免疫耐受叙利亚仓鼠人胃癌异种移植模型的免疫耐受检测

目的：研究免疫耐受叙利亚仓鼠人胃癌异种移植模型对移植瘤的免疫耐受状态。方法：给新生24小时之内叙利亚仓鼠直接腹腔接种活的人瘤细胞建立模型，然后进行淋巴细胞转化试验、补体依赖性细胞毒试验，观察移植瘤内宿主淋巴细胞浸润情况。经给新生叙利亚仓鼠腹腔接种死的人瘤细胞诱导免疫耐受后，再于3周龄时皮下接种活的人瘤细胞观察致瘤情况，研究移植瘤的生长是否与肿瘤分泌的免疫抑制因子直接相关。结果：移植瘤细胞不能刺激荷瘤鼠淋巴细胞转化；荷瘤鼠血清不能引起对移植瘤细胞的补体依赖性细胞毒效应；移植瘤内未见宿主淋巴细胞浸润；新生叙利亚仓鼠腹腔接种死的人胃癌细胞诱导免疫耐受后，再于3周龄时接种活的人胃癌细胞可以致瘤。结论：本模型动物对移植瘤产生了特异性免疫耐受。     

芒柄花黄素通过TLR4/NF-κB通路抑制肝癌荷瘤小鼠肿瘤免疫逃逸北大核心CSCD

目的:揭示芒柄花黄素对肝癌免疫逃逸的作用机制。方法:采用不同浓度(50、100、200μg/ml)芒柄花黄素处理人肝癌细胞系HepG2 24 h,CCK-8检测细胞增殖,Annexin V-FITC/PI试剂盒检测细胞凋亡。雄性C57BL/6小鼠皮下接种HepG2细胞(4×105个)建立荷瘤小鼠模型,10/50 mg(/kg·d)芒柄花黄素治疗28 d,计算抑瘤率。qRT-PCR、Western blot检测小鼠肝癌组织和HepG2细胞TLR4和NF-κB p65表达,ELISA检测小鼠血清和HepG2细胞TNF-α、IL-1β和IL-10水平。结果:芒柄花黄素可提高HepG2细胞增殖抑制率和凋亡率(P<0.05),降低荷瘤小鼠血清和HepG2细胞TNF-α、IL-1β和IL-10水平(P<0.05),提高荷瘤小鼠抑瘤率(P<0.05),降低荷瘤小鼠肿瘤组织和HepG2细胞TLR4和细胞核NF-κB p65蛋白表达(P<0.05),且呈剂量依赖性。结论:芒柄花黄素通过抑制TLR4/NF-κB通路介导的肿瘤免疫逃逸抑制肝癌发生发展。     

移植耐受诱导的异基因胎肝移植小鼠的免疫功能重建

本文用门静脉注射异型脾细胞加腹腔注射环磷酰胺方法，成功地诱导了成年Ｂａｌｂ／ｃ小鼠（Ｈ－２ｄ）对Ｃ５７ＢＬ／６（Ｈ－２ｂ）小鼠的免疫耐受。致死照射的耐受Ｂａｌｂ／ｃ小鼠用Ｃ５７ＢＬ／６（Ｂ６）小鼠的胎肝细胞移植后，无移植排斥产生。嵌合状态分析的结果表明，在胎肝移植后９０ｄ和２４０ｄ，重建的Ｂａｌｂ／ｃ小鼠的脾细胞分别有７４．４％和８３．７％来自于供体Ｂ６小鼠．证明Ｂ６小鼠胎肝造血干细胞已经在致死照射的Ｂａｌｂ／ｃ小鼠体内稳定植入。免疫功能检测的结果表明，在胎肝移植后９０ｄ，照射Ｂａｌｂ／ｃ小鼠的免疫功能已经重建。     

