抗人VEGF的单克隆抗体对乳腺癌生长及转移的影响

目的用具有中和活性的抗人血管内皮细胞生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＶＥＧＦ）单抗进行抑瘤动物实验，探讨ＶＥＧＦ在肿瘤生长及转移中的作用。方法采用杂交瘤技术制备抗ＶＥＧＦ的单抗，经亲和层析纯化单抗，在ＩＶＴ２ＭＡ－８９１津白ＩＩ小鼠自发乳腺癌伴肺转移动物模型中进行实验研究。结果Ｄ７、Ｅ５、Ｇ１、Ｃ５、Ｄ１１及Ｈ５６株单抗都能显著地抑制肿瘤的肺转移，对肺转移灶数的抑制率分别为４４％，４５％，６２％，７０％，７２％和７４％。对肺转移灶大小的抑制率为７９％～１００％。其中Ｄ７、Ｃ５、Ｇ１、Ｄ１１４株单抗也能明显地抑制原发瘤的生长。其抑制率分别为４１％，４６％，５３％和８３％。结论ＶＥＧＦ单抗能阻断肿瘤血管的形成，显著地抑制原发瘤的生长和转移，在肿瘤的治疗中显示出潜在的应用价值。     

鼠抗粒细胞集落刺激因子单克隆抗体的研制

本文运用杂交瘤技术，成功地建立了６株能稳定分泌小鼠抗重组人粒细胞集落刺激因子（Ｇ－ＣＳＦ）单克隆抗体的杂交瘤细胞１Ｂ９Ｄ１０、１Ｇ５Ｆ１０、２Ｃ８Ｃ１、２Ｅ４Ｆ６、３Ｃ６Ｅ１１、７Ｄ２Ｃ７）。试验结果表明，６株单抗均为ｌｇＧ类，且特异性强，能分别识别不同的抗原结合位点，相对亲和力２Ｅ４Ｆ６单抗最高，１Ｇ５Ｆ１０最低，为今后Ｇ－ＣＳＦ单抗的应用打下了基础。     

抗rhG—CSF单克隆抗体的研制及初步鉴定

利用淋巴细胞杂交瘤技术，以ｒｈＧ－ＣＳＦ为免疫原，建立了６株稳定分泌抗Ｇ－ＣＳＦ单抗的杂交瘤细胞株，并研究了其分泌抗体的某些生物学特性。１材料与方法１．１抗原ｒｈＧ－ＣＳＦ，人用制剂，由北京医科大学免疫学系马大龙教授赠送。１．２细胞及动物Ｂａｌｂ／ｃ...     

抗人重组SCF单克隆抗体杂交瘤细胞系的建立及初步鉴定

本研究利用ＰＥＸ３１Ｂ作为载体，在大肠杆菌表达出重组人ＳＣＦ融合蛋白。用该蛋白作为抗原，免疫ＢＡＬＢ／ｃ小鼠，通过鼠－鼠杂交瘤技术，成功地获得４株分泌抗人重组ＷＳＣＦ单克隆抗体的杂交瘤细胞系。经检测，它们所分泌的抗体亚类均为ＩｇＧ１，免疫印迹试验和抗体特异性鉴定证实，４株单抗均能特异性地识别可溶性ＳＣＦ。抗ＳＣＦ单抗杂交瘤细胞系的建立，为深入研究ＳＣＦ的生物学特性、功能以及ＳＣＦ产品的纯化，提供了     

鼠抗粒细胞集落刺激因子单克隆抗体的研制

本文运用杂支瘤技术，成功地建立了６株能稳定分泌小鼠杭重组人粒细胞集落刺激因子（Ｇ－ＣＳＦ）单克隆抗体的杂交瘤细胞１Ｂ９Ｄ１０、１ＧＳＦ１０、２Ｃ８Ｃ１、２Ｅ４Ｆ６、３Ｃ６Ｅ１１、７Ｄ２Ｃ７）。试验结果表明，６株单抗均为１ｇＧ类，且特异性强，能分别识别不同的抗原结合位点，相对亲和力２Ｅ４Ｆ６单抗最高，１ＧＳＦ１０最低，为今后Ｇ－ＣＳＦ单抗的应用打下了基础。     

