP-9的表达提高、Bax表达降低均得到显著扭转。结论 HMGB1分子可以削弱HPV重组腺病毒载体疫苗的免疫效果,其机制可能是HMGB1在调节免疫平衡状态及控制肿瘤细胞的凋亡、浸润、转移等多层面均能参与并发挥重要作用。     

人乳头瘤病毒重组蛋白疫苗酶联免疫检测法的建立及其应用

目的建立人乳头瘤病毒重组蛋白疫苗(HPV16 L2E6E7)酶联免疫的检测方法,用于疫苗发酵过程中的重组蛋白表达的监控。方法用常规方法制备兔抗HPV16 L2E6E7多克隆抗体作为包被抗体,商品化小鼠抗HPV16 E7单克隆抗体为检测抗体,夹心ELISA方法检测HPV16 L2E6E7抗原参考品,建立抗原标准曲线,确定线性范围及检测限,同时验证该方法的特异性。结果建立了检测HPV16 L2E6E7抗原含量的夹心ELISA方法,抗原参考品系列浓度在19.53~1 250 ng·m L-1内有很好的线性(R2>0.99)和回收率(90%~110%);特异性好,不受发酵液中宿主菌蛋白的干扰。结论建立了人乳头瘤病毒重组蛋白疫苗重组抗原含量的测定方法,为该疫苗的发酵工艺的质控提供了有效技术手段。     

B7-1基因修饰的胃癌细胞诱导抗胃癌主动免疫研究

目的　探讨B7 1基因转染胃癌细胞是否具有抗肿瘤主动免疫增强作用。方法　采用重组腺病毒载体将B7 1基因导入人胃癌细胞SGC 790 1,G4 18阳性克隆筛选 ,流式细胞分析显示B7 1的表达 ;将携带B7 1基因的胃癌细胞 (命名为SGC 790 1/B7 1)接种于C5 7BL/ 6小鼠背部皮下 ,观测其致瘤性能 ;SGC 790 1/B7 1致敏的小鼠对野生型瘤细胞是否具有免疫保护作用 ;用SGC 790 1和SGC 790 1/B7 1细胞分别经腹腔免疫小鼠 ,得到腹腔浸润淋巴细胞及致敏脾细胞 ,MTT法检测其体外杀伤实验。结果　B7 1基因在胃癌细胞中获得高表达 ;SGC 790 1/B7 1诱导的CTL(cytotoxicTlymphocyte)对SGC 790 1的杀伤活性显著高于野生型SGC 790 1诱导的CTL对相同靶细胞的杀伤活性 (P <0 0 5 ) ;SGC 790 1/B7 1诱导的CTL对SGC 790 1/B7 1的杀伤率显著高于对野生型SGC 790 1的杀伤率 (P <0 0 5 )。结论　B7 1促进抗胃癌CTL的增殖、活化 ,能诱导抗胃癌主动免疫功能。     

HPVl8 E7联合SLC基因疫苗的构建及在真核细胞中的表达

目的：构建人乳头瘤病毒（human papilloma virus，HPV）18型E7联合小鼠次级淋巴组织趋化因子（Secondary lymphoid tissue chemokine，SLC）真核共表达基因，以获得高效性治疗HPVl8感染及相关肿瘤的DNA疫苗。方法：分别以HPVl8型的中国野毒株、pDsRed2-N1-SLC为模板，利用PCR克隆技术制备HPVl8E7和SLC基因，分别双酶切后胶回收获得HPVl8E7、SLC基因片段，分别插入到真核表达载体pIRES的多克隆位点B（MCSB）和多克隆位点A（MCSA）中，构建pIRES-E7-SLC真核共表达质粒。酶切及核酸序列分析鉴定质粒。脂质体转染人脐静脉内皮细胞（ECV）细胞，以Western bolt方法鉴定真核表达质粒E7的表达，用ELISA的方法鉴定真核表达质粒SLC的表达。结果：酶切及核酸序列测定表明真核表达质粒pIRES-E7-SIC的成功构建。RT-PCR和Western-Blotting方法证实E7及SLC蛋白在真核细胞的正确表达。结论：成功地构建真核表达质粒plRES-E7-SLC，用它转染ECV细胞后，能表达E7和SLC蛋白，为下一步研究HPVl8感染及相关肿瘤疫苗奠定了基础。     