同基因造血干细胞移植延长同种异体小鼠皮肤移植物存活时间的实验研究

为了研究同基因造血干细胞移植诱导器官移植免疫耐受的可行性。建立小鼠异基因皮肤移植模型,术后2周给予FK506腹腔注射,3周起行全身照射及同基因骨髓移植,观察记录小鼠和移植物存活情况,以流式细胞检测受体GFP嵌合表达,混合淋巴细胞反应、迟发型超敏反应检测诱导耐受的特异性和效能。实验组小鼠移植物存活时间达(29.14±4.92)d,显著长于对照组(P<0.05);GFP在BMT后4周、6周嵌合程度达到82%、91%;实验组MLR、DTH结果与对照组差异显著,提示诱导耐受具有高度特异性和高效性。同基因造血干细胞移植联合免疫抑制剂治疗可以有效诱导小鼠皮肤移植的免疫耐受。     

过继转输的胚胎抗原耐受T、B细胞在受体孕鼠体内的归巢和分析

目的 :观察过继转输的胚胎抗原耐受T、B细胞在受体孕鼠体内的归巢和分布 ,以探讨免疫耐受T细胞在诱导受体母 胎免疫耐受中的作用机制。方法 :于孕 4天 (着床期 )给小鼠自然流产模型CBA J×DBA 2孕鼠腹腔注射抗CD80和CD86mAb ,以诱导母 胎免疫耐受。孕 9天应用免疫磁珠分选孕鼠脾脏T、B细胞 ,并用CFSE体外荧光标记。将标记的T、B细胞分别转输至孕 4天的CBA J×DBA 2孕鼠 ,36小时后在双光子共聚焦显微镜下观察T、B细胞在受体体内脾脏、子宫引流淋巴结及母 胎界面组织中的分布。结果 :过继转输的T、B细胞分布在孕鼠体内脾脏和子宫引流淋巴结 ,但并不留驻在母 胎界面。结论 :过继转输的胚胎抗原耐受T细胞定居于外周免疫器官 ,从而介导受体孕鼠对相应父系抗原的免疫耐受。     

电穿孔法转染GFP标记H22和S180的实验研究

采用电转染法将绿色荧光蛋白(GFP)基因导入小鼠肝癌H22和肉瘤S180细胞,通过G418筛选,建立了稳定表达该蛋白的小鼠肝癌H22细胞株和肉瘤S180细胞株;光镜和电镜观察其细胞形态、超微结构及生长状况没有发生显著改变;建立相应的皮下和腹腔荷瘤动物模型,观察到其皮下和腹腔成瘤时间与腹腔荷瘤生存时间无显著改变(P＞0.05),在活体荧光成像系统能观察到其荷瘤部位荧光.期望利用gfpH22和gfpS180阳性细胞株,运用免疫荧光技术、活体荧光成像系统和激光共聚焦系统对肿瘤进行体内外直观、可视和半定量的深入研究.     

抗人DR5单克隆抗体对人肝癌细胞系HepG2的凋亡作用

探讨抗人DR5单克隆抗体(mDRA-6)对人肝癌细胞系HepG2致凋亡的作用。常规培养人肝癌细胞系HepG2,流式细胞术定量分析检测HepG2细胞表面DR5的表达。MTT法检测mDRA-6的细胞毒性作用;Annexin V-FITC/PI双色标记HepG2细胞,流式细胞术定量分析检测细胞凋亡率;在荧光显微镜下观察mDRA-6对HepG2细胞形态变化的影响。结果显示,HepG2细胞表面有DR5表达,其平均表达百分率为40%。MTT法检测显示在40 mg/L mDRA-6浓度下可杀伤42%的细胞;Hoechst33258染色证实杀伤作用是通过细胞凋亡实现的经流式细胞术检测显示,3 mg/L的mDRA-6作用HepG2细胞6 h导致24.61%的细胞发生凋亡;在荧光显微镜下可观察到mDRA-6诱导导致HepG2细胞呈现典型细胞凋亡的形态特征。上述结果表明,mDRA-6能够诱导人肝癌细胞系HepG2凋亡。     

