抗人生长转化因子15单克隆抗体制备及鉴定

目的:制备抗人生长转化因子15(GDF15)单克隆抗体(mAb)并鉴定其特性。方法:构建GDF15原核表达载体pGEX-4T-2-gdf15,诱导表达并纯化GST-GDF15融合蛋白。以该融合蛋白免疫BALB/c小鼠,取小鼠的脾细胞与同系小鼠Sp2/0骨髓瘤细胞常规融合,依次进行阳性杂交瘤细胞株的筛选及亚克隆,最终获得稳定分泌抗GDF15 mAb杂交瘤细胞株。以间接ELISA法检测抗体效价;Western blot鉴定抗体特异性;免疫共沉淀法初步检测在肝癌患者血清中GDF15表达水平。结果:获得1株稳定分泌抗人GDF15 mAb杂交瘤细胞株,命名为9G3;抗体免疫球蛋白亚类为IgG1,轻链为κ型;免疫共沉淀及质谱分析证实抗GDF15 mAb9G3能与肝癌患者血清中GDF15蛋白特异性结合并且GDF15水平在肝癌患者血清中较正常人显著升高。结论:成功制备了抗人GDF15mAb,为后期肝癌早期诊断试剂盒的开发奠定了良好的基础。     

抗ARL-1单克隆抗体的制备及初步应用

目的: 制备抗醛糖还原酶相似蛋白(aldosereductaselikeprotein, ARL- 1)的单克隆抗体 (mAb)并进行初步应用。方法: 用纯化的ARL- GST蛋白免疫BALB/c小鼠, 采用杂交瘤技术制备mAb。间接ELISA法、Westernblot及免疫组织化学染色法, 对mAb进行鉴定及初步应用。结果: 获得 1株可稳定分泌抗ARL -1蛋白mAb的杂交瘤细胞系。mAb的Ig亚类为IgG2b, 轻链属于κ型。腹水mAb的效价为: 1∶4. 096×105。Westernblot的结果表明, ARL 1蛋白在被检测的肝癌组织中呈高表达, 而在相应的癌旁组织中不表达。免疫组织化学染色的结果表明, ARL -1蛋白在肝癌组织中呈高表达且主要定位在胞质内。结论: 成功地制备 1株抗ARL -1蛋白的mAb。初步应用的结果表明, ARL- 1蛋白在肝癌组织中呈高表达, 为进一步研究ARL -1蛋白的功能, 以及其与肝癌的确切关系奠定了基础。     

肝癌候选标志物HCCR单克隆抗体的制备和鉴定

目的:制备肝癌候选标志物HCCR蛋白的单克隆抗体(mAb).方法:重组表达HCCR-1167-360蛋白用于动物免疫,用含有HCCR蛋白抗原表位YLGTRR的重组蛋白(Ep-HCCR)检测血清抗体效价及筛选阳性克隆.通过ELISA、Western blot、免疫荧光和免疫组化方法鉴定制备的HCCR抗体的性质.结果:获得1株分泌HCCR抗体杂交瘤细胞株,通过ELISA方法测定抗体的亲和力常数为5.4×106L/mol;Western blot结果显示,该抗体能特异识别肝癌HepG2细胞中HCCR-1和HCCR-2蛋白;免疫荧光检测显示,HCCR蛋白主要分布在细胞质和细胞膜中;免疫组化检测到肝癌组织中有HCCR蛋白表达但在正常组织中没有.结论:成功制备了1株抗HCCR特异性mAb,为建立基于HCCR的肝癌检测方法奠定了基础.     

癌基因STAT5在肝细胞肝癌中的表达和意义

目的 检测STAT5在人肝癌组织中的表达并探讨其在肝癌发生发病中的意义.方法 采用免疫组织化学技术,分析58例肝癌标本,27例肝硬化组织标本,18例肝脏血管瘤标本,12例正常肝组织标本中STAT5蛋白的表达并进行统计学分析比较.结果 STATS在58例肝癌标本中有49例阳性表达(84.48%),27例肝硬化标本中4例阳性表达(14.81%),30例肝血管瘤和正常肝组织中无阳性表达(P＜0.01).结论 STAT5在肝癌组织中表达率高,可能在肝脏恶性肿瘤的发病中有重要意义.     

