特异性转移因子对人AFP促进小鼠H22腹水肝癌细胞RNA合成的拮抗效应

用亲和层析法从3～5月龄引产胎儿肌肉组织中提纯AFP.用纯化AFP免疫小鼠,取其脾脏按匀浆透析法制备AFP特异转移因子(SP-TF),取正常未免疫鼠脾脏制备非特异转移因子(N—TF),从预接种小鼠腹水中分离Hz2腹水肝癌细胞(H22细胞),体外用[3H]—UR参入方法,从转录水平观察SP—TF对AFP促进小鼠H22细胞生长的拮抗效应。结果表明:SP—TF对AFP促进H22细胞RNA合成具有非常显著的拮抗效应。此效应具有抗原特异性和供体依赖性。     

必需脂肪酸对BEL-7402人肝癌细胞生长和甲胎蛋白分泌抑制的研究

本实验用放射免疫法研究了必需脂肪酸亚麻酸和亚油酸对BEL-7042人肝癌细胞甲胎蛋白(AFP)的分泌的影响,并观察它们对肝癌细胞生长的抑制作用.实验结果表明,42～50μg/ml亚麻酸能明显地抑制肝癌细胞AFP分泌(P<0.01),亚油酸则作用较弱,与对照相比无差异(P>0.05),我们对亚麻酸的抗癌作用的研究为癌症病人的保健和治疗提供实验依据.     

人AFP腺病毒载体感染的树突状细胞诱导小鼠抗肝癌免疫

目的探讨复制缺损型腺病毒载体(Ad)介导异种AFP修饰的树突状细胞(DCs)诱发抗肝癌免疫、打破肿瘤免疫耐受的效果。方法从HepG2和Hepa16细胞中克隆人和小鼠AFP,插入Ad中构建AdhAFP和AdmAFP。用AdhAFP或AdmAFP感染小鼠骨髓来源的DC后,在无或有删除CD4或CD8情况下免疫C57BL/6小鼠,7d后取脾细胞行51Cr释放实验,检测特异性CTL杀伤活性;或给免疫小鼠接种Hepa16肝癌细胞,观察荷瘤小鼠成活情况。结果AdhAFP/DCs免疫小鼠1周后其细胞毒性T淋巴细胞杀伤活性明显强于AdmAFP/DCs。AdhAFP/DCs免疫小鼠后1周接种5×106Hepa16肝瘤细胞,2个月后仍然有80%的小鼠无瘤生长;而接种1×106Hepa16细胞至AdmAFP/DCs免疫小鼠,2个月后小鼠成活率为20%。删除小鼠CD4或CD8T细胞均使AdhAFP/DCs诱发的抗肿瘤免疫反应消失。结论Ad介导异种AFP修饰的DCs能有效地打破肿瘤的免疫耐受,诱发强烈的抗原特异性细胞免疫反应,这种特异性细胞免疫反应是CD4和CD8依赖性的。     

腺病毒介导CD/5FC“自杀基因”疗法对人肝癌裸鼠和小鼠结肠腺癌的治疗作用研究

“自杀基因”疗法应用于肿瘤治疗时,通常是在肿瘤局部直接转染“自杀基因”,以期达到在肿瘤局部发挥抗肿瘤作用而避免全身毒性.在本实验中,我们观察了腺病毒介导的“自杀基因”疗法(CD/5FC系统)对人肝细胞癌SMMC-7721及小鼠结肠腺癌CT26的生长抑制作用,证实了腺病毒介导的CD/5FC系统在体外能杀伤人肝癌细胞SMMC-7721,并且能明显地抑制裸鼠SMMC-7721肿瘤模型的生长,对小鼠的结肠腺癌CT26也有一定的治疗作用.同时观察了以AFP启动子驱动的CD基因合并5FC的使用对AFP阳性肝癌细胞的特异性杀伤作用,证实了以肝癌特异启动子驱动的CD基因能在高表达AFP的肝癌细胞HepG2中特异表达,合并5FC的使用能在体外特异地杀伤HepG2细胞.这一组织特异性的“自杀基因”系统能特异地抑制高水平分泌AFP的人肝细胞癌裸鼠模型7721AFP( )的生长,上述研究结果表明,腺病毒介导的CD/5FC系统是一个有效的治疗方案,组织特异性的表达调控更能体现该疗法的实用价值.     