家蝇抗菌肽defensin对人肝癌细胞HepG2的增殖、凋亡和周期的影响

采用四甲基偶氮噻唑蓝（MTT）比色法,分析家蝇抗菌肽defensin对人肝癌细胞HepG2体外生长增殖与凋亡的影响,用倒置光学显微镜检测defensin对人肝癌细胞HepG2和人正常心肌细胞H9C2生长增殖情况的影响,并采用流式细胞术检测defensin对人肝癌细胞HepG2细胞凋亡及细胞周期的影响。结果显示,家蝇抗菌肽defensin对人肝癌细胞HepG2的生长有抑制作用,对人正常心肌细胞H9C2无抑制作用。通过显微镜观察HepG2细胞凋亡的形态变化,MTT检测发现defensin对肿瘤细胞的增殖抑制率呈剂量反应关系和时间反应关系。defensin能诱导人肝癌细胞HepG2发生凋亡,并且影响人肝癌细胞HepG2的细胞周期。家蝇抗菌肽defensin对人肝癌细胞HepG2具有增殖抑制作用及凋亡作用,对正常人心肌细胞H9C2基本无抑制作用。     

重组腺病毒载体mIL-12治疗小鼠黑色素瘤的实验研究

目的： 观察重组腺病毒载体AdmIL-12对小鼠黑色素瘤的治疗效应.方法： 皮下注射B16黑色素瘤细胞给C57BL/6小鼠建立荷瘤模型.于瘤内注射重组腺病毒载体AdmIL-12治疗, 观察皮下肿瘤的生长及荷瘤小鼠的存活期.采用乳酸脱氢酶（LDH）释放法检测荷瘤小鼠脾细胞的杀伤活性.结果： 重组腺病毒载体AdmIL-12治疗后, 能显著抑制荷瘤小鼠肿瘤的生长, 明显延长荷瘤小鼠的存活时间, 增强荷瘤小鼠脾细胞的杀伤活性.结论： 重组腺病毒载体AdmIL-12对小鼠黑色素瘤有显著的治疗效应.     

环孢素A诱导自然流产模型孕鼠脾脏CD4+CD25+Foxp3+的调节性T细胞扩增及外周母胎免疫耐受

目的探讨环孢素A(Cyclosporin A,CsA)对小鼠自然流产模型妊娠预后及外周母胎免疫耐受的影响.方法以正常妊娠模型CBA/J×BALB/c为对照组,自然流产模型CBA/J×DBA/2为研究组,CBA/J孕鼠于妊娠第4天分别腹腔注射5 mg/kg CsA.单向混合淋巴细胞反应分析孕鼠脾脏免疫细胞对父系抗原的增殖能力,ELISA分析上清液IL-2分泌水平,流式细胞术分析脾细胞CD4 CD25 Foxp3 调节性T细胞扩增,并观察两种模型各组胚胎吸收率.结果于妊娠第4天腹腔注射CsA显著降低自然流产模型孕鼠胚胎吸收率以及孕鼠外周免疫细胞对父系抗原的增殖能力与IL-2分泌(P＜0.01,P＜0.01,P＜0.01),促进脾脏CD4 CD25 Foxp3 调节性T细胞亚群扩增(P＜0.01).结论孕早期腹腔注射CsA可诱导自然流产模型孕鼠外周免疫细胞对父系抗原的特异性免疫耐受,从而使自然流产模型孕鼠的妊娠结局达到正常妊娠水平,提示CsA可能成为治疗妊娠失败的有效药物.     

脂质体介导的p53基因对肝癌细胞生长的抑制作用

目的 观察外源野生型ｐ５３基因在肝癌基因治疗方面的可行性。方法 将载有人野生型ｐ５３－ｃＤＮＡ的真核表达质粒ｐ５３－ｐｃＤＮＡ３,用阳离子脂质体介导转染人肝癌细胞系ＨｅｐＧ２,用流式细胞仪检测ｐ５３－ｐｃＤＮＡ３对ＨｅｐＧ２细胞生长的影响。结果 通过观察细胞生长曲线与流式细胞仪检测细胞周期和细胞的凋亡指数发现,ＨｅｐＧ２细胞生长受到明显的抑制。结论 脂质体介导的ｐ５３基因可在Ｈ细胞中表达,且明显抑     

Effects of granulocyte/macrophage colony-stimulating factor on the development and differentiation of CD5-positive macrophages and their potential derivation from a CD5-positive B-cell lineage in mice.

