免疫亲和层析法纯化新的骨肉瘤相关抗原

目的 用自行制备的抗人骨肉瘤单克隆抗体mAb5D3纯化其相应的抗原5D3Ag。方法 联合应用饱和硫酸铵沉淀法和蛋白A柱层析法，从小鼠腹水中纯化骨肉瘤单克隆抗体mAb5D3，将此单体共价交联到溴化氰活化的Sepharose—4B上，制备成免疫亲和层析柱；再将骨肉瘤细胞OS—9607的裂解液过此免疫亲和层析柱，纯化mAb5D3相应的抗原5D3Ag。结果 从骨肉瘤细胞OS—9607的裂解液中纯化出一分子量为43KD的骨肉瘤相关分子5D3Ag，经SDS—PAGE分析5D3Ag达到了电泳纯。结论 能够用免疫亲和层析的方法从骨肉瘤细胞裂解液中纯化出mAb5D3对应的抗原5D3Ag，其分子量与以往献所报道的骨肉瘤相关抗原的分子量都不相同，是一种新的骨肉瘤相关抗原。     

mGM-CSF重组质粒的构建、表达及活性鉴定

目的 构建pc—mGM—CSF重组质粒载体，为mGM—CSF基因治疗肿瘤的研究奠定基础。方法 采用RT—PCR方法从小鼠脾脏中获得目的基因mGM—CSF，克隆于pcDNA3.1／Myc-His(-)(A)质粒上，成为pc-mGM-CSF，用PCR、酶切进行鉴定，然后用脂质体转染小鼠骨髓瘤细胞SP2／0，用G418筛选后通过RT—PCR和SDS—PAGE鉴定，将转染SP2／0上清加入NFS-60细胞，检测蛋白活性。结果 重组质粒中含有mGM—CSF基因，在SP2／0中有表达，且表达产物能分泌到肿瘤细胞外，分泌到细胞外的产物用mGM—CSF依赖株NFS-60细胞检测证明具有生物学活性。结论 成功构建含mGM—CSF真核表达重组质粒，有助于进一步研究其抗肿瘤作用。     

β-catenin协同BMP9诱导骨肉瘤TE85细胞成骨分化

目的:检测β-连环蛋白(β-catenin)联合骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)对骨肉瘤TE85细胞成骨分化的影响。方法 :采用构建有β-catenin和BMP9基因的重组腺病毒Adβ-catenin和Ad BMP9,单独或联合感染人骨肉瘤TE85细胞,并用半定量RT-PCR法检测β-catenin和BMP9 m RNA在TE85细胞中表达水平的改变;荧光素酶报告基因系统检测β-catenin/TCF4相对活性的变化,碱性磷酸酶(alkaline phosphatase,ALP)染色和读数法分析TE85细胞的早期成骨能力,茜素红染色法检测细胞的晚期成骨能力;半定量RT-PCR法检测骨保护蛋白(osteoprotegerin,OPG)以及晚期成骨分化指标骨钙蛋白(osteocalcin,OC)和骨桥蛋白(osteopontin,OPN)m RNA的表达水平,蛋白质印迹法检测OC和OPN蛋白的表达水平。结果:重组腺病毒Adβ-catenin与AdBMP9感染TE85细胞后能显著提高外源性β-catenin与BMP9 mRNA的表达水平(P值均<0.05),Adβ-catenin能明显增强β-catenin/TCF4的相对活性(P<0.05)。Adβ-catenin与AdBMP9能分别诱导骨肉瘤细胞TE85早期成骨分化指标ALP活性增加(P<0.05),但增加的程度有限;对晚期成骨指标OC和OPN mRNA和蛋白表达以及钙盐沉积无明显上调的作用。Adβ-catenin与AdBMP9联合作用之后,上述各项早晚期成骨指标的表达均显著增强,差异均有统计学意义(P值均<0.05)。结论:β-catenin和BMP9单独诱导骨肉瘤细胞TE85成骨分化的能力有限,而联合作用后诱导成骨分化的能力显著增强,β-catenin能协同BMP9诱导骨肉瘤细胞TE85成骨分化。     