TNF-α单抗Fab段基因的克隆及其在大肠杆菌的表达

目的进行ＴＮＦ－α单抗Ｆａｂ段基因的克隆并在大肠杆菌中表达。方法用逆转录－聚合酶链反应技术（ＲＴ－ＰＣＲ）从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆重链Ｆｄ段和κ链基因；并用ＥＬＩＳＡ和免疫印迹分析证明表达的Ｆａｂ段特异结合ＴＮＦ－α。结果从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆出了重链Ｆｄ段和κ基因。经ＤＮＡ序列测定表明，ＶＨ、Ｄ、ＪＨ分别属于ＶＨ３Ｄ、ＤＳＰ２和ＪＨ４；Ｖκ和Ｊκ分别属于Ｖκ１和Ｊκ１。将该Ｆｄ和κ链ｃＤＮＡ克隆到表达载体ｐＣｏｍｂ３Ｈ中，在大肠杆菌中获得了表达。结论ＥＬＩＳＡ和免疫印迹分析表明，表达的Ｆａｂ段可特异地和ＴＮＦ－α结合。     

血管内皮生长因子的免疫学测定法

目的：检测肿瘤细胞条件培养液中ＶＥＧＦ的表达量。方法：在表达及分离纯化ＶＥＧＦ的基础上,成功地制备了ＶＥＧＦ抗体,以夹心法测定ＶＥＧＦ,ＶＥＧＦ测定范围为０．０２～２００．００ｎｇ／ｍｌ,灵敏度可达０．０２ｎｇ／ｍｌ,批内及批间变异均小于１０％,回收率平均为１０２．４％。其中人胃癌细胞ＭＧＣ８０３、ＢＧＣ８２３、乳癌ＮＭＣＦ－７及肝癌ＢＥＬ－７４０２分泌上清中ＶＥＧＦ的含量分别为６．５０、０．４５     

抗胃癌鼠单抗3G9 Fab段基因的克隆及其在大肠杆菌…

本文报道用聚合酶链延伸反应（ＰＣＲ）从分泌抗胃癌鼠单抗的杂交瘤细胞系３Ｇ９中克隆出了重链Ｆｄ段和ｋ链的基因。ＤＮＡ序列测定表明３Ｇ９的ＶＨ、Ｄ、ＪＨ分别属于ＶＨ７１８３、ＤＦＬ１６．１和ＪＨ３；Ｖｋ和Ｊｋ分别属于ＶｋⅡ和Ｊｋ２。将３Ｇ９Ｆｄ段和ｋ链ｃＤＮＡ克隆到表达载体ｐＣｏｍｂ３中，在大肠杆菌中获得了表达，用还原和非还原的ＳＤＳ－ＰＡＧＥ电泳作免疫印迹实验，证明了Ｆａｂ段的表达。并用酶联免过滤实     

抗人甲胎蛋白单克隆抗体的制备及鉴定

利用细胞融合技术建立了５株分泌抗人甲胎蛋白（ＡＦＰ）单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，快速酶联免疫分析法测得其亲和常数为５×１０７～２×１０９Ｍ－１之间；单抗的Ｉｇ亚类１Ｄ６和１Ｅ６为鼠ＩｇＧ２ａ（κ），３Ａ８、５Ａ７和７Ｈ１１为ＩｇＧ１（κ）。免疫组化中５株单抗均在肝癌细胞的胞浆显色，３Ａ８和１Ｄ６还能结合在肝癌细胞膜上。应用方阵配对实验初步证实，５株单抗至少针对ＡＦＰ分子上３个不同表位，并分析了单抗在ＥＬＩＳＡ反应中的特性。     