重组4-1BB配体对治疗性重组人乳头瘤病毒16型蛋白疫苗的佐剂作用

目的 研究重组4-1BB配体(recombinant 4-1BB ligand,r4-1BBL)作为佐剂对治疗性重组人乳头瘤病毒16型(human papillomavirus-16,HPV16)蛋白疫苗(HPV16蛋白疫苗)免疫效果的影响.方法 对4-1BBL胞外功能区进行密码子优化,并通过NdeⅠ和XhoⅠ酶切位点定向插入pET28a表达载体.将获得的重组表达载体转化至大肠埃希菌BL21 (DE3),并用异丙基-β-D-硫代半乳糖苷诱导重组蛋白表达.用蛋白质印迹法对重组蛋白进行鉴定,并用非还原性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电子显微镜分析重组蛋白结构.用CCK-8试剂盒检测纯化r4-1BBL对小鼠T细胞体外增殖的影响.同时将r4-1BBL与HPV16蛋白疫苗联合免疫C57BL/6小鼠,用酶联免疫斑点试验检测小鼠的细胞免疫应答水平,观察r4-1BBL对HPV16蛋白疫苗抑制肿瘤生长的影响.结果 密码子优化的4-1BBL胞外功能区能在重组表达载体中表达,且表达产物同时以包涵体和可溶性形式存在.可溶性产物的结构主要为二聚体,电子显微镜下可以观察到类似亚单位的结构.纯化r4-1BBL可促进小鼠脾淋巴细胞的体外增殖.与单纯HPV16蛋白疫苗相比,r4-1BBL联合HPV16蛋白疫苗能诱导更强的特异性细胞免疫应答(t=3.525,P=0.024)和产生更明显的肿瘤生长抑制作用(t=2.534,P=0.021).结论 r4-1BBL能在小鼠中提高HPV16蛋白疫苗的免疫效果,具有作为HPV16蛋白疫苗佐剂的潜力.     

肿瘤细胞裂解物抗小鼠B16F10黑色素瘤的作用研究

反复冻融B16F10肿瘤细胞制备裂解物,以白喉毒素(Diphtheria toxin,DT)为载体,OK432和来源于结核分枝杆菌(Mycobacterium tuberculosis)热休克蛋白70(HSP70)第407-426(mHSP70407～426,M)的两段串联重复序列M2为佐剂,制备了肿瘤细胞疫苗B16F10-DT-M2-OK432(BDTMOK),探讨其能否抑制小鼠B16黑色素瘤,并且对其抗肿瘤的作用机理进行部分探讨。以制备的BDTMOK免疫C57BL/6小鼠,分别检测体液免疫应答和细胞免疫应答。通过ELISA法,从血清中检测到高滴度的抗B16肿瘤细胞裂解物(B16 tumor cell lysate,B16TCL)类抗体。淋巴细胞增殖实验的结果显示,BDTMOK的免疫能够有效的刺激脾淋巴细胞的增殖。预防结合治疗性实验的结果显示,BDTMOK激发的免疫应答对于B16肿瘤攻击起到有效的保护作用,与PBS阴性对照组比较,皮下注射BDTMOK可以延长皮下移植瘤发生的潜伏期(P<0.05),并且平均瘤重显著降低(P<0.05);抑制了小鼠皮内肿瘤模型中的血管新生(P<0.01)。疫苗BDTMOK能有效的抑制小鼠B16黑色素瘤的生长。     

