抗兔辅酶Ⅱ依赖性视黄醇脱氢/还原酶单克隆抗体的制备和鉴定

背景与目的:制备抗兔辅酶Ⅱ依赖性视黄醇脱氢/还原酶[NADP(H)-dependent retinol dehydrogenase/reductase,NRDR]的单克隆抗体(mAb),并鉴定其特性.材料与方法:以基因工程重组兔NRDR为抗原免疫BALB/c小鼠,用杂交瘤技术建立稳定分泌兔NRDR mAb的细胞株.以间接ELISA法筛选阳性克隆、鉴定Ig亚类、细胞培养上清及腹水效价,同时采用Western blot方法检测mAb的特异性.结果:获得3株可分泌特异性mAb的杂交瘤细胞(NR1、NR2和NR5).其抗体亚类均为IgG1,细胞培养上清效价依次为1∶20、1∶40和1∶20,腹水效价分别为1∶106、1∶107和1∶106.Western blot结果显示NR1、NR2和NR5抗体均能有效识别兔肝组织中的NRDR及重组表达的兔NRDR.结论:成功地建立了稳定分泌兔NRDR mAb的杂交瘤细胞株,为NRDR生物学功能的进一步研究打下了基础.     

正常肝和肝癌细胞中DHRS4L2基因选择性剪接亚型的克隆及其表达调控研究

目的：鉴定正常肝细胞HL-7702和肝癌细胞Hep-G2中DHRS4L2基因的选择性剪接新亚型,并探讨其表达调控机制。方法：应用RT-PCR,3′RACE和生物信息学方法发现并鉴定DHRS4L2基因的选择性剪接新亚型;去甲基化药物5-aza-dC处理HL-7702和Hep-G2细胞,通过RT-PCR和real-time PCR方法检测DHRS4L2的表达情况。结果：发现了2个新的DHRS4L2剪接亚型DHRS4L2A和DHRS4L2A3,2个新亚型均出现了新的第1个外显子,命名为Ea,前者缺失外显子1,后者缺失外显子1和3;在正常肝细胞中去甲基化后含DHRS4L2外显子1的转录产物比处理前明显增加,在肝癌细胞中则比处理前减少。结论：获得2个新的DHRS4L2剪接亚型,并发现DHRS4L2的表达和甲基化调控相关,且甲基化的调控作用在正常肝与肝癌细胞之间存在差异,其机制尚待进一步探讨。     

细胞角蛋白20在宫颈鳞癌中的表达及意义

目的:检测宫颈鳞癌癌灶组织中细胞角蛋白20（CK20）的表达并探讨其临床意义。方法:采用免疫组化SP方法,检测43例宫颈鳞癌癌灶组织和30例正常宫颈组织中CK20的表达,并分析其表达与临床病理因素之间的关系。结果:CK20在宫颈鳞癌癌灶组织的阳性表达率(46.5%)明显高于对照组（P<0.01）。CK20的高表达与临床分期、淋巴结转移有相关性,与病理分化程度无相关性（P>0.05）。结论:检测CK20对宫颈鳞癌的临床分期及判断淋巴结转移等有一定参考价值。     