In co-cultures of either the murine pre-B cell line J13, fetal liver cells, or adult peritoneal or bone marrow cells with ST2 mouse bone marrow stromal cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), the development of CD5+ macrophages was demonstrated by immunohistochemical staining and flow cytometry. Although CD5+ macrophages were not present in the peritoneal cavities of normal mice, approximately 30% of the peritoneal macrophages in viable motheaten (mev/mev) mice, deficient in SHP-1 protein tyrosine phosphatase, expressed cell surface CD5 and B220, markers for B cells. In the mev/mev mice, GM-CSF level in peritoneal fluid was increased significantly. At 5 days after daily intravenous injection with GM-CSF, many CD5+ macrophages appeared in the peritoneal cavity and in omental milky spots of normal mice but fewer in osteopetrosis (op) mutant mice, deficient in macrophage (M)-CSF. These results indicate that GM-CSF, in combination with M-CSF, induces the development and differentiation of CD5+ macrophages in the peritoneal cavity, particularly in the omental milky spots of mice. In the peritoneal cavity of GM-CSF-treated mice, the percentages of hematopoietic progenitor cells doubly positive for CD5 and CD34 or c-kit and of macrophage precursor cells doubly positive for CD5 and ER-MP58 or ER-MP20 were increased significantly during the development of CD5+ macrophages and CD5 B cells, suggesting that CD5+ macrophages and B cells may share a bipotential progenitor in vivo.     

Peritoneal metastasis inhibition by linoleic acid with activation of PPARγ in human gastrointestinal cancer cells

The effect on peritoneal metastasis of linoleic acid (LA) was examined using in vitro treatment of cancer cells and mouse peritoneal metastasis models. Firstly, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by LA in a dose-dependent manner with increment of apoptosis. LA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor gamma (PPARγ) or 15-lipoxygenase-1, which converts LA to PPARγ ligands. LA significantly inhibited invasion into type-IV collagen-coated membrane of MKN28 and Colo320 cells (p<0.05). BALB/c nu/nu mice inoculated with MKN28 and Colo320 cells into their peritoneal cavities were administrated with LA intraperitoneally (weekly, four times). The LA treatment significantly diminished the number of metastatic foci of both cells in the peritoneal cavity (p<0.05). Protein production in MKN28 and Colo320 cells treated with LA showed a decrease of epidermal growth factor receptor and an increase of Bax. These findings suggest that LA inhibits invasion and metastasis of human gastric and colon cancer cells by nondietary administration.     

瘤内注射MIP-3α对小鼠肝癌生长抑制作用的研究

目的:探讨瘤内注射巨噬细胞炎症蛋白-3α(macrophage inflammatory protein-3α,MIP-3α)能否趋化外周树突状细胞(Dendritic cells,DCs)至肿瘤组织内,诱导特异性免疫应答。方法:成功建立小鼠皮下肝癌模型后随机分为3组,第10天起分别向MIP-3α治疗组、PBS对照组的小鼠皮下肿瘤内注射MIP-3α溶液及PBS,空白对照组小鼠不予任何处理。20天后取肿瘤组织,免疫组化法检测肿瘤内DCs、CD4+、CD8+细胞浸润情况,流式细胞术检测肿瘤内DCs浸润数量及其表型。另外,每组各10只小鼠持续观察,用于绘制肿瘤生长曲线并观察生存时间。结果:①MIP-3α治疗组的小鼠肿瘤内浸润的CD4+、CD8+细胞及DCs数量均显著高于其他两组。②MIP-3α治疗组小鼠肿瘤内浸润性DCs的CD80、CD86表达率显著高于其他两组(P<0.05)。③MIP-3α治疗组小鼠肿瘤生长速率显著低于对照组(P<0.001),生存时间较对照组明显延长(P<0.05)。结论:①瘤内注射MIP-3α可在小鼠肝癌病灶内趋化、募集外周的树突状细胞,使其摄取并提呈肿瘤抗原,有效诱导针对肝癌细胞的特异性免疫应答。②MIP-3α在小鼠皮下肝癌模型的局部微环境下可能具有促进树突状细胞成熟的作用。     