跨膜型超抗原金黄色葡萄球菌肠毒素A融合蛋白的抗肿瘤作用

目的　为扩大超抗原金黄色葡萄球菌肠毒素A(SEA)的抗瘤谱 ,制备跨膜型SEA融合蛋白 ,研究该蛋白制备的肿瘤疫苗的抗肿瘤作用。方法　在荷B16黑色素瘤的C5 7BL/ 6小鼠上 ,观察跨膜型SEA融合蛋白制备的肿瘤疫苗对荷瘤小鼠的免疫治疗作用和免疫保护作用 ,并通过乳酸脱氢酶 (LDH)释放法检测治疗组和免疫组小鼠脾细胞的天然杀伤细胞(NK)和细胞毒性T细胞 (CTL)活性。结果　融合蛋白制备的肿瘤疫苗能够显著抑制荷瘤小鼠肿瘤的生长 ,并延长其生存期 ,其脾细胞的NK和CTL活性显著增强。同时 ,该肿瘤疫苗对同种肿瘤细胞攻击可产生较强的免疫保护作用。结论　跨膜型SEA融合蛋白制备的肿瘤疫苗具有显著的抗肿瘤作用 ,可有效激发荷瘤小鼠机体的特异性和非特异性抗肿瘤免疫应答 ,增强CTL和NK活性。     

Expression and activity of cytochromes P450 2E1, 2A, and 2B in the mouse ovary: the effect of 4-vinylcyclohexene and its diepoxide metabolite.

4-Vinylcyclohexene (VCH), an occupational chemical, causes destruction of small preantral follicles (F1) in mice. Previous studies suggested that VCH is bioactivated via cytochromes P450 (CYP450) to the ovotoxic, diepoxide metabolite, VCD. Whereas hepatic CYP450 isoforms 2E1, 2A, and 2B can metabolize VCH, the role of ovarian metabolism is unknown. This study investigated expression of these isoforms in isolated ovarian fractions (F1, 25-100 microm; F2, 100-250 microm; F3, >250 microm; interstitial cells, Int) from B6C3F1 mice dosed daily (15 days; ip) with vehicle, VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day). Ovaries were removed and either isolated into specific ovarian compartments for mRNA analysis, fixed for immunohistochemistry, or prepared for enzymatic assays. mRNA and protein for all isoforms were expressed/distributed in all ovarian fractions from vehicle-treated mice. In the targeted F1 follicles, VCH or VCD dosing increased (p < 0.05) mRNA encoding CYP2E1 (645 +/- 14% VCH; 582 +/- 16% VCD), CYP2A (689 +/- 8% VCH; 730 +/- 22% VCD), and CYP2B (246 +/- 7% VCH) above control. VCH dosing altered (p < 0.05) mRNA encoding CYP2E1 in nontargeted F3 follicles (168 +/- 7%) and CYP2A in Int (207 +/- 19%) above control. Immunohistochemical analysis revealed the greatest staining intensity for all CYP isoforms in the Int. VCH dosing altered (p < 0.05) staining intensity in Int for CYP2E1 (19 +/- 2.4% below control) and CYP2A (39 +/- 5% above control). Staining intensity for CYP2B was increased (p < 0.05) above control in granulosa cells of small preantral (187 +/- 42%) and antral (63 +/- 8%) follicles. Catalytic assays in ovarian homogenates revealed that CYP2E1 and CYP2B were functional. Only CYP2E1 activity was increased (149 +/- 12% above control; p < 0.05) by VCH dosing. The results demonstrate that mRNA and protein for CYP isoforms known to bioactivate VCH are expressed in the mouse ovary and are modulated by in vivo exposure to VCH and VCD. Interestingly, there is high expression of these isoforms in the Int. Thus, the ovary may contribute to ovotoxicity by promoting bioactivation of VCH to the toxic metabolite, VCD.     