过氧化物还原酶Ⅱ在胃癌中的表达及临床病理意义

背景与目的:过氧化物还原酶Ⅱ(peroxiredoxinⅡ,PrxⅡ)是具有过氧化物酶活性的蛋白,相关研究提示其参与多种恶性肿瘤的发生、发展.该实验通过研究人类胃癌组织及细胞中的PrxⅡ的表达情况,分析其与胃癌临床病理特征的关系,探讨其与胃癌发生、发展及预后的关系.方法:采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测45例胃癌患者的癌组织及相应的癌旁组织、胃正常细胞(GES-1)及胃腺癌细胞(MGC-803、MKN-45和MKN-28)的PrxⅡmRNA及蛋白表达情况.应用免疫组织化学S-P法检测胃癌组织芯片中116例胃癌患者癌组织及癌旁组织的PrxⅡ的表达量,分析PrxⅡ表达与胃癌临床病理特征的关系并做生存分析.结果:RTFQ-PCR和Western blot检测结果显示,胃癌组织中PrxⅡmRNA及蛋白表达水平明显高于相应的癌旁组织(P<0.05).胃癌细胞中PrxⅡmRNA及蛋白表达水平明显高于胃正常细胞(P<0.01).免疫组织化学结果显示,胃癌组织中PrxⅡ蛋白的表达阳性率(76.7％)同样明显高于相应癌旁组织(30.1％,P<0.01).PrxⅡ蛋白表达与胃癌的肿瘤大小、分化程度、浸润深度、TNM分期及淋巴结转移密切相关(P<0.05),而与患者的性别、年龄、肿瘤部位及远处转移无关(P>0.05).PrxⅡ蛋白高表达者的生存时间要显著低于低表达者(P<0.01),PrxⅡ是影响胃癌预后的独立危险因素.结论:PrxⅡ促进胃癌的发生、发展,是胃癌的不良预后因素,其研究可能为胃癌的治疗提供新的靶点.     

KAI1在宫颈鳞癌中的表达及临床意义

目的探讨KAI1在宫颈鳞癌组织中的表达, 及其与宫颈癌侵袭、转移的关系。方法 采用免疫组化S-P法、逆转录-聚合酶链反应(RT-PCR)法和Western blot方法, 检测92例宫颈鳞癌组织中KAI1的表达。结果 免疫组化结果显示:KAI1在宫颈癌组织中的表达率明显低于宫颈正常上皮组和宫颈上皮内瘤变组, 差异有统计学意义(χ²=29.51, P＜0.01;χ²=22.38, P＜0.01), 且有淋巴结转移的癌变组织其表达情况明显低于未转移的宫颈癌组织, 差异显著(χ²=11.15, P＜0.01), KAI1在宫颈癌中的阳性表达与临床分期无关, 但随着病理分期的增加阳性表达率逐渐减少, 且有统计学意义(χ²=7.16, P＜0.05)。RT-PCR及Western blot同样证实:KAI1在宫颈癌组中的相对表达量明显低于宫颈正常上皮组(F=6.6, P＜0.01;F=9.73, P＜0.01)。结论 宫颈癌中KAI1的异常表达与宫颈癌的恶变程度有关。     

TCAB1在宫颈鳞状上皮内病变及宫颈鳞癌中的表达

摘 要：［目的］ 探讨TCAB1在宫颈鳞状上皮内病变及宫颈鳞癌的表达及意义。［方法］选取2016年1月至2018年8月海南医学院第二附属医院病理科宫颈鳞状细胞癌组织60例,低级别鳞状上皮内病变组织30例,高级别鳞状上皮内病变40例组织及正常宫颈组织30例,采用免疫组化法检测各组的TCAB1表达。［结果］ 低级别鳞状上皮内病变、高级别鳞状上皮内病变和宫颈鳞癌组TCAB1阳性比例逐渐升高,依次为40.0％(12/30)、75.0％(30/40)和81.7％(49/60）,明显高于对照组的0.7％(2/30)(P<0.05)。［结论］ TCAB1可能参与宫颈鳞状细胞癌的发生发展。     

S100A4蛋白在宫颈鳞癌中的表达及意义

目的　研究S10 0A4蛋白在宫颈鳞癌组织中的表达及其临床意义。方法　应用免疫组织化学SP法检测 65例宫颈鳞癌组织和10例正常宫颈组织中S10 0A4蛋白。结果　在正常宫颈组织中S10 0A4蛋白不表达 ;在宫颈鳞癌组织中S10 0A4蛋白的表达率为3 5 .4% ( 2 3 /65 ) ;与正常宫颈组织比较 ,差异具有显著性 (P <0 .0 1)。S10 0A4蛋白在宫颈鳞癌中的表达与临床分期和淋巴结转移有关 (P <0 .0 1) ,与组织学分级无关 (P >0 .0 5 ) ;阳性细胞在血管平滑肌细胞以及淋巴细胞中亦有表达。结论　S10 0A4蛋白和宫颈鳞癌的侵袭和转移密切相关 ;S10 0A4蛋白可作为判定宫颈鳞癌临床病理特征的重要指标     

S100A4蛋白在宫颈鳞痛中的表达及意义

目的研究S100A4蛋白在宫颈鳞癌组织中的表达及其临床意义.方法应用免疫组织化学SP法检测65例宫颈鳞癌组织和10例正常宫颈组织中S100A4蛋白.结果在正常宫颈组织中S100A4蛋白不表达;在宫颈鳞癌组织中S100A4蛋白的表达率为35.4%(23/65);与正常宫颈组织比较,差异具有显著性(P＜0.01).S100A4蛋白在宫颈鳞癌中的表达与临床分期和淋巴结转移有关(P＜0.01),与组织学分级无关(P＞0.05);阳性细胞在血管平滑肌细胞以及淋巴细胞中亦有表达.结论S100A4蛋白和宫颈鳞癌的侵袭和转移密切相关;S100A4蛋白可作为判定宫颈鳞癌临床病理特征的重要指标.     

CD_(44v6)及C-myc在宫颈鳞癌中的表达及意义

目的探讨CD44v6及C-myc在宫颈鳞癌发生、发展中的作用和意义以及二者之间的关系。方法选取30例正常宫颈组织和60例宫颈鳞癌组织应用组织芯片和免疫组化技术分别检测CD44v6及C-myc蛋白的表达情况。结果CD44v6在Ⅰ￣Ⅱ期宫颈鳞癌中的表达与病理分级、累及颈管肌层深度、淋巴结转移相关,而与年龄、临床分期无关。C-myc的表达与临床分期、累及颈管肌层深度、淋巴结转移相关,而与年龄、病理分级无关。结论CD44v6及C-myc在宫颈鳞癌中的表达可作为预后指标;二者有协同作用,共同检测有助于预测宫颈癌的侵袭、转移、预后。     

一种人神经母细胞瘤辅酶II-依赖性视黄醇脱氢酶cDNA的克隆及特征分析

背景与目的:检测神经母细胞瘤中新的辅酶II_依赖性视黄醇脱氢/还原酶[NADP(H)_dependen tretinold ehydrogenase/reductase,NRDR]选择性剪接亚型。材料与方法:我们用NRDR特异引物,从人神经母细胞瘤细胞系SK_N_SH和SK_SY_5YcDNA中分别经PCR扩增出635bp和429bpDNA片段,测序证实635bp片段是NRDR,而429bp片段是选择性剪接新亚型。再用快速cDNA末端扩增(Rapidamplification of cDN Aends,RACE)方法得到429bp片段cDNA全长序列。用绿色荧光蛋白与新亚型的融合蛋白做亚细胞定位。结果:得到新亚型全长并命名为humanNRDRA2(hum NRDRA2)(AY616182)。已知NRDR有8个外显子,而新亚型hum NRDRA2由选择性剪接造成了第4和第6外显子丢失,从第5外显子开始读码框架前移1bp,导致随后的编码区框码漂移(frameshift),同时蛋白翻译终止信号提前出现,产生仅有188个氨基酸的蛋白,其中第137氨基酸以后的序列相对于NRDR完全发生改变,同时C_末端的过氧化物酶体定位信号_SRL丢失,却在160～176氨基酸处出现了一个细胞核定位信号。我们在ESTs库中未找到与其相同的剪接形式,提示它可能是神经母细胞瘤特有的一种NRDR剪接亚型。用GFP_NRDRA2融合蛋白做该蛋白的亚细胞定位,发现发绿色荧光的细胞均已悬浮,但悬浮状态不佳,而作为对照的NRDR另一亚型则能定位成功,提示NRDRA2蛋白可能具有一定的细胞毒性。结论:人神经母细胞瘤NRDRA2选择性剪接亚型羧基端框移突变并有核定位序列;该蛋白可能是一种细胞毒性蛋白。     

Expression of a novel alternatively spliced variant of NADP(H)-dependent retinol dehydrogenase/reductase with deletion of exon 3 in cervical squamous carcinoma

NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) plays an important role in maintaining the homeostasis of retinoid. Aberrations in retinoid metabolism are considered as early events in carcinogenesis. We identified a novel alternatively spliced variant, NRDRB1, in HeLa cell and human cervical squamous carcinoma tissues, which is characterized by a complete deletion of exon 3. The latter resulted in changes in subcellular localization of NRDRB1 when compared with the peroxisomal localization of NRDR. To clarify the clinical significance of NRDRB1, we investigated its mRNA and protein expressions in normal cervical and cervical squamous carcinoma tissues, using RT-PCR, quantitative real-time PCR, Gateway expressing system, immunoprecipitation, immunoblotting, MALDI-TOF mass spectrometry and immunohistochemistry. We detected NRDRB1 mRNA in 14 of 26 (53.9%) cervical cancer tissues, but in none of the 12 normal cervical tissues. NRDRB1 protein was expressed in NRDRB1 mRNA-positive cases. While the full-length NRDR mRNA was observed in both normal and neoplastic cervical tissues, its protein was only expressed in normal cervical epithelium. The results presented here provide evidence that metabolic disturbances of retinal and retinoic acid, due to abnormal splicing and functional disorder of NRDR, may be involved in cervical tumorigenesis.     

宫颈鳞癌组织中NRDR选择性剪接新亚型的鉴定及意义

目的:探讨NRDR选择性剪接新亚型NRDRB1 mRNA和蛋白在宫颈鳞癌组织中的表达及其临床意义。方法:采用RT-PCR、免疫荧光、免疫沉淀、免疫印迹、MALDI-TOF质谱分析等技术,检测正常宫颈上皮及宫颈鳞癌组织中NRDRB1mRNA及蛋白表达。结果:NRDRB1亚型全长1 171 bp,其蛋白保留了SDR家族的N末端辅酶结合模序、C末端底物结合模序以及过氧化物酶体定位信号。因第3外显子的缺失,导致SDR家族典型的“LVSNAA”折叠的丢失,使NRDRB1蛋白空间构象发生改变,从而影响该蛋白的细胞内定位和功能,NRDRB1蛋白的亚细胞定位明显不同于NRDR,酶活性亦明显低于NRDR。NRDRB1 mRNA及蛋白在宫颈鳞癌组织中表达(15/27,55.6%),明显高于正常宫颈上皮组织(P<0.05),正常宫颈上皮除NRDR外,未检测到NRDRB1 mRNA及其蛋白。结论:NRDR选择性剪接的异常,以及新的剪接亚型NRDRB1细胞内定位及蛋白酶活性的改变可能与宫颈鳞癌的发生发展密切相关。     

宫颈癌细胞中NRDR选择性剪接新亚型表达质粒的构建及原核表达

背景与目的:构建宫颈癌细胞中视黄醇脱氢/还原酶-辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NRDR)及其选择性剪接亚型NRDRB1原核表达载体,并在BL21-AI大肠杆菌中表达融合蛋白. 材料与方法:用RT-PCR检测宫颈癌组织中NRDR、NRDRB1表达,RACE方法克隆NRDRB1全长cDNA,运用Gateway表达系统将NRDR、NRDRB1编码区序列构建到表达载体,转化到大肠杆菌中表达,表达的融合蛋白通过金属离子亲和层析纯化. 结果:在宫颈鳞癌组织中发现NRDR新的选择性剪接亚型NRDRB1,构建了NRDR、NRDRB1原核表达载体,在BL21-AI中表达出氨基端含6×His标签的重组蛋白,诱导表达4 h后目的蛋白可占总蛋白量的30%～50%,经一步亲和层析得到了高纯度的NRDR及NRDRB1重组蛋白. 结论:在大肠杆菌中获得高效表达的NRDR、NRDRB1蛋白,为进一步研究其功能提供了实验材料.     

CDKN2 (MTS1/p16INK4A) Gene Alterations in Adult T-Cell Leukemia/Lymphoma

p16^INK4A is a cyclin-dependent kinase inhibitor (CDKI), and regulates the cell cycle negatively. Recently, p16^INK4A protein was shown to be encoded by the CDKN2 gene, which is identical to multiple tumor suppressor gene 1 (MTS1) on chromosome 9p21, where genetic alterations occur frequently in many malignant tumors. As the loss of p16^INK4A function by genetic alterations leads to inappropriate progression of the cell cycle, the CDKN2 gene has been investigated intensively as a new candidate tumor suppressor gene in many malignant tumors. Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy associated with human T-cell lymphotrophic virus type 1 (HTLV-1). As the development to ATL is believed to require not only HTLV-1 infection but also accumulation of genetic alterations, we investigated the relationship between alterations in the CDKN2 gene and ATL. Alterations in the CDKN2 gene were detected in approximately 15 to 20% of ATL patients. Interestingly, most of the patients with CDKN2 gene alterations had the aggressive form of ATL. The CDKN2 gene appears to be the major tumor suppressor gene on chromosome 9p21, and alteration in this gene may play an important role during late stages in the transformation process induced by HTLV-1.     

3-氯-4-二氯甲基-5-羟基-2(5氢)-呋喃酮诱导人胚胎肝细胞ras基因突变

背景与目的:研究饮水氯化消毒副产物3-氯-4-二氯甲基-5-羟基-2(5氢)-呋喃酮(3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone,MX)对体外培养的人胚胎肝细胞(L-02细胞)ras基因突变的诱导。材料与方法:MX染毒剂量为300μmol/L,以二甲基亚砜(DMSO)做溶剂对照,将L-02细胞连续染毒培养12d后,收获细胞提取基因组DNA,应用PCR-克隆测序法检测ras基因(K-ras、H-ras、N-ras)12、13、61密码子是否存在突变。结果:MX染毒组H-ras基因57密码子的GAT置换成GGT,未检测到K-ras、N-ras及H-ras12、13、61密码子突变,DMSO溶剂对照组相应的ras基因目的片段均未检测到突变。结论:MX可能诱导L-02细胞ras基因突变。     

Inhibitory Effects of (—)-Epigallocatechin Gallate on Spontaneous Hepatoma in C3H/HeNCrj Mice and Human Hepatoma-derived PLC/PRF/5 Cells

The inhibitory effect of (—)-epigallocatechin gallate (EGCG), a main constituent of Japanese green tea, on spontaneous hepatoma in C3H/HeNCrj mice was investigated. A total of 72 mice were divided into three groups; the control group without EGCG, and two experimental groups receiving 0.05% (w/w) or 0.1% EGCG in drinking water. EGCG reduced the incidence of hepatoma-bearing mice from 83.3% (control) to 56.0% (0.05% EGCG) and 52.2% (0.1% EGCG), and also reduced the average number of hepatomas per mouse from 1.83 (control) to 0.72 (0.05% EGCG) and 0.91 (0.1% EGCG) at week 65. Ridit analysis of the distribution of the number of hepatomas in each group revealed that EGCG significantly increased the rate of mice without hepatoma in the two EGCG groups as compared to the control. EGCG did not affect body weight gain, food consumption or any serum biochemical parameter. EGCG inhibited the growth and secretion of α-fetoprotein by human hepatoma-derived PLC/PRF/5 cells without decreasing their viability. These results indicate that EGCG may be a practical, nontoxic preventive agent against human hepatoma.     

Effects of K-ras Gene Mutations in the Development of Lung Lesions Induced by 4-(N- Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone in A/J Mice

The relationship between the development of peripheral lung lesions induced by tobacco-specific 4-( N -methyl- N -nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and K- ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K- ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K- ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and dideoxy sequencing methods. K- ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42 (75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K- ras gene mutation was a G-to-A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K- ras gene mutations. Thus, K- ras gene mutation is closely associated with the development of NNK-induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.