人T细胞受体ζ链基因在昆虫细胞中的表达

目的：克隆人T细胞受体 (TCR) ζ链基因，运用昆虫杆状病毒系统表达该蛋白。方法：用RTPCR从人外周血单个核细胞（PBMC）中克隆TCR ζ链cDNA，构建到昆虫杆状病毒系统专用的Transfer载体中，并与杆状病毒共转染昆虫sf 9细胞。用SDSPAGE电泳和用小鼠抗人ζ链蛋白的单抗在流式细胞仪中鉴定重组蛋白的表达。结果：克隆的人TCR ζ链cDNA插入昆虫杆状病毒载体，并在昆虫sf 9细胞中特异地表达了蛋白。表达量约为细胞裂解上清液总蛋白的11%。经SDSPAGE分析，其分子量大小与预期的重组ζ链蛋白一致。用胞内标记流式细胞仪检测证实，转染重组杆状病毒的昆虫细胞含有人的ζ链蛋白。结论： 从人PBMC中成功克隆了T细胞受体ζ链的基因，并在昆虫细胞中获得高效表达。用生物工程技术获得TCR ζ链蛋白有利于深入研究其生物学功能     

骨折修复过程中骨形态发生蛋白2对趋化因子12表达的调控及其意义

目的探讨骨形态发生蛋白2(bone morphogenetic protein,BMP2)对趋化因子12(chemokine C-X-C motif-ligand-12,CXCL12)表达调节及其在骨折修复过程中的意义。方法构建BMP2敲除鼠BMP2~(cKO/+)和BMP2~(cKO/cKO)。免疫组化分析正常小鼠、BMP2~(cKO/+)小鼠及BMP2~(cKO/cKO)小鼠胫骨骨折模型中骨内膜细胞CXCL12的表达及其随时间的变化。实时定量PCR(RT-q PCR)比较对照组与BMP2~(cKO/+)小鼠骨内膜细胞及其分化细胞CXCL12、骨钙蛋白、α-SMA表达差异。结果BMP2~(cKO/+)和BMP2~(cKO/cKO)小鼠BMP2显著低于对照组。小鼠骨折修复过程中骨内膜细胞和成骨细胞CXCL12表达明显升高,且表达CXCL12细胞先增多再减少;BMP2~(cKO/+)小鼠骨折修复过程中表达CXCL12的骨内膜细胞和成骨细胞逐渐增加。BMP2~(cKO/+)小鼠分离的骨内膜细胞CXCL12表达显著高于正常小鼠。在诱导分化的小鼠骨内膜细胞中添加rh BMP2,CXCL12的表达减少,骨钙蛋白和α-SMA表达显著增加。诱导分化的BMP2~(cKO/cKO)骨内膜细胞CXCL12表达显著高于正常小鼠,而骨钙蛋白的表达则显著降低。CXCL12受体拮抗剂AMD3100处理诱导分化的BMP2~(cKO/cKO)骨内膜细胞,骨钙蛋白和α-SMA表达显著增加。结论骨折修复过程中,BMP2下调CXCL12的表达有助于成骨细胞的分化从而促进骨折的修复。     

人可溶性4-1BBL在毕氏酵母中的表达及其协同刺激效应的研究

目的：通过甲醇营养型毕氏酵母蛋白质表达系统，表达出具有生物活性的人可溶性4－1BBL重组蛋白。方法：PCR方法从XG－4－1BBL转基因细胞中获得4－1BBL的胞外段基因；利用酵母表达载体pPICZαA构建重组质粒pPICZαA－s4-1BBL,经线性化后电转化导入毕氏酵母GS115中，甲醇诱导表达；SDS－PAGE蛋白电泳和Western blot分析发酵上清中hs4－1BBL蛋白表达水平及其特异性；体外T细胞增殖试验（^3H-TdR掺入法）观察rhs4－1BBL蛋白的生物学活性。结果：（1）成功地扩增到4－1BBL的胞外段基因，酶切鉴定及测序结果与已知的基因序列一致；（2）SDS－PAGE蛋白电泳和Western blot结果显示所获重组蛋白的分子量与预期分子量（21kD)相同，并可被4－1BBL特异性抗体识别；（3）重组蛋白在体外具有协同促进T细胞增殖的效应。结论：成功地在毕氏酵母表达系统中表达出具有生物学活性的人可溶性4－1BBL重组蛋白，为进一步研究其功能奠定了物质基础。     