登革病毒对人血管内皮细胞分泌ET-1及PGI2的影响

目的　研究登革病毒感染对人血管内皮细胞分泌重要的血管活性物质ＥＴ１ 及ＰＧＩ２ 的影响,以了解登革出血热及登革休克综合征（ＤＨＦＤＳＳ）的发病机制。方法　用登革病毒Ⅱ型,感染人脐静脉内皮细胞（ＨＵＶＥＣ） ,于感染后４ 、２４ 、４８ 、７２ 及９６ 小时,分别收集病毒感染上清液,用放射免疫检测法测定ＥＴ１ 及ＰＧＩ２ 的含量。结果　登革病毒感染可使ＨＵＶＥＣ分泌ＥＴ１ 及ＰＧＩ２ 的能力受到明显抑制。在病毒感染早期（４ 小时）,ＨＵＶＥＣ分泌ＥＴ１ 及ＰＧＩ２ 的能力即受到明显抑制。登革病毒对ＨＵＶＥＣ分泌ＥＴ１ 抑制作用强烈而持久,至感染后９６ 小时,ＨＵＶＥＣ分泌ＥＴ１ 的能力与未受感染的阴性对照组比较,差异仍有显著性。然而,登革病毒对ＨＵＶＥＣ 分泌ＰＧＩ２ 的抑制作用,可随时间的推移而减弱,至感染后９６ 小时,ＨＵＶＥＣ分泌ＰＧＩ２ 的能力已达正常水平。结论　登革病毒感染可影响血管内皮细胞分泌血管活性物质ＥＴ１ 及ＰＧＩ２ 的功能,导致血管通透性增加和凝血、止血功能障碍。因此,登革病毒所致的血管内皮细胞功能障碍,可能是ＤＨＦＤＳＳ重要的发病机制     

无蛋白培养中杂交瘤细胞的生长与单克隆抗体分泌

目的：建立应用无蛋白培养基大量制备单克隆抗体（McAb)的生产方法。方法：本实验以3株猪瘟病毒特异单抗杂交瘤细胞为研究对象，通过与常规的有血清培养法比较，研究了无蛋白培养基中杂交瘤的细胞生长特性、抗体分泌效率、活性和纯度。结果：无蛋白培养基中单抗的产量与效价比有血清培养的单抗高2－4倍，而且单抗的纯度显著提高。结论：无蛋白培养基可完全取代常规的有血清培养基，用于生产高滴度、高纯度单克隆抗体。     

抗Epstein－Bars病毒壳抗原gp125单克隆抗体的研制

用重组痘苗病毒在１４３细胞上表达的ｇＰ１２５粗提物免疫Ｂａｌｂ／Ｃ小鼠，用杂交瘤技术获得７株稳定分泌抗Ｅｐｓｔｅｉｎ－Ｂａｒｓ（ＥＢ）病毒ｇｐ１２５单克隆抗体的杂交瘤细胞株。对杂交瘤细胞及单克隆抗体进行一系列分析和鉴定，初步建立了用表达产物及特异性单克隆抗体检测ＥＢ病毒ＩｇＡ／ｇｐ１２５抗体的三步ＥＬＩＳＡ法。用此法检测ＩｇＡ／ＶＣＡ阳性的鼻咽癌病人血清及ＩｇＡ／ＶＣＡ阴性的正常人血清，结果完全吻合。     

COX-2/VEGF-dependent facilitation of tumor-associated angiogenesis and tumor growth in vivo

Nonsteroidal anti-inflammatory drugs are known to suppress the occurrence and progression of malignancies such as colorectal cancers. However, the precise mechanism of these actions remains unknown. We have evaluated the role of an inducible cyclo-oxygenase (COX-2) in tumor-associated angiogenesis and tumor growth, and identified the downstream molecules involved using a ddy mouse model of sponge angiogenesis, which mimics tumor angiogenesis and is COX-2 and vascular endothelial growth factor (VEGF) dependent. In this model, VEGF expression was down-regulated by selective COX-2 inhibition with NS-398. To find out the involvement of COX-2/VEGF pathway in tumor-associated angiogenesis, we estimated angiogenesis occurring around implanted Millipore chambers containing sarcoma-180 (S-180) cells or Lewis lung carcinoma cells. Daily oral administration of NS-398 or of aspirin, a nonselective COX inhibitor, suppressed angiogenesis seen around the Millipore chambers. S-180 cells implanted in ddy mice formed substantial tumors with extensive angiogenesis markedly suppressed by aspirin and COX-2 inhibitors NS-398 and JTE522, but not by mofezolac, an inhibitor of constitutive COX-1. Tumor-associated angiogenesis was also significantly suppressed by a neutralizing antibody against VEGF. S-180 tumor growth in the subcutaneous tissues was also suppressed by aspirin, COX-2 selective inhibitors, and the VEGF antibody, but not by the COX-1 inhibitor. These results demonstrate that the inhibition of the COX-2/VEGF-dependent pathway was effective in tumor-associated angiogenesis, tumor growth, and tumor metastasis.     

抗杜氏利什曼原虫犬分离株单克隆抗体的研制及用于病犬血清循环抗原的检测

本文报道用杜氏利什曼原虫大分离株前鞭毛体膜抗原免疫BALB/c小鼠的脾细胞与Sp2/0瘤细胞融合,得到多株分泌抗体较高且稳定的细胞株。进一步筛选出McAb C11-G10-A4可用于检测病犬血清循环抗原。用McAb-AST检测采自甘肃省黑热病流行区的30份犬血清,阳性率30％。与骨髓涂片查病原体的总符合率为86.7％,阳性符合率达100％。69份非流行区对照大血清检测结果,仅1份出现阳性反应,可见本试验的敏感性较高,特异性较强(98.55％)。     

CAPE suppresses VEGFR-2 activation, and tumor neovascularization and growth

The growth and metastasis of human solid tumors and the development of conditions such as diabetic retinopathy, rheumatoid arthritis, inflammatory psoriasis, and others are regulated by the balance between angiogenic stimulators and inhibitors released in the angiogenic–pathological microenvironment. Vascular endothelial growth factor (VEGF), an angiogenic factor, is a potent endothelial-specific mitogen that activates endothelial cells in pathological angiogenesis. Recently, we demonstrated that caffeic acid phenethyl ester (CAPE) inhibits tumor growth, invasion, and metastasis. However, the precise molecular mechanism underlying the inhibitory effect of CAPE on VEGF-mediated angiogenesis remains unknown. Here, we show that CAPE suppressed VEGF-induced proliferation, tube formation, migration, the formation of actin stress fibers and loss of VE-cadherin at cell–cell contacts in endothelial cells, indicating the inhibition of VEGF-mediated VEGF receptor-2 (VEGFR-2) and its downstream signal activation in vitro. CAPE blocked VEGF-stimulated neovascularization in the Matrigel plugs assay, and reduced vascular permeability in mouse skin capillaries in vivo. CAPE inhibited the growth and neovascularization of primary tumor cells in C57BL/6 and BALB/c mice inoculated with Lewis lung carcinoma, colon carcinoma, and melanoma cells. These results suggest that CAPE negatively modulates VEGF-induced angiogenesis by suppressing VEGFR-2 activation, and might be a therapeutic avenue for anti-angiogenesis.     

O1群小川型霍乱弧菌单克隆抗体的制备与鉴定

目的制备抗O1群小川型霍乱弧菌(VibriocholeraeO1serotypeOgawa)特异性单克隆抗体(McAb),为霍乱的早期快速诊断提供有力的抗体工具。方法以灭活的O1群小川型霍乱弧菌免疫Balbc小鼠,通过杂交瘤技术制备针对O1群小川型霍乱弧菌的McAb,以间接ELISA法对所需的杂交瘤细胞株进行筛选,分析其亚类,检测其效价及相对亲和力,以间接ELISA和Westernblot鉴定McAb特异性,并进行McAb结合表位分析。结果融合了602株能分泌抗O1群小川型霍乱弧菌McAb的杂交瘤细胞株,最后得到5株能稳定分泌特异性的针对该McAb的细胞株,其抗体亚类分别为3株IgG1,1株IgG2b,1株IgG3;腹水效价均达1×10-6;亲和常数在1×108～1×109之间。间接ELISA法及Westernblot证实所获的McAb可与O1群小川型霍乱弧菌发生特异性反应。ELISA相加实验结果显示除有2株McAb识别相同的抗原表位外,其余均识别不同的抗原表位。结论获得霍乱弧菌O1群小川型特异性McAb,为O1群小川型霍乱早期快速诊断和发病机理的研究提供基础。     

抗大肠杆菌J5株脂多糖小鼠McAb的制备及初步鉴定

以热杀大肠杆菌Ｊ５株为抗原，免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞和Ｓｐ２／０骨髓瘤细胞融合，获得４株能稳定分泌抗菌体脂多糖单克隆抗体的杂交瘤细胞系。鉴定了其中３株单克隆抗体和８种革兰氏阴性菌的交叉反应性，为进一步研究打下了基础。     

人肝再生增强因子单克隆抗体的制备:以非纯化的大肠杆菌表达抗原筛选杂交瘤

目的 :利用杂交瘤技术、以非纯化的大肠杆菌表达重组蛋白作为筛选抗原 ,制备小鼠抗人肝再生增强因子(hALR)的单克隆抗体。方法 :用实验室纯化的重组hALR 硫氧环蛋白融合蛋白免疫BALB c小鼠 ;小鼠脾细胞与SP2 0骨髓瘤细胞融合后经HAT选择培养基筛选杂交瘤 ;以重组质粒pQE30 hALR与空质粒pQE30在大肠杆菌中诱导表达后的细菌裂解产物作为筛选抗原和对照抗原 ,用ELISA方法筛选能分泌抗hALR单克隆抗体的阳性杂交瘤细胞克隆 ;进一步以ELISA方法和免疫印迹方法检测该杂交瘤细胞产生的抗体对真核细胞表达的重组hALR及人体血清中天然hALR的反应性。结果 :成功筛选出一株能稳定分泌抗hALR单克隆抗体的杂交瘤细胞 ;其产生的抗体能对真核细胞表达的重组hALR及人体血清中天然的hALR发生特异的抗原抗体反应。结论 :大肠杆菌表达的重组蛋白在以空质粒表达产物作为对照下 ,不经过任何纯化步骤也能够用于单克隆抗体制备中的杂交瘤筛选 ;hALR单克隆抗体为深入研究hALR提供了研究手段。     

Gamma-secretase inhibitor DAPT suppresses glioblastoma growth via uncoupling of tumor vessel density from vessel function

The objective of the current study was to investigate the regulation of VEGF signaling and tumor angiogenesis by gamma-secretase inhibitor DAPT in glioblastoma. Effects of DAPT on VEGFR1, VEGFR2, endothelial cell proliferation and vessel function were evaluated using mouse microvascular endothelial H5V cell line and U87MG xenograft mouse models. We found that DAPT efficiently inhibited Notch signaling, increased VEGFR2 expression, but decreased VEGFR1 expression. DAPT treatment enhanced endothelial cell proliferation when used combined with VEGF, but exerted no effect if used alone. In U87MG xenograft mouse models, DAPT treatment increased tumor vessel density but compromised vessel function, as evidenced by poor perfusion and aggravated hypoxia. Therefore, DAPT treatment results in an uncoupling of tumor vessel density from vessel function and suppresses glioblastoma growth; disturbance of angiogenesis with DAPT presents a novel therapeutic approach for glioblastoma.     

Correlation between tumor vascularity, vascular endothelial growth factor production by tumor cells, serum vascular endothelial growth factor levels, and serum angiogenic activity in patients with breast carcinoma.

Angiogenesis is an important prognostic factor in invasive breast carcinoma. We analyzed sera and tumor samples from 36 patients with primary breast carcinomas to determine the relationship between tumor vascularity, vascular endothelial growth factor (VEGF) production by tumor cells, levels of circulating VEGF (measured by ELISA assay), and levels of endothelial growth factors analyzed by a functional test of human umbilical vein endothelial cells (HUVEC) proliferation. Tumor vascularity was correlated directly with VEGF production by the tumor, indicating that VEGF production is a relevant factor in determining angiogenesis in primary tumor. No correlation was found either between the number of vessels in the tumor or the production of VEGF by tumor cells and the levels of serum angiogenic factors including VEGF. On the contrary, the two serum tests correlated together because a high serum level of VEGF is more frequent in cases with the presence of HUVEC-stimulating growth factors. These data indicate that the principal source of factors stimulating angiogenesis in the primary tumor is the tumor itself. This is an important issue in the context of anti-angiogenic therapeutic approaches, which should be planned to interfere with tumor production of angiogenic factors rather than with circulating angiogenic factors. In conclusion, whereas the vessel count and VEGF production by tumor cells are parameters that give direct information on tumor angiogenesis, long-term follow-up is necessary to determine the clinical significance of the determination of serum HUVEC-stimulating factors in the progression of breast carcinoma.