Sephadex induces eosinophil migration to the rat and mouse peritoneal cavity: involvement of mast cells,LTB4, TNF-α, IL-8 and PAF

OBJECTIVE AND DESIGN: In this study we investigated the chemotactic mediators involved in the Sephadex-induced eosinophil migration into the peritoneal cavities of rats and mice, and which resident peritoneal cells release these mediators. MATERIALS AND METHODS: Sephadex suspension was injected into the peritoneal cavities of rats or mice which were pretreated, or not, with specific drugs that inhibit synthesis or production of the inflammatory mediators and eosinophil chemotactic activities were observed. To investigate the role of resident peritoneal cells as a source of these chemotactic factors, the macrophage population was enhanced or the mast cell population was depleted. The resident cells were also stimulated, in vitro, with Sephadex and the chemotactic activity of the supernatants was determined. RESULTS: Sephadex induced dose and time dependent eosinophil migration in rats and mouse, which were inhibited by dexamethasone and MK 886. BN 52021 only affected the eosinophil migration into the mouse peritoneal cavity. An increase in the macrophage population did not alter the eosinophil migration induced by Sephadex in rat or mouse. However, mast cell population depletion reduced eosinophil migration in rats, but did not alter the migration in mice. Sephadex-stimulated rat mast cells released an eosinophil chemotactic factor whose release was inhibited by dexamethasone and MK 886. Anti-TNF-alpha and anti-IL-8 Abs inhibited the chemotactic activity of the mast cell supernatant. CONCLUSION: Sephadex-induced eosinophil migration into the rat peritoneal cavity is dependent on mast cells, which release LTB4, TNF-alpha and CINC-1. Conversely, Sephadex-induced eosinophil migration into the mouse peritoneal cavity is mediated by PAF and LTB4, which are not released from resident macrophages or mast cells.     

采用HepG2细胞株建立裸鼠肝癌模型的方法

目的 :探讨采用HepG2 细胞株建立裸鼠肝癌模型的方法。方法 :采用皮下注射HepG2 细胞 (1 2× 1 0 6)建立裸鼠肝癌模型 ,观察种植细胞后 1 4天内裸鼠的死亡率 ,并在 1 4天时活杀动物测定瘤体重量、直径及血清AFP浓度。结果 :该法造模成功率达 1 0 0 %。结论 :该方法操作简便 ,周期短 ,成功率高等可作为肝癌模型应用     

量子点探针对人肝癌裸鼠模型的体内靶向成像研究

目的研究量子点标记靶向探针对人肝癌裸鼠模型的体内成像技术。方法将巯基乙酸修饰的水溶性量子点结合鼠抗人甲胎蛋白（AFP）单克隆抗体制备成水溶性量子点-AFP-Ab复合物探针。荧光、紫外光光谱分析及透射电镜研究其特性。通过直接免疫荧光法,用该复合物探针特异性识别肝癌细胞株HCCLM6 AFP抗原。将体外培养的肝癌细胞株HCCLM6通过皮下接种和尾静脉注射裸鼠分别建立人肝癌裸鼠模型和肺转移模型。尾静脉注射量子点-AFP-Ab探针,用蓝光二极管照射获得活体荧光成像;用掺Ti蓝宝石激光器照射,对肿瘤部位和正常部位进行光谱分析。取血清检测丙氨酸转氨酶、天冬氨酸转氨酶、尿素氮和肌苷水平。取裸鼠肝、脾、肾、肺、心和脑6种主要实质性器官行振荡切片,共聚焦显微镜观察,研究量子点-AFP-Ab探针在裸鼠体内的非特异性摄取。结果量子点-AFP-Ab复合物探针具有激发光谱宽、荧光强度高的特点,能特异性与肝癌细胞AFP抗原高亲和力结合,在体内能特异性靶向肿瘤组织进行活体成像,无明显急性毒性。光谱分析显示量子点-AFP-Ab复合物探针主要分布于肿瘤的外周部位,少数该探针被肝、脾和肺非特异性摄取。结论量子点-AFP-Ab复合物探针具有优良的光学特性和生物相容性,能够进行肝癌体内靶向成像,将有助于肝癌的分子靶向研究。     