抗人S100A4单克隆抗体的制备、鉴定及初步应用

目的:制备抗人S100A4单克隆抗体(mAb),建立检测肿瘤样品中S100A4蛋白的可靠方法.方法:采用标准杂交瘤技术得到抗重组人S100A4的mAb.以ELISA、Western blot和免疫组织化学法对抗体进行鉴定.并将抗体应用于临床人乳癌和结肠癌样品的免疫组织化学检测.结果:获得了1株稳定分泌抗人S100A4蛋白mAb的杂交瘤细胞株D101.在免疫印迹实验中,该mAb较多抗特异性更好.该mAb适用于来源于人组织的样品S100A4表达的检测,在少量乳癌和结肠癌样品的检测中给出的结果与商品化多抗的结果一致.结论:该mAb是S100A4特异的,在临床应用中有更好的重复性.     

抗SARS冠状病毒N蛋白单克隆抗体的制备和初步应用

目的:研制抗SARSCoVN蛋白的单克隆抗体(mAb)。方法:以纯化的GSTN免疫BALB/c小鼠,采用杂交瘤技术制备抗SARSCoVN蛋白mAb;用免疫双扩散鉴定Ig亚类;Westernblot和免疫组化鉴定mAb的特异性;间接ELISA检测mAb的腹水效价、相对亲和常数。结果:获得1株可分泌特异性mAb的抗SARSCoVN蛋白的杂交瘤细胞系3E10H,Ig亚类为IgG2b;其腹水效价为8×10-5;其相对亲和力1.725×10-10mol/L,Westernblot和免疫组化阳性。结论:获得特异性抗SARSCoVN蛋白的mAb,为进一步用于临床诊断和实验研究创造了条件。     

抗人白细胞介素15单克隆抗体的制备及特性鉴定

目的 :制备鼠抗人白细胞介素 15(hIL 15)单克隆抗体 (mAb) ,并鉴定其特性。方法 :自重组人白细胞介素 15(rhIL 15)基因工程菌中 ,提取融合蛋白GST IL 15,以 12 0g/LSDS PAGE分离鉴定 ,切取含有目的条带的凝胶 ,免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2 / 0骨髓瘤细胞常规融合 ,依次进行HAT选择培养 ,间接ELISA法筛选抗体阳性的杂交瘤细胞及克隆化。对杂交瘤细胞株的稳定性及其分泌的mAb的特性进行鉴定。另外 ,以rhIL 15包涵体蛋白 (rhIL 15IBP)免疫新西兰白兔 ,制备抗hIL 15的多克隆抗体 (多抗 )。用抗hIL 15的mAb与多抗建立双抗体夹心间接ELISA。结果 :获得 1株可稳定分泌特异性抗hIL 15mAb的杂交瘤细胞。建立了双抗体夹心间接ELISA ,检测rhIL 15蛋白的敏感性达 10 μg/L。结论 :成功地制备抗hIL 15mAb ,并建立了一种可用于hIL 15检测的双抗体夹心间接ELISA。     

抗铜绿假单胞菌外膜蛋白F单克隆抗体制备及夹心ELISA检测方法的建立

目的:制备抗铜绿假单胞菌外膜蛋白F(OprF)的单克隆抗体(mAb),并建立双mAb夹心ELISA检测方法。方法:利用分子生物学方法克隆OprF基因,诱导表达并纯化OprF。用OprF免疫BALB/c小鼠后通过杂交瘤技术制备特异性的mAb,并用ELISA法测定mAb的免疫活性;建立检测铜绿假单胞菌的双mAb夹心ELISA方法,并对其敏感性、特异性进行初步评价。结果:成功地克隆OprF基因,经诱导表达获得铜绿假单胞菌的OprF。通过杂交瘤技术筛选出4株mAb:6D8F7、2H2B6、3C2F5和7B5D9,经鉴定mAb 6D8F7可作为捕获抗体,mAb 7B5D9被HRP标记后可作为检测抗体,以这2株mAb建立的双mAb夹心ELISA方法重复性好、特异性强,敏感性高达到1×103集落形成单位(CFU/mL)。检测的线性范围为1×103～108CFU/mL。与传统的细菌分离方法比较,检测临床标本的符合率高达94.7%。结论:通过杂交瘤技术制备抗铜绿假单胞菌OprF的mAb,并建立了高特异性、高敏感性检测铜绿假单胞菌抗原的双mAb夹心ELISA方法,可用于临床检测铜绿假单胞菌的感染。     