Ginkgo biloba exocarp extracts inhibits metastasis and its effects on TGF-β1/ERK1/2/MMP-9 signaling pathway in B16-F10 melanoma

OBJECTIVE To study the inhibition and mechanism of Ginkgo biloba exocarp extracts(GBEE)for the metastasis of B16-F10 melanoma in C57 BL/6 J mice. METHODS The metastasis model of B16-F10 in C57BL/6J mice was set up. The C57 BL/6 J mice were randomly separated into these groups: positive control,model control, normal control and GBEE treatment groups, n=10. The mice in positive group wereintraperitoneal(ip) injectioncis-dichlorodiamineplatinum(Ⅱ) at a dose of 5 mg·kg~(-1), twice a day for 7 d; model group and normal group were both intragastric gavage(ig) normal saline(NS) in a volume of 0.1 m L/10 g, once a day for17 d; the GBEE treatment groups were respectively ig GBEE 50, 100 and 200 mg · kg~(-1), once a day for 17 d.After the administration, the lung tissue was removed and the lung surface metastasis was observed; the rate of lung metastasis and anti-metastasis were calculated;the degree of lung metastasis was observed by HE staining; in vitro, the effect of GBEE on the migration rate of B16-F10 cel s was detected by wound healing test; Western Blot was used to detect the expression of TGF-β_1, ERK1/2,p-ERK1/2 and MMP-9 protein in B16-F10 cel s. RESULTS In vivo, we discovered that GBEE(50, 100, 200 mg·kg~(-1))cansuppress tumor lung metastasis of B16-F10 melanoma in C57 BL/6 J mice in a dose-dependent way. In vitro, we found that GBEE(20, 40, 80 mg·L~(-1)) can significantly inhibit B16-F10 cells treated for 24 h and 48 h migration in a time-and concentration-dependent way. GBEE(20, 40,80 mg ·L~(-1)) can suppressed TGF-β_1, p-ERK1/2/ERK1/2 and MMP-9 protein expression level in a concentrationdependent way. CONCLUSION GBEE significantly inhibit the metastasis of B16-F10 melanoma, and its mechanism of action is related to the inhibition of extracellular matrix degradation and cell migration through the TGF-β_1/ERK1/2/MMP-9 signaling pathway.     

黄芪甲苷对病毒性心肌炎小鼠心肌纤维化的影响(英文)

目的 :探讨黄芪活性成分黄芪甲苷对小鼠病毒性心肌炎心肌纤维化的影响。方法 :BALB/C小鼠 10 0只 ,随机分成 6组。非感染小鼠腹腔无菌注射病毒培养液 ,分为正常对照组 (A组 ,12只 ,以生理盐水 0 .1mL灌胃 7d)、9%黄芪甲苷对照组(B组 ,16只 ,9%黄芪甲苷 0 .1mL灌胃 7d) ;余 72只小鼠以柯萨奇病毒 (CVB3)腹腔无菌注射制作病毒性心肌炎模型 ,模型小鼠随机分为心肌炎对照组和 1% ,3% ,9%黄芪甲苷干预心肌炎组 ,分别以生理盐水 ,1% ,3%和 9%黄芪甲苷 0 .1mL灌胃 7d (分别为C ,D ,E ,F组 ,每组 18只 )。 14d后处死小鼠 ,以ELISA法检测血清胶原前肽 (PIP和PII INP)浓度 ,心脏组织用天狼星红染色后测定胶原容积积分 (CVF) ,以流式细胞仪检测心肌细胞凋亡指数。结果 :9%黄芪甲苷干预心肌炎组小鼠较心肌炎对照组死亡率明显降低 [2 2 % (4/ 18)vs 4 4%(8/ 18) ,P <0 .0 5 ],9%黄芪甲苷对照组无小鼠死亡 ;与心肌炎对照组相比 ,CVF以及血清PIP和PIIINP浓度在 9%黄芪甲苷干预心肌炎组显著下降 (分别为 (10±s 3) %vs (2 1± 8) % ,(1.4±0 .6 )mg·L- 1vs (3.0± 1.9)mg·L- 1,(2 2± 4 )mg·L- 1vs (2 9± 5 )mg·L- 1,均P <0 .0 1) ,而 1%和 3%黄芪甲苷对心肌炎小鼠生存率和血清胶原前肽无明显影响 ;心肌细胞凋亡指?     