人血管抑素克隆表达及抑制血管生成的研究

目的　研究并获得重组人血管抑素 (angiostatin) ,探讨其临床应用价值。方法　采用逆转录 -聚合酶链式反应 (RT PCR )方法 ,从人胚胎肝细胞中扩增出人全长血管抑素cDNA片段。用原核细胞表达载体构建 pBV 2 2 0 /angiostatin重组质粒 ,并将其转化大肠杆菌DH 5α进行表达 ,初步纯化。应用鸡胚尿囊膜血管生成实验及脐静脉内皮细胞增殖实验检测其活性。结果　经SDS PAGE分析 ,表达产物相对分子量约 3 8kD ,薄层扫描显示表达产物可达细菌总蛋白的 3 0 %。活性实验显示 ,重组蛋白能够抑制血管形成。结论　重组人Angiostatin在大肠杆菌中实现高效表达 ,并具有很好的生物学活性     

牛垂体bFGF的纯化及生物、生化性能检测

目的 bFGF能刺激毛细血管内皮细胞的增殖与分化,与肿瘤的生长有密切的关系,其在血或局部组织中的含量对肿瘤的诊断及治疗监测有意义。方法 用(NH_4)SO_4沉淀、CM—Sephadex C—50离子交换层析、肝素—Sepharose亲和层析方法分离提纯出BFGF,用HUVC和成纤维细胞测定了活性,并进行了PAGE和SDS—PAGE分析。结果 产物PI>8.9,分子量为16400,ED_(50)为83pg/ml,最终纯化倍数为220000,产率为0.1mg每公斤垂体。结论 纯化产品为单一成份,纯度较高,且回收率达到国外文献报道的高值。     

厌氧棒菌苗诱导小鼠产生内源性肿瘤坏死因子的条件及其特性

本文采用正交设计,初步探讨了厌氧棒菌苗(CP)诱导内源性肿瘤坏死因子(TNF)活性的条件。结果表明:仅用CP一种调节剂,按照一定方式对小鼠进行三次刺激.便可使其血清具有显著的内源性TNF活性。有TNF活性的部分经SDS—PAGE和凝胶过滤测得分子量分别约为60,000dal和55,000dal;醋酸纤维膜电泳显示为α球蛋白;TNF活性经56℃处理30分钟仍稳定,70℃,10分钟即丧失;在pH6～9之间、活性稳定。     

表达增强型绿色荧光蛋白的小鼠骨肉瘤体外和体内荷瘤模型的建立

背景与目的：小鼠骨肉瘤模型在骨恶性肿瘤的研究中应用较为广泛，良好的活细胞标记方法将有利于研究骨肉瘤的侵袭和转移生物学行为，本研究拟建立稳定表达增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因的小鼠骨肉瘤S180细胞株，同时对EGFP-S180细胞进行体内和体外的初步研究。方法：采用电穿孔转染法将EGFP基因导入小鼠骨肉瘤S180细胞，通过G418筛选,建立稳定表达EGFP蛋白的小鼠骨肉瘤EGFP-S180细胞株；激光共聚焦显微镜检测转染前后癌基因c-Myc的表达；建立相应的皮下接种和腹腔接种荷瘤小鼠模型。结果：建立了稳定表达EGFP的EGFP-S180细胞株，转染与未转染细胞的c-Myc表达差异无统计学意义（P〉0.05），小鼠皮下接种和腹腔接种成瘤的时间无显著性差异（P〉0.05）。结论：稳定表达EGFP的S180细胞株没有显著改变肿瘤细胞S180的生物学行为，建立稳定表达EGFP的S180细胞株在体内外研究的方法是可行的，为骨肉瘤体内外的示踪研究提供了实验基础。     

Expression of bone morphogenetic protein and its receptors in osteosarcoma and malignant fibrous histiocytoma