负载人AFP肽段的树突细胞在体内外对小鼠肝癌的抑制作用

背景与目的:甲胎蛋白(alpha-fetoprotein,AFP)是原发性肝细胞癌(hepatocellular carcinoma,HCC)免疫治疗的一个良好的靶分子,如何克服对自身抗原的免疫耐受状态是诱导有效抗肿瘤免疫反应的关键.本研究探讨异种同源蛋白疫苗负载人AFP肽段的树突细胞(human AFP-derived peptide-pulsed dendritic cells,hAFP-DCs)对小鼠肝癌的体外杀伤作用和体内抑瘤效应.方法:传统方法制备骨髓来源的DCs.MTT法检测hAFP-DCs诱导的CTL对小鼠肝癌细胞Hepal-6的体外杀伤活性.建立Hepal-6细胞C57BL/6小鼠移植瘤模型,分别瘤内注射hAFP-DCs、DCs和PBS(每周两次),观察小鼠肿瘤体积和荷瘤存活时间.结果:成功制备小鼠骨髓来源的DCs.体外杀伤实验显示,hAFP-DCs刺激组和单纯DCs刺激组CTL对Hepal-6细胞的杀伤作用强于PBS组,但组间差异无统计学意义(P>0.05).体内实验表明.每个C57BL/6小鼠接种7×106个Hepal-6细胞31 d后,hAFP-DCs、DCs和PBS组小鼠平均移植瘤体积分别为(195.04±155.22)mm3、(360.65±209.02)mm3和(756.19±503.24)mm3,组间比较差异有显著性(P<0.001).在40 d的观察期内,小鼠的累积存活率分别为100%、90%和50%(P=0.008).结论:负载人AFP抗原肽的DEs疫苗在体外和体内均能有效抑制小鼠肝癌的生长.     

高强度聚焦超声对前列腺癌小鼠的疗效及T细胞亚群的影响

目的探讨高强度聚焦超声（high intensity focused ultrasound, HIFU）对前列腺癌荷瘤小鼠的疗效及T细胞亚群的影响。方法RM-1细胞接种于C57BL6小鼠建立小鼠前列腺癌皮下移植瘤模型,随机分为肿瘤对照组、HIFU组、放疗组和HIFU联合放疗组。观察治疗后各组的肿瘤生长曲线,计算肿瘤体积倍增时间（DT）。2周后,通过流式细胞仪检测正常小鼠和荷瘤小鼠外周血CD3⁺、CD3⁺CD4⁺、CD3⁺CD8⁺ 细胞占白细胞总数的百分比。结果与肿瘤对照组相比,HIFU组、放疗组肿瘤生长延缓,DT显著延长。HIFU组小鼠的CD3⁺(%)、CD4⁺(%)显著高于肿瘤对照组,而放疗组与肿瘤对照组差异无统计学意义。HIFU联合放疗组的DT较HIFU组显著延长,小鼠的CD3⁺(%)、CD4⁺(%)显著高于HIFU组,且CD4⁺(%)显著高于正常小鼠。结论HIFU治疗前列腺癌不仅能局部抑制肿瘤生长,而且能通过对T细胞亚群的影响提高机体的细胞免疫水平。HIFU联合放疗能进一步提高疗效。     

赖氨匹林对C3H小鼠移植性乳腺癌的抑制作用

背景与目的:在肿瘤的发生、发展中,环氧合酶(cyclooxygenase,COX)起了很重要的作用,而多种COX抑制剂可以抑制肿瘤细胞增殖,诱导肿瘤细胞凋亡。本实验观察不同浓度赖氨匹林(Aspisol)对小鼠移植性乳腺癌的抑制作用以及对肿瘤细胞凋亡和对肿瘤组织环氧合酶-2(COX-2)、Caspase-3蛋白的表达的影响,并探讨其抑制乳腺癌生长的作用机制。方法:用细胞悬液法将肿瘤细胞接种于C3H小鼠前肢腋窝皮下,制备可移植肿瘤模型。次日给予不同浓度的Aspisol腹腔注射,每天一次,共28天,用氟尿嘧啶(5-fluorouracil,5-FU)和生理盐水分别作为阳性对照和阴性对照。计算Aspisol的抑瘤率;原位凋亡检测法(TUNEL)检测Aspisol对肿瘤细胞凋亡的影响;免疫组化法检测Aspisol对小鼠肿瘤组织的COX-2、Caspase-3表达的影响。结果:175、350、700mg/kgAspisol对小鼠乳腺癌的抑瘤率分别为38.9%、48.2%、47.0%,10mg/kg5-FU的抑瘤率为60.4%。各给药组肿瘤细胞均呈现明显凋亡形态改变,不同浓度Aspisol均明显下调小鼠肿瘤组织COX-2的表达,增强Caspase-3表达。结论:Aspisol对C3H小鼠移植性乳腺癌的生长具有抑制作用;能诱导肿瘤细胞的凋亡,其机制可能与抑制COX-2表达、增强Caspase-3表达有关。     