The cellular cancer resistance of the SR/CR mouse

The SR/CR mouse phenotype, first described in 1999 in BALB/c and later bred into C57BL/6 mice, is resistant to cancer formation following high doses of cancer cells administered intraperitoneally. The tumor cell targeting and destruction mechanisms have not been identified. By fluorescence‐activated cell sorting analysis, the immune response of SR/CR mice after intraperitoneal injection of cancer cells was investigated and compared with parent strain mice. A massive influx of leukocytes into the peritoneal cavity was found. A large fraction of these leukocytes were polymorphonuclear granulocytes, macrophages and natural killer cells. A relative decrease in influx of B‐cells compared with controls was demonstrated. Increased proportions of leukocytes belonging to the innate immune system were also demonstrated in splenocytes of SR/CR mice. Cytospins of peritoneal fluid from SR/CR mice after cancer cell injection showed formations of immune cells morphologically resembling polymorphonuclear granulocytes and macrophages adjoining the cancer cells. The results point to the potential involvement of innate immune cells in cancer immunology. Our data support migration of polymorphonuclear granulocytes, macrophages and NK cells into the peritoneum of the SR/CR mouse in response to intraperitoneal injection of S180 cancer cells. The cell composition of spleens of SR/CR mice reflected the differential regulation of the innate immune cells in peritoneal exudates. Both peritoneal exudates and the spleens of SR/CR mice contained decreased proportions of B‐cells compared with BALB/c and C57BL/6 mice. We reproduce important aspects of previous published data and further extend them by showing differentially regulated populations of splenocytes including B‐lymphocytes in SR/CR mice compared with parent strain controls. Importantly, this differentially regulated immune response of SR/CR mice could not be found in response to challenge with the lymphoma cell line EL‐4.     

AFP启动子驱动的yCD/TK双自杀基因靶向杀伤肝癌细胞的体内外研究

目的： 研究携带甲胎蛋白（AFP）启动子的酵母菌胞嘧啶脱氨酶/胸苷激酶（yCDglyTK）双自杀基因体内外靶向性杀伤肝癌细胞的效果和机制。方法: 构建携带AFP启动子的yCD/TK双自杀基因表达质粒。通过阳离子脂质体将携带AFP启动子的yCD/TK双自杀基因转染HepG2和SMMC7721细胞,用MTT法测定不同浓度氟胞嘧啶（5-FC）、更昔洛韦（GCV）及联合治疗的杀伤作用,用流式细胞仪检测细胞周期。建立裸鼠肝癌皮下种植瘤模型,观察自杀基因体内杀瘤效果以及细胞凋亡的情况。结果: 成功构建的携带AFP启动子的yCD/TK双自杀基因靶向性地在AFP阳性的HepG2细胞上表达,而AFP阴性的SMMC7721细胞无表达,GCV、5-FC及两者联合可有效抑制HepG2细胞生长,随药物浓度的增高而杀伤作用增强,药物间抑瘤效果比较是GCV＋5-FC＞5-FC＞GCV,而SMMC7721细胞的生长未受影响。体内实验可见GCV、5-FC及两药联合对转染后的HepG2细胞种植瘤有明显的抑制效果,并检测到明显的细胞凋亡,而对SMMC7721细胞种植瘤的生长无影响,种植瘤内极少凋亡细胞。结论: 携带AFP启动子的yCD/TK双自杀基因能有效地靶向性地杀伤AFP阳性的肝癌细胞,细胞凋亡可能是其杀伤的重要机制之一。