新型表位基因疫苗的构建及对小鼠黑色素瘤的影响

目的：构建以MAGE-1（161—169）为表位的癌症基因疫苗并检测其抗小鼠黑色素瘤效果。方法：构建基因疫苗pcDNA—HSP70-MAGE—l（161-169）,并免疫C57BL／6小鼠。最后一次免疫后第2周,接种小鼠黑色素瘤细胞。于肿瘤细胞接种后14d,处死全部动物,称量肿瘤的重量。采用ELISA法对小鼠血清中抗MAGE-1-IgG类抗体及小鼠原代脾淋巴细胞IFN-7释放水平进行检测。结果：pcDNA-HSP70-MAGE-1（161—169）表位基因疫苗能够成功地诱发机体产生抗MAGE-1的特异性抗体,并能在体内起到抗小鼠黑色素瘤作用,抑瘤率为40．7％。同时还能提高约3．05倍的小鼠原代脾淋巴细胞IFN-7释放量。结论：pcDNA-HSP70-MAGE-1（161-169）有望成为有效的抗小鼠黑色素瘤基因疫苗。     

Antimetastatic effect of an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B,Z-100, through the production of interleukin-12

In this study, the role of interleukin (IL)-12 on the antimetastatic effect of Z-100 was investigated using wild-type C57BL/6 mice or IL-12p40 knockout (IL-12p40 KO) mice inoculated with highly metastatic B16F10 melanoma. When C57BL/6 mice were inoculated with B16F10 melanoma (2x10(5) cells/mouse i.v.), Z-100 (10 mg/kg i.p.) significantly suppressed the pulmonary metastasis of B16F10 melanoma 14 d after tumor inoculation. On the other hand, the antimetastatic effect of Z-100 was not observed in IL-12p40 KO mice inoculated with B16F10 melanoma. These results indicate that IL-12 is essentially required for the appearance of the antimetastatic effect of Z-100. Since helper T (Th) 2 cell responses have been reported to have a role in tumor metastasis, the regulatory effect of Z-100 on the immune balance of Th1/Th2 cell responses was investigated. In both C57BL/6 mice and IL-12p40 KO mice bearing B16F10 melanoma, Th1 cytokine production (IL-2, interferon-gamma) was significantly suppressed as compared with those in normal mice. On the other hand, Th2 cytokine production (IL-4, IL-10) in these mice was increased. The administration of Z-100 (10 mg/kg i.p.) in C57BL/6 mice bearing B16F10 melanoma improved the balance of Th1/Th2 cell responses from the Th2-dominant state to the normal state. However, the improvement of Th1/Th2 cell responses by Z-100 was not observed in IL-12p40 KO mice bearing the same tumors. In addition, Z-100 significantly increased IL-12 production by macrophages in a concentration-dependent manner, while Z-100 significantly decreased IL-10 production by these cells in vitro. These results suggested that up-regulation of IL-12 production and down-regulation of IL-10 production by Z-100 are related to the improvement of Th1/Th2 cell responses from the Th2-dominant state to the normal state, which resulted in suppression of tumor metastasis.     