抑癌因子对小鼠实体瘤生长抑制作用的研究

目的　观察人抑癌因子直接瘤内注射对小鼠实体瘤生长的影响。方法　小鼠右腋皮下分别接种S180 和肝癌细胞 ,成瘤后 ,让小鼠带瘤生长半月 ,再于瘤内注射抑癌因子 ,并设注射生理盐水为对照组 ,观察小鼠的肿瘤生长、瘤体变化及生存情况。结果　抑癌因子能强烈地抑制小鼠肿瘤的生长 ,使肿瘤体积明显缩小 ,实验组与对照组比较 ,差异非常显著 (P <0 .0 1 )。实验组小鼠的寿命显著延长 ,S180 和肝癌小鼠的寿命延长率分别为 1 6 3%和 1 4 8%。结论　抑癌因子可能通过某种机制启动细胞凋亡信号传递系统 ,诱导并加速癌细胞的凋亡 。     

小鼠AFPcDNA真核表达载体的构建、表达及体外抗肝癌免疫活性的检测

背景与目的:探索肝癌细胞的突变基因产物——具有潜在抗原性的异常蛋白质而无法形成有效免疫原的机理,寻找肝癌的特异性抗原,并在此基础上研制出治疗肝癌的DNA疫苗将对肝癌的免疫学治疗产生积极的影响。本实验构建BALB/c小鼠具有分泌性信号肽的AFP1cDNA和去掉信号肽的AFP2cDNA真核表达载体,分别在小鼠树突细胞(dendriticcells,DCs)中表达,并观察其在体外抗肝癌免疫中的作用。方法:采用RT-PCR方法自小鼠肝癌细胞株H22中扩增出具有分泌性信号肽的AFP1cDNA和去掉分泌性信号肽的AFP2cDNA,将该基因定向插入真核表达载体PEGF-N3中;同时培养经rmGM-CSF、rmIL-4诱导的小鼠骨髓细胞,体外获取大量DCs,脂质体转染PEGF-N3/AFP1、PEGF-N3/AFP2至DCs进行表达、鉴定。将DCs疫苗与同源小鼠脾淋巴细胞混合培养,ELISA方法测定脾细胞γ-干扰素(IFN-γ)分泌活性,51Cr释放法测定脾淋巴细胞的特异性杀伤活性。结果:从H22中克隆到AFP1cDNA和AFP2cDNA,经测序完全正确,所构建的真核表达质粒PEGF-N3/AFP1和PEGF-N3/AFP2在小鼠DCs中获得稳定高效表达,其中PEGF-N3/AFP2明显刺激T细胞增殖,刺激指数(SI)为5.12±1.46,明显高于空质粒组(1.42±0.73)。AFP2/DC疫苗对H22细胞的特异性杀伤活性[(88.15±16.47)%]明显高于空质粒组[(12.72±5.45)%]。结论:成功地构建了真核表达载体PEGF-N3/AFP1和PEGF-N3/AFP2,编码去掉分泌性信号肽的PEGF-N3/AFP2转染的DC,能够诱导较强的特异性抗肝癌免疫效应。其机制可能为诱导肝癌细胞凋亡。     

莲房原花青素对黑色素瘤B16细胞的诱导分化作用

背景与目的:探讨莲房原花青素(LSPC)对荷瘤小鼠体内黑色素瘤B16细胞的诱导分化作用.材料与方法:以荷黑色素瘤B16小鼠为模型,随机分LSPC 3个剂量(60、120、240 mg/kg)组和阴性对照组,每组10只,实验组用LSPC、阴性对照组用等体积溶剂分别灌胃13 d后取材,检测黑色素瘤B16细胞中酪氨酸酶活力、黑色素含量、S-100蛋白表达,并用光学显微镜和透射电镜观察其细胞形态的变化.结果:60～120 mg/kg的LSPC能抑制黑色素瘤B16细胞在小鼠体内生长,且呈浓度依赖关系.其中,120 mg/kg LSPC组作用最显著(P＜0.01),抑瘤率达55.3%,并使B16细胞内酪氨酸酶活力和黑色素含量分别提高28.5%和23.8%,与阴性对照比较,差异有统计学意义(P＜0.05),并使S-100蛋白表达下降.结论:LSPC显著抑制B16细胞在小鼠体内的生长,并从形态上和功能上诱导B16细胞分化.     