人精子蛋白17单克隆抗体的制备及特性鉴定

目的:制备抗精子蛋白17(Sp17)的单克隆抗体(mAb)并鉴定其特性。方法:克隆人Sp17cDNA,表达带有6His标记的重组Sp17,用纯化的重组Sp17免疫BALB/c小鼠制备mAb。用ELISA筛选抗体阳性的细胞克隆。用免疫组化染色法及阻断试验鉴定mAb的特异性。结果:获得2株杂交瘤细胞系3C12和3D6,其分泌的mAb的Ig亚类(型)分别为IgG1和IgM(κ),杂交瘤细胞培养上清的ELISA效价分别为1∶64和1∶32;腹水mAb的效价分别为1∶1×105和1∶5×104。用人和大鼠睾丸组织以及人精液精子免疫组化染色及阻断试验证明,抗Sp17mAb具有良好的特异性。抗Sp17mAb也可识别卵巢癌组织中异常表达的Sp17。结论:成功地制备特异性的抗Sp17mAb,为研究该蛋白的功能、天然分布及异常表达奠定了基础。     

抗3种标签蛋白单克隆抗体的制备及特性鉴定

目的：制备并鉴定抗GST、His x6及FLAG的单克隆抗体（mAb）。方法：分别以含GST、His x6及FLAG标签的融合蛋白为抗原免疫BALB／c小鼠，通过细胞融合制备抗相应标签蛋白的mAb。用Western blot检测mAb对变性融合蛋白的反应性。用流式细胞术（FCM）检测抗FLAG mAb对细胞膜表面融合蛋白的反应性。结果：其获得12株可稳定分泌抗3种标签蛋白mAb的杂交瘤细胞（其中5株可分泌抗GST mAb，5株可分泌抗His x6 mAb，2株可分泌抗FLAG mAb）。11株mAb均可用于相应融合蛋白的Western blot检测。1株抗FLAG mAb检测细胞膜表面融合蛋白的阳性率，与商品化的mAb M2相接近。用1株抗His x6 mAb制备亲和层析柱，并用于纯化IL-8-His融合蛋白，也获得较满意的结果。结论：成功地制备了抗GST、抗His x6及抗FLAG的mAb，为含标签的融合蛋白的研究和应用提供了重要的工具。     

Expressions of HSP70 and HSP27 in hepatocellular carcinoma

The heat shock proteins (HSPs) are ubiquitous molecules induced in cells exposed to various stress conditions, including carcinogenesis. The HSP70 and HSP27 among HSPs are of special relevance in human cancer inhibiting apoptosis. The aim of this study is to investigate the expressions of HSP70 and HSP27 in hepatocellular carcinoma (HCC) in association to tumor cell proliferation and apoptosis. We examined the expressions of HSP70 and HSP27 by immunohistochemical staining in 71 cases of HCC, and then related their expressions to clinicopathologic parameters and expressions of p53, Ki-67 and Apotag. HSP70 and HSP27 were frequently stained in the cytoplasm and nuclei of tumor cells, but not in the non-neoplastic hepatocytes. Immunoreactivities of HSP70 and HSP27 were observed in 56.3% and 61.9% of HCCs, respectively. HSP70 immunoreactivity correlated with high Ki-67 labeling indices (LIs) (p=0.0159), large tumor size (p=0.0129), presence of portal vein invasion (p=0.0231), and high tumor stage (p=0.0392). HSP27 immunoreactivity significantly related with the subgroup of HBV-associated HCCs (p=0.0003), but not with the others. Both HSP70 and HSP27 immunoreactivities showed no relation to Apotag LIs or p53 immunoreactivity. In conclusion, expressions of HSP70 and HSP27 may play an important role in hepatocarcinogenesis, and especially HSP70 showed a close relationship to the pathological parameters associated with tumor progression and high Ki-67 LIs. Our results could be additional evidence that HSP70 expressions can contribute to not only hepatocarcinogenesis but also tumor progression by promoting tumor cell proliferation.     