厄洛替尼对肺癌模型小鼠的时辰药理学作用及相关机制研究

目的:研究厄洛替尼按不同时辰给药的抗肿瘤作用,并探讨其可能的作用机制。方法:建立Levis肺癌细胞皮下移植瘤小鼠模型,随机分为A、B、C、D、E、F 6个厄洛替尼用药组和模型组,A^F组小鼠分别在对应的8:00、12:00、16:00、20:00、24:00、次日4:00灌胃给予厄洛替尼30 mg/kg,模型组小鼠给予同体积的羧甲基纤维素钠溶液。检测20 d内各组小鼠肿瘤体积及第21天时肿瘤瘤质量和肿瘤组织中表皮生长因子受体(EGFR)及其下游信号分子蛋白激酶B(PKB)、周期素依赖性激酶4(CDK-4)和细胞周期蛋白D1(Cyclin D1)mRNA的表达水平。结果:与模型组比较,AF组小鼠分别在对应的8:00、12:00、16:00、20:00、24:00、次日4:00灌胃给予厄洛替尼30 mg/kg,模型组小鼠给予同体积的羧甲基纤维素钠溶液。检测20 d内各组小鼠肿瘤体积及第21天时肿瘤瘤质量和肿瘤组织中表皮生长因子受体(EGFR)及其下游信号分子蛋白激酶B(PKB)、周期素依赖性激酶4(CDK-4)和细胞周期蛋白D1(Cyclin D1)mRNA的表达水平。结果:与模型组比较,A^F组小鼠肿瘤生长较缓慢(P<0.05),明期(8:00-16:00)肿瘤生长较暗期(20:00-次日4:00)缓慢,其中C组小鼠肿瘤生长最慢,E组小鼠肿瘤生长最快(P<0.05);AF组小鼠肿瘤生长较缓慢(P<0.05),明期(8:00-16:00)肿瘤生长较暗期(20:00-次日4:00)缓慢,其中C组小鼠肿瘤生长最慢,E组小鼠肿瘤生长最快(P<0.05);A^F组小鼠瘤质量均降低,其中A、B、C组比D、E、F组降低明显,C组最明显;A、B、C组小鼠EGFR、PKB、Cyclin D1 mRNA表达较D、E、F组降低明显。结论:厄洛替尼对肺癌模型小鼠的抗肿瘤作用初步认为明期比暗期明显,其作用机制可能与EGFR/PKB/Cyclin D1/CDK-4介导的凋亡通路有关。     

五步蛇毒抗肿瘤组分X对小鼠生殖毒性及F1子代的影响

目的研究蛇毒的抗肿瘤组分X(FX)对小鼠的生殖毒性及F1子代的影响。方法选状态良好、发育正常的雄性小鼠120只,雌性120只,分别将雌雄小鼠进行随机分组,雄性4组,每组30只,雌性4组,每组30只。给药按照低剂量组为2.5mg/kg;中剂量组为5mg/kg;高剂量组为10mg/kg;第4组为生理盐水阴性对照组(CK),观察对雄鼠、孕鼠一般生殖毒性、围产期生殖毒性的影响。结果低剂量FX对小鼠的一般生殖毒性和围产期生殖毒性影响与对照组比较无显著性差异,高剂量FX对小鼠的一般生殖毒性和围产期生殖毒性影响与对照组比较有显著性差异。结论结果提示5mg/kg剂量用于临床可能是安全的。     

Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis strain Aoyama B, augments anti-tumor activities of X-ray irradiation against B16 melanoma in association with the improvement of type 1T cell responses

In this study, the effects of combination therapy consisting of X-ray irradiation and Z-100 on the survival time of C57BL/6 mice inoculated with B16F10 melanoma were investigated. Survival time was significantly prolonged in B16F10 melanoma-bearing mice treated with the X-ray irradiation (5 Gy) and Z-100 (10 mg/kg s.c.) combination therapy compared with mice irradiated with X-rays alone. The weight of primary tumors and number of metastatic colonies were also significantly suppressed by the combination therapy compared with that in the X-ray irradiation group. These results indicated that Z-100 could enhance the anti-tumor effects of radiotherapy against B16F10 melanoma. On the other hand, the survival time of CD4 knockout mice bearing the same tumors was not prolonged by the combination therapy compared with mice irradiated with X-rays alone, suggesting that CD4+ cells are partly involved in augmentation of the anti-tumor effect of radiotherapy by Z-100. In addition, type 1 cytokine (IL-2, IFN-gamma) production was significantly increased and type 2 cytokine (IL-4, IL-10) production was significantly suppressed in the tumor-bearing mice treated with the combination therapy compared with the X-ray irradiation group. Moreover, interleukin-12 production by CD11c+ cells was also significantly increased in mice treated with the combination therapy compared with the X-ray irradiation group. These results indicate that Z-100 augmented the anti-tumor effects of X-ray irradiation. Moreover, we demonstrated that the effects of Z-100 were expressed at least in part, by the improvement of the T cell responses from type 2-dominant to type 1-dominant via up-regulation of IL-12 production.     

Efficient expression of modified human papillomavirus 16 e6/e7 fusion protein and the antitumor efficacy in a mouse model

Infection with human papillomavirus, particularly type 16 (HPV16), is highly associated with the development of cervical intraepithelial neoplasia and cervical cancer. The two early viral oncogenes, E6 and E7, are selectively retained and constitutively expressed in tumor cells and are therefore attractive immunotherapeutic targets. Thus a vaccine strategy based on recombinant HPV16 E6/E7 fusion protein represents an efficient approach against HPV16-associated tumors. Although the expression level of HPV16 E6/E7 fusion protein was presumed to be low, direct experimental proof in vivo was lacking. To enhance the expression level and investigate its antitumor efficacy in vivo, we constructed a modified HPV16 E6/E7 fusion gene with three point-mutations and expressed it in Escherichia coli. The encoded protein, denoted mE6(1-120)/mE7(1-60), comprises 120 N-terminus amino acids of E6 and 60 N-terminus amino acids of E7 plus a histine tag, was purified on an affinity column, and subsequently characterized by Western blotting. Immunization of mice with mE6(1-120)/mE7(1-60) completely protected them against subsequent challenge and rechallenge with TC-1 tumor cells expressing HPV16 E6 and E7 proteins. In the therapeutic experiments, most mice eliminated the preexisting tumors and had a long-term protection. Consistent with the results of in vivo experiments, the splenocytes from immunized mice elicited cytotoxic T lymphocytes and specifically lysed TC-1 cells in vitro. More importantly, the expression level of mE6(1-120)/mE7(1-60) was significantly improved, meeting the necessary quantity required for a vaccine clinical trial. In conclusion, these data provide a scientific basis for the use of modified mE6(1-120)/mE7(1-60) in future human trials.     

Serotonin 5-HT2 receptor antagonist does not reverse established ethanol-induced sensitization but blocks its development and expression

Several substances that inhibit the induction or expression of behavioral sensitization have been proposed, but patients who present for treatment often have already an established sensitized drug response. Serotonergic agents, including serotonin-2 (5-HT(2)) antagonists, reverse cocaine sensitization, but there is no evidence for the same effect with ethanol, although serotonin involvement in ethanol sensitization has been well reported. To evaluate a 5-HT(2C) antagonist effect on reversing established ethanol sensitization, three experiments were performed assessing locomotor activity of mice under different treatments. First, mice received daily intraperitoneal saline (S), mianserin 10 (M1) or 20 mg/kg (M2), ethanol 2 g/kg (E), or ethanol+mianserin for 21 days. Then, each treatment was withdrawn for 3 days, and mice were randomly challenged with S, E, M1, or M2. During the next 7 days, S and E groups were subjected to daily treatment with S, E, M1, or M2. On the eighth day, all rats were tested under ethanol challenge. The saline group expressed sensitization under ethanol challenge similarly to the ethanol group. Mianserin+ethanol blocked the development of sensitization, suggesting an involvement of the 5-HT(2C) receptor subtype on ethanol-induced sensitization. Ethanol challenge to the chronic mianserin group did not express sensitization, implicating a role for mianserin in protection against stress. Mianserin did not reverse established ethanol sensitization, suggesting that cocaine- and ethanol-induced sensitization involved different mechanisms.