Stimulation of tumor-cell growth by alpha-fetoprotein

It has been recognized that alpha-fetoprotein (AFP), as an oncofetal antigen, is re-expressed in large amounts in adult tumor cells and serves clinically useful purposes in tumor-marker assays. However, its biological activities are still undefined. In the present study, the ability of AFP to stimulate tumor-cell growth was observed by an in vitro experimental system. Mouse ascites cancer cells derived from hepatoma-22(H-22) or Ehrlich ascites carcinoma(EAC) were extracted intraperitoneally and cultured in RPMI 1640 medium containing 10% newborn calf serum for 48 hr. Cell growth was quantitated by a colorimetric assay using a MTT microculture tetrazolium dye. The results demonstrated that AFP significantly increased H-22-cell proliferation, with stimulation percents of 122 to 156%. A similar growth-promoting effect of AFP was observed using EAC cells, with stimulation percents of 86 to 210%. Moreover, the growth-stimulatory activity of AFP could be abrogated with anti-AFP antibodies. In addition, 5-fluorouracil could obviously inhibit AFP-induced proliferation of H-22 or EAC cells in vitro. These results suggest that AFP is associated with tumor-cell growth and may serve as an important target of tumor therapy. Int. J. Cancer 75:596–599, 1998. © 1998 Wiley-Liss, Inc.     

氧化赖氨酸的抗肿瘤作用及其免疫学活性

目的研究氧代赖氨酸（ＯＸＬ）抗肿瘤作用及其免疫学活性。方法用 用ＭＴＴ法体外观察小鼠腹水肝癌－２（Ｈ－２２）细胞的生长及其脾淋巴细胞增殖。结果ＯＬＸ９６．３－２００μｇ／ｍｌ）单用对比Ｈ－２２细胞生物抑制作用较小;但与４－氟脲嘧啶（５－Ｆｕ）合用,可协同抑制Ｈ－２２细胞生长、抑制率由单用ＯＸＬ（２５μｇ／ｍｌ）时的１３．８％或单用５－Ｆｕ（３．１μｇ／ｍｌ）时的１８．９％,提高到合用的４３．１％。     

Characterization of "H-2K,D-like structures" on MM2 X mouse L cell hybrids. I. "H-2K,D-like" alloantigen encoded by the D region and/or to the right of the D region of the H-2 gene complex

It was found that anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, detected molecules of 44-46,000 and 12,000 daltons on EL4 leukemic cells and on C57BL/6 lymph node cells, as well as on somatic cell hybrids between the MM2 and mouse L cells. Experiments with a known rabbit anti-mouse beta2-microglobulin serum showed that the two molecules detected by anti-MM2 serum were hydrophobically associated with each other in membrane extracts; thus, the antigen detected by anti-MM2 serum was structurally similar to H-2K and D antigens. Preclearing of lysates from C57BL6 lymph node cells with a mixture of anti-H-22, anti-H-2.33, and anti-H-2.28 sera did not remove the "H-2-like" antigen, indicating that the antigen was distinct from the H-2Kb and Db molecules. Neither C3H/He nor B10.BR lymph node cells expressed the "H-2-like" antigen, but B10.A(4R) and B10.AM lymph node cells did possess the antigen. Absorption of anti-MM2 serum with EL4 cells abolished the capacity of the absorbed serum to precipitate the "H-2-like" antigen activity on C57BL/6 lymph node cell extracts and reduced the "H-2-like" radioactive peaks of the hybrid cells. These results indicate that there are at least two components being recognized by the anti-MM2 serum in hybrid cells between MM2 tumor and mouse L cells, both of which had originated in the C3H/He mouse (H-2k). One is the same as or cross-reactive with an "H-2-like" alloantigen of normal C57BL/6 lymph node cells (H-2d) and the other is another "H-2-like" antigen. Experiments with recombinant mice show that the "H-2-like" alloantigen on lymph node cells is coded for by the D region and/or to the right of the D region of the major histocompatibility complex (MHC).     

Selection of a low antigenic variant subline from the TA3St ascites carcinoma by repeated passage of antibody-coated cells through anti-immunoglobulin columns.