Survivin在肝细胞癌中的表达特点及其与预后的关系

目的: 研究原发性肝细胞癌组织中Survivin的表达及其与肝癌细胞的生物学行为及预后的关系。 方法: 应用免疫组织化学染色对83例肝癌及相应癌旁组织中Survivin蛋白的表达情况进行检测，应用半定量RT-PCR技术对11例肝癌及相应癌旁组织中Survivin mRNA的表达情况进行检测，结合临床病理资料分析。 结果: 肝癌及癌旁组织中Survivin蛋白的表达率分别为63.9%(53例)和39.6%(21例)。肝癌组织中，41.5%(22例)表达于肝癌细胞核，45.3%(24例)表达于肝癌细胞浆，13.2%(7例)在胞核、胞浆中均有表达。统计学分析表明，Survivin蛋白阳性反应定位于胞核的病例，其肿瘤的包膜侵犯率和转移率更高 (P<0.05)；且术后生存期<2年的肝癌组中的核表达率显著高于术后生存期≥2年者(P<0.01)。RT-PCR结果显示，11例肝癌组织均表达Survivin mRNA，而癌旁组织只有45.5%(5例)表达。 结论: Survivin在肝癌中呈现高表达，其核表达与肝癌的恶性生物学行为及预后密切相关。     

Aberrant expression of p53 gene product in malignant melanoma.

According to the current concept of carcinogenesis, the alterations of p53 tumor suppressor gene have been the most frequently detected in both human cancer cell lines and cancer tissues freshly isolated. This study was conducted to investigate the p53 gene alteration in malignant melanoma. Nineteen tumor tissues were obtained from 19 patients with malignant melanoma and examined for the expression of p53 protein by immunohistochemical staining with mouse monoclonal anti-p53 antibody, NCL-p53-DO-7. Twelve out of 19 cases (63%) showed positive reactions for p53 protein: 26, 21 and 16% of which had low, intermediate and high reactivity, respectively. p53 alteration more frequently expressed in female (10/12) than male patients (2/7) with malignant melanoma (p < 0.05). The incidence of expression of p53 protein was compared according to the stages and the sites of tissue obtained. The positive rate for p53 protein was not significantly different between the stages. The positive rates for p53 protein were five out of five (100%), one out of two (50%) and six out of twelve (50%) in tissues obtained from the metastatic, lymph node, and primary sites, respectively. The difference in the positive rates, however, is not statistically significant. These results suggest that p53 gene is a frequent target for mutation in the development of malignant melanoma.     

MDM2和P53蛋白表达与原发性肝细胞肝癌发生的关系

目的:研究MDM2和突变型P53蛋白表达与原发性肝细胞肝癌(HCC)发生的关系。方法: 用免疫组织化学SP法,检测55例HCC癌组织、23例癌旁组织、10例正常肝组织MDM2和突变型P53蛋白表达情况。结果: 55例HCC中MDM2蛋白阳性表达17例(30.9%),突变型P53蛋白阳性表达23例(41.8%),二者均阳性表达11例(20%),MDM2和突变型P53蛋白表达有相关性(r=0.310, P＜0.05)。23例癌旁组织MDM2蛋白阳性1例,突变型P53蛋白阳性表达2例,肝癌组织和癌旁组织MDM2和突变型P53蛋白表达有差异(P＜0.05)。10例正常肝组织无MDM2和突变型P53蛋白表达。结论: 肝细胞癌组织有MDM2和突变型P53过量表达,MDM2蛋白和/或 p53 基因突变使 p53 基因功能失活在HCC发生中可能有重要作用。     

Use of specific ELISA for the detection of antibodies directed against p53 protein in patients with hepatocellular carcinoma.

AIMS: To analyse the significance of antibodies to p53 protein as a serological marker for changes in p53 gene expression in patients with hepatocellular carcinoma. METHODS: Thirty eight patients with hepatocellular carcinoma, 19 showing accumulation of p53 protein by immunohistochemistry and 19 having no accumulation, were studied. The presence of anti-p53 was tested using a novel ELISA utilising a recombinant p53 protein as a capture system and verified by western blotting. p53 gene mutations were sought by single strand conformational polymorphism and DNA sequencing analyses. RESULTS: Of 19 patients with p53 protein accumulation in tumour tissue, 10 (52%) had antibodies to p53 in serum by ELISA. Four patients with p53 negative immunohistochemistry also had detectable anti-p53. Western blot analysis confirmed the specificity of the ELISA positive serum samples. The presence of anti-p53 was independent of serum alpha-fetoprotein and was detected in 50% of small tumours while only 8% were alpha-fetoprotein positive. Mutations affecting exons 5 and 6 seem to be more frequently associated with development of anti-p53, than mutations in exons 7 or 8. CONCLUSIONS: The ELISA for anti-p53 is a convenient and specific tet for the detection of humoral response to alterations in p53 gene expression and could be of value in the diagnosis and characterisation of patients with hepatocellular carcinoma.     

肝细胞癌组织中Glypican-3基因表达及其调控机制

目的：探讨肝细胞癌中Glypican-3（GPC3）基因表达及调控机制。方法:采用RT-PCR和聚合酶链反应-单链构象多态性(PCR-SSCP)方法分别检测48例肝细胞癌的癌组织、39例癌旁组织和31例正常肝组织中GPC3 mRNA的表达和基因突变；免疫组化S-P法检测GPC3、P53和PCNA蛋白的表达。结果:GPC3 mRNA在肝细胞癌的癌组织中的阳性表达率为77.1%，但其在癌旁和正常肝组织中不表达；肝细胞癌的癌组织中未发现GPC3基因突变；GPC3与P53蛋白表达无相关性（r＝-0.12574，P＞0.05），肝细胞癌的癌组织中GPC3 表达阳性和阴性的增殖细胞核抗原(PCNA)平均指数分别为（46.32±27.54）%、（39.83±21.47）%,P＞0.05。结论:肝细胞癌组织中GPC3 mRNA高表达与基因突变无关，没有直接影响P53和PCNA蛋白表达。     

细胞外HSP70/HSP70-PCs对人肝癌HepG2细胞上皮-间充质转化的影响及机制研究

 目的：探讨细胞外热休克蛋白70(HSP70)/HSP70肽复合物(HSP70-PCs)对肝癌细胞上皮-间充质转化(EMT)的影响和可能机制。方法：将HepG2细胞分为3组：正常对照组、HSP70/HSP70-PCs组（终浓度2 mg/L）和LY294002+HSP70/HSP70-PCs组。应用real-time RT-PCR与Western blotting法检测上皮细胞表面标志E-cadherin和间质细胞表面标志α-平滑肌肌动蛋白（α-SMA）的表达变化,以及磷脂酰肌醇3-激酶（PI3K）和缺氧诱导因子1α(HIF1-α)的表达变化。结果：细胞外HSP70/HSP70-PCs可以促进HepG2细胞EMT的发生。HepG2细胞的EMT过程伴随HIF-1α和PI3K表达增加。应用LY294002阻断PI3K后,HepG2细胞没有发生EMT,同时细胞外HSP70/HSP70-PCs上调HIF-1α表达的作用消失。结论：细胞外HSP70/HSP70-PCs可以通过PI3K/HIF-1α促进肝癌细胞发生EMT。     

Double immunostaining for p53 and molecular chaperone hsp72/73 in gastric carcinoma.