BACKGROUND: Bone morphogenetic protein (BMP) activity has been found in cases of malignant fibrous histiocytoma (MFH) and osteosarcoma but only tumors in the latter category show evidence of ossification. The aim of this study was to try to understand this difference by examination of the distribution of BMP and its receptors (BMPR) for this bone inducing protein in these tumors. METHODS: Sections of 11 osteosarcoma and 10 MFH were analyzed immunohistochemically for BMP and BMPRs by use of the avidin-biotin peroxidase method. RESULTS: Nine out of 11 osteosarcoma cases (80.1%) showed positive staining for both BMP and BMPRs. Two cases of chondroblastic type osteosarcoma did not show any significant staining for BMP and BMPRs. In eight out of 10 MFH cases (80%) there was positive staining for BMP. No immunoreactivity for BMPRs was found in any case of MFH. CONCLUSIONS: MFH does not express BMPRs and this may be the reason why-MFH tumors do not ossify, even in the presence of BMP.     

A factor inducing differentiation of the human monocytic cell line U-937 produced by 12-O-tetradecanoylphorbol 13-acetate-treated U-937

A new factor capable of inducing differentiation of human leukemic cell line U-937 into macrophages was found and partially purified from the conditioned medium of U-937 previously treated with 12-O-tetradecanoylphorbol 13-acetate. The purification procedure included ultrafiltration, DEAE-Sephacel and butyl-Toyopearl column chromatography. The purified factor gave a major band of protein with a molecular weight of 67,000 daltons which coincided with the biological activity of differentiation-inducing factor, and it was not adsorbed on a concanavalin A column. These results suggest that this factor is distinct from other differentiation-inducing factors.     

抗人双特异性蛋白磷酸化酶18单克隆抗体的制备及鉴定

目的制备高亲和力、高特异性的抗人双特异性磷酸化酶18（Dusp18）单克隆抗体,并对其生物学特性进行鉴定,为Dusp18功能的研究奠定基础。方法重组Dusp18免疫BALB／c小鼠,取其脾细胞与SP2／0细胞融合,经筛选建立可稳定分泌抗Dusp18的单抗细胞株,制备腹腔积液,ProteinG纯化所得腹腔积液,ELISA及Westernblotting检测抗体的效价和亲和力,用亚型鉴定试纸条鉴定单克隆抗体的亚型。应用免疫组织化学检测Dusp18在肝癌组织中的表达情况。结果获得两株（F003和F004）单抗,上清效价分别为1：5120和1：10240,腹腔积液效价分别为1：25600和1：51200,Westernblotting结果均在预期位置出现阳性条带。利用两株抗体均可在肝癌组织中检测到Dusp18的表达。两株抗体亚型均为IgG1型,轻链均为k型。结论成功制备能特异性识别Dusp18的单克隆抗体,为进一步研究Dusp1的生物学功能,揭示其与肿瘤发生、发展之间的关系奠定了基础。     

Human tumor cells synthesize and secrete alpha-2-macroglobulin in vitro

In previous studies we showed that human sarcoma and melanoma cell lines synthesize and secrete into culture medium a glycoprotein, migrating in urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 140,000. It is not detected in cultures of the corresponding normal cells. Conditioned medium of the melanoma cell line HMB-2, producing among the cell lines tested the largest amounts of this glycoprotein, has now been used as a source for purification of the protein. NH2-terminal amino-acid sequence determination of the purified glycoprotein showed that it is identical to human alpha 2-macroglobulin (alpha 2M). Rabbit antibodies raised against the glycoprotein specifically reacted in immunoblotting and immunodiffusion tests with alpha 2M present in human plasma. Likewise, these antibodies immunoprecipitated from the conditioned media of 35S-methionine-labelled melanoma and osteosarcoma cell lines the protein which had a molecular weight corresponding to alpha 2M. alpha 2M was also synthesized and secreted by 2 strains of fetal lung fibroblasts but not by fetal skin fibroblasts or adult skin fibroblasts autologous to the osteosarcoma cell line.     