TA3St mouse ascites carcinoma cells of strain A origin (H-2a) were coated with anti-H-2a serum and passaged through mouse immunoglobulin-anti-immunoglobulin columns repeatedly prior to each in vivo passage. After six passages in vivo, a variant cell line emerged with significantly decreased H-2a expression and an increased ability to grow progressively across H-2 allograft barriers. Chromosome analysis of the variant cell line revealed no major changes in karyotype. The application of the method for studies on the generation of immunoresistant tumor cell variants and on the relationship between antibody-binding sites and rejection target sites is discussed.     

Inhibition of sensitization of T-cells by alpha-fetoprotein.

A two-phase model of allograft immunity was studied. In the first phase, specific immune T-cells were generated by incubation of responder cells with mitomycin-C treated allogeneic stimulator cells of the same H-2 type as mouse mastocytoma tumor cells. In the second step, the ability of the sensitized cells to kill Cr51-labelled P-815 mastocytoma cells was assayed. Alpha-fetoprotein (AFP) was shown to inhibit the generation of immune cytotoxic T-cells at low concentrations (1-100 ng/ml) when added at the beginning of the first phase. When added at the end of the first phase or in the second (killing) phase, AFP was found to have no significant effect on cytotoxicity, indicating that it did not inhibit the killer T-cell once it was generated.     

Effect of the tumor-bearing host serum on the leukocyte adherence inhibition reaction mediated by immune RNA

51Cr-labelled leucocyte adherence inhibition (LAI) assay was used as an index to study the activity of xenogenic immune RNA (iRNA) in inducing normal LACA mouse spleen lymphocytes to mediate tumor specific response in vitro. RNA was extracted from the spleen and lymph nodes of the guinea pigs immunized by S180 or H22 ascitic tumor cells and from those of the normal guinea pigs. Extracts of S180 or H22 tumor cells and that of normal LACA mouse livers were prepared by PBS method respectively. Three kinds of RNA and three kinds of cell (tissue) extracts were grouped into nine combinations. A defined number of 51Cr-labelled lymphocytes was added to each group. The results showed that only in the combination of two kinds of iRNA and the corresponding tumor cell extract, adherence of normal lymphocyte was significantly inhibited. Serum from tumor bearing host inhibited LAI reaction mediated by iRNA. The "blocking" effect was specific since it occurred only when the serum and tumor extract in the reaction system were taken from the same tumor-bearing donor.     

人甲胎蛋白基因启动子siRNA表达载体的构建及效应鉴定

目的：探索基于人甲胎蛋白基因启动子构建的siRNA表达载体介导目的基因RNA干扰的可行性，为实现肝癌细胞靶向RNAi治疗作一初步研究。方法：PCR法扩增获得人甲胎蛋白基因启动子并插入pSilencer1．0-U6中以置换其U6启动子；依据hLRH-1基因RNAi靶位点设计出siRNA模板DNA，体外合成相应寡核苷酸片段后经退火形成短双链，再克隆构建成靶向人hLRH-1基因siRNA表达载体；脂质体转染法将上述重组质粒转入HepG2细胞中，实时定量PCR法初步分析所建系统在AFP表达细胞中触发靶基因hLRH-1的RNAi效应，以及hLRH-1调控下游靶基因的表达改变。结果：获得人甲胎蛋白基因启动子驱动的siRNA表达载体pSiAFP。所建的RNAi载体系统可在表达AFP的HepG2细胞中有效诱导靶基因hLRH-1表达下调，抑制率约达85％；同时调控hLRH-1下游靶基因CyclinE 1与Oct4的表达相应下调。结论：人甲胎蛋白基因启动子驱动的siRNA表达载体可在表达AFP的HepG2细胞中有效介导靶基因的RNAi效应。     

Alpha fetoprotein: effect of heterologous antiserum on hepatoma cells in vitro.

Hepatoma cells derived from The Jackson Laboratory mouse hepatoma BW7756 synthesized alpha fetoprotein (AFP) in vitro. The AFP was immunologically identical to that circulating in the sera of hepatoma-bearing mice. An in vitro cytotoxic effect of rabbit antiserum to AFP was studied in hepatoma cells obtained both from fresh cell suspensions and short-term cell culture. The use of intact and/or inactivated anti-AFP serum inhibited the growth of the AFP-producing cells. The cytotoxic effects of the antiserum depended on exposure time and serum concentration. The cytotoxicity was complement independent, as demonstrated by studies with heat-deactivated serum devoid of extrinsic complement. The control target cells included fresh cell suspensions of normal mouse liver and mouse muscle fibroblasts grown in short-term culture. Specificity of the antisera for the target cells was demonstrated by absorption with purified mouse AFP. The results could be explained by the presence of AFP on the hepatoma cell surface.