AIMS: To examine the relation between the expression of p53 protein and the chaperone heat shock protein (hsp)72/73 in a population at high risk for gastric carcinoma, using single and double immunohistochemistry, and to compare the expression of these two proteins with clinicopathological features. METHODS: Monoclonal antibodies were used to investigate the expression of p53 protein and hsp72/73 in 46 human gastric carcinomas. A double immunohistochemical technique was used in cases that showed p53/hsp72/73 coexpression. RESULTS: p53 immunoreactivity was present in 11 tumours (24%), and hsp72/73 immunostaining was observed in 22 cases (48%). p53 expression was observed as nuclear staining in tumoral cells. hsp72/73 expression was demonstrated mainly as cytoplasmic staining, but six tumours also showed focal weak nuclear staining. Seven cases showed p53 and hsp72/73 coexpression with immunoreactivity for both proteins in the same neoplastic cells, three of them with focal areas of nuclear coexpression. p53 expression was seen more frequently in cases that showed a high intensity (+ + +) of hsp72/73 staining. No significant association was observed between the expression of the two proteins and clinicopathological features. CONCLUSIONS: More than half of our cases may have some impairment in p53 protein growth suppressive function, as a result of p53 gene alterations or complex formation. The positive correlation between p53 expression and intensity of hsp72/73 supports the postulate of a p53 regulating function for the chaperone hsp72/73. A high intensity of hsp72/73 immunohistochemical staining could be used as an indirect marker of p53 gene abnormalities.     

Apoptosis in human hepatocellular carcinoma and in liver cell dysplasia is correlated with p53 protein immunoreactivity.

AIMS: To investigate the prevalence of apoptosis in human hepatocellular carcinomas (HCC) of different types and grades and in liver cell dysplasia, and to test whether the apoptotic rate is correlated with the p53 protein status. METHODS: 37 HCC and 66 six liver samples with liver cell dysplasia were analysed for apoptosis using in situ DNA end labelling (ISEL), and for p53 protein expression by immunohistochemistry. In HCCs, proliferative activity was quantitatively assessed using proliferating cell nuclear antigen labelling. RESULTS: The apoptotic index in HCC as based on ISEL ranged from 0.1 to 13.5 per 1000 cells analysed and was not related to type or grade. No nuclear staining was observed in multinuclear tumour cells. There was a significant correlation between the apoptotic rate and both the proliferative activity and p53 protein reactivity. In liver samples containing p53 protein positive liver cell dysplasia cells, there was a significantly higher apoptotic rate of these cells. CONCLUSIONS: Apoptosis is detectable in HCC, and is not related to type and grade. There is a highly significant positive correlation between the apoptotic rate in HCC and both the proliferative activity and p53 protein expression. A similar phenomenon occurs for putative cancer precursors. The findings support the role of p53 in regulating apoptosis in preneoplastic and neoplastic liver lesions.     

热休克蛋白70、90在子宫内膜癌中的表达

目的:探讨子宫内膜癌组织中热休克蛋白(HSP)70、90的表达及意义。方法:用免疫组化Envision法及图象分析仪,检测 30例正常子宫内膜、30例增生过长子宫内膜和53例子宫内膜癌中HSP70、HSP90的表达。结果:子宫内膜癌中HSP70表达的灰度值为(209.06±5.36),明显高于正常内膜[(145.21±4.09),P<0.01]和增生过长内膜[(148.59±4.23),P<0.01];子宫内膜癌中HSP90表达的灰度值为(166.98±5.71),明显低于正常子宫内膜[(208.57±31.14),P<0.05]和增生过长子宫内膜[(249.73±4.94),P<0.01]。子宫内膜癌中HSP70的表达随肿瘤病理分级的增加而增强(P<0.01),非内膜样癌(229.90±3.77)较内膜样癌表达强[(198.37±3.15),P<0.01];子宫内膜癌中HSP90表达随肿瘤病理分级的升高而表达减弱,非内膜样癌(140.21±3.22)较内膜样癌表达减弱[(176.59±2.79),P<0.01]。子宫内膜癌中HSP70、90的表达与肌层浸润深度、临床分期及淋巴结转移未见显著相关性(P>0.05)。结论:HSP70、90可能与子宫内膜癌的发生及预后有关。