131I标记抗人成骨肉瘤单抗荷瘤裸鼠体内分布和放射免疫显像研究

目的 :研究13 1I标记抗人成骨肉瘤单克隆抗体在荷人成骨肉瘤裸鼠体内分布和放射免疫定位显像。方法 :采用Iodogen固相法标记制备13 1I HOSMcAb。 2 5只荷瘤裸鼠随机分为 5组 ,分别腹腔注射13 1I HOSMcAb后 ,于6、12、2 4、48和 72h 5个时间段进行裸鼠的体内分布研究 ;对 5只荷瘤裸鼠分别腹腔注射13 1I HOSMcAb后 ,于 6、12、2 4、48和 72h 5个时间段进行荷瘤裸鼠的放射免疫定位显像研究。结果 :在荷人成骨肉瘤裸鼠腹腔注射13 1I HOSMcAb后 ,12h肿瘤与血的T/NT比值为1 3 7,2 4h为 3 75 ,48h达到最高为 5 2 4。腹腔注射13 1I HOSMcAb后 ,12h肿瘤部位即可见明显放射性浓聚 ,48h本底明显降低 ,肿瘤呈放射性热区。结论 :13 1I HOSMcAb对成骨肉瘤定向性较好 ,对放射免疫定位显像有利 ,为进一步放射免疫治疗研究提供了理论基础     

Response of cultured hepatocytes to a hepatomitogen after initiation by conditioned medium or other factors

Injection of a substantially purified hepatomitogen into recipient rats that had 40% of their liver removed resulted in a significant stimulation of hepatic DNA synthesis as determined by the labeling index and the mitotic index. Normal or sham-operated rats did not respond to the injection of the mitogen. The extraction and partial purification of this hepatomitogen have previously been reported (A. Francavilla et al., Cancer Res., 47:5600-5605, 1987). Addition of the factor to an epithelial-like liver-derived cell line in culture (clone 9) or to a hepatoma cell line (HTC-SR) resulted in a dose-dependent stimulation of DNA synthesis. Hepatocytes in primary culture, on the other hand, were not stimulated by the addition of the factor. However, when the mitogen was added to hepatocytes in primary culture, together with conditioned medium, obtained from the responsive cell lines, a significant stimulation of DNA synthesis could be demonstrated in hepatocytes in culture. The stimulation was dose dependent with respect to the mitogen, was abolished by 10 mM hydroxyurea, and was independent of epidermal growth factor. The conditioned medium could be replaced by a protein factor extracted from the two cell lines as previously reported (P. Ove et al., J. Cell. Physiol., 131: 165-174, 1987). It appears that a cofactor is provided by the conditioned medium or by the cell extract, enabling the hepatomitogen to act on hepatocytes in primary culture.     

人类骨肉瘤细胞的血管形成因子研究

Objective To partially purify the angiogenesis factor of human osteosarcoma(HuOs) and study its biological features. Methods The active peptide with a molecular weight of 8000-10000 Da in the conditioned medium obtained from the cultivation of Hu-Os cells(osteoblastic osteosarcoma) was partially purified by ultrafiltration, chromatography and dialysis.The angiogenic effects of the fractions were assessed by proliferation assay of human umbilical vein and pig thoracic aorta endothelial cells. Results The chromatography fractions 4-6 could significantly promote the proliferation of the endothelial cells.Conclusion The HuOs cells could synthesize and secrete angiogenesis factor with a molecular weight of 8000-10000 Da.     

骨形态发生蛋白在骨肉瘤中的临床意义及评价

目的为了解骨形态发生蛋白在骨肉瘤的临床诊断、侵袭、转移中的意义。方法应用免疫组化方法检测3l例骨肉瘤标本的BMP表达，结合临床病理资料进行综合分析。结果AKP升高病例组中95％的病例BMP阳性表达，临床X线成骨性骨肉瘤病例组中88％的病例BMP阳性表达，肺转移病例组中90％的病例BMP阳性表达。P值分别为0．0016和0．0238，统计学上有显著性差异。结论BMP阳性表达合并AKP值升高，可作为成骨性骨肉瘤临床筛选检测；BMP较临床X线对骨肉瘤的预后判定更准确；BMP阳性表达与肺转移呈正相关。     

Copurification of osteolytic and transforming growth factor beta activities produced by human lung tumor cells associated with humoral hypercalcemia of malignancy

The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.     

Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors.