脂质体介导外源DNA体外转染金黄地鼠卵母细胞的研究

背景与目的:研究外源DNA在金黄地鼠卵母细胞染色体上的整合率,探索卵母细胞作为外源基因载体的可行性.材料与方法:在体外,用脂质体包裹外源NF-Luc质粒,将其与金黄地鼠卵母细胞共培养后收获细胞,分别用斑点杂交、Southern杂交以及荧光原位杂交分析检测Luc 基因在卵母细胞中的整合.结果:Southrn杂交分析,在13个样本DNA中发现9个样本有Luc 基因特异性条带扩增;阳性率为69.2%(48.2%～94.5%),荧光原位杂交分析显示,在253个转染后卵母细胞中发现,2例中期相染色体及3例间期核上有明显的Luc 基因阳性杂交信号.结论:在国内首次证实金黄地鼠卵母细胞可被脂质体包裹的外源DNA转染,即金黄地鼠卵母细胞可以作为外源基因的载体,这一新途径为研究外源基因在金黄地鼠卵母细胞中的表达提供了实验基础.     

HIV-1 gag基因经卵母细胞垂直传递模型的建立

目的：建立HIV-1gag基因经卵母细胞垂直传递的模型,为HIV通过雌性生殖细胞垂直传递的研究奠定实验基础。方法：将含有HIV-1gag基因的重组质粒pIRES2-EGFP-gag注射到小鼠双侧卵巢,转染生殖细胞。应用超排获得实验用卵母细胞,收集显示绿色荧光的卵母细胞,将一部分呈现绿色荧光的卵母细胞裂解,提取基因组DNA,采用PCR检测gagDNA是否存在卵母细胞中;再将另一部分带有绿色荧光的卵母细胞常规制备中期染色体片,用荧光原位杂交（FISH）技术检测HIV-1gag基因在成熟卵母细胞染色体上的整合。结果：转染pIRES2-EGFP-gag后,在荧光显微镜下能够很清楚地看到带有绿色荧光的卵母细胞;PCR结果显示在带有绿色荧光的卵母细胞中可检测到HIV-1gag基因特异的阳性条带;FISH检测结果显示在卵母细胞的中期染色体上检测到HIV-1gagDNA的阳性信号。结论：HIV-1gag基因能够通过卵透明带和细胞膜并整合到卵母细胞基因组内,本研究为HIV-1gag基因可经过雌性生殖细胞垂直传递给子代奠定了实验基础。     

精子携带的乙肝病毒基因在金黄地鼠胚胎中的复制与表达

背景与目的:乙型肝炎是危害人类健康的全球性疾病。本研究旨在探索乙肝病毒(HepatitisBvirus,HBV)通过精子垂直传播的可能性。方法:将金黄地鼠精子与pBR322-HBVDNA重组质粒进行共培养后,与金黄地鼠正常卵母细胞受精,取2-细胞胚分别制片和提取RNA,用荧光原位杂交(Fluorescenceinsituhybridization,FISH)、RT-PCR和免疫荧光检测技术研究精子携带的HBV基因在胚胎细胞中的复制与表达。结果:①FISH:用全长HBVDNA探针与128个2-细胞胚进行FISH,其中3个胚胎FISH结果阳性,每个胚胎的2个间期核上均有HBVDNA杂交信号;②RT-PCR:在检测样本中可见特异的HBxDNA阳性条带,阴性对照1(模板为灭菌水)和阴性对照2(未加反转录酶)均为阴性;③免疫荧光检测:2-细胞胚经血清封闭后,分成两组。一组作检测样本,另一组用PBS代替一抗(小鼠抗人HBsAg)作为阴性对照,检测样本中可见阳性免疫荧光信号,阴性对照未见信号。结论:以精子为载体,携带到卵内的HBV基因能够在胚胎细胞中复制和表达,提示HBV能够通过男性生殖细胞由父亲垂直传递给子代。     

逆转录制备cDNA探针对人胃癌MGC80—3细胞系多种癌基因转录的同步检测

本文报道利用人胃癌MGC80-3细胞系总polyA ~+RNA,逆转录制备cDNA探针,并与多种克隆癌基因的质粒DNA斑点杂交,同步检测倍增生长的人胃癌MGC 80-3细胞系不同原癌基因转录本的丰度。结果发现,V-sis、c-erbB-2、V-abl、H-ras、K-ras和c-myc质粒DNA的斑点出现阳性杂交信号,其中V-sis、c-erbB-2的斑点信号最强。说明人胃癌MGC80-3细胞系中,多种原癌基因协同地进行表达。     

转染人突变dhfr基因的小鼠骨髓细胞对小鼠造血功能的长期保护作用

目的:将转染了人突变dhfr基因的第二代小鼠骨髓,移植给经致死剂量照射的第三代小鼠,观察该基因对小鼠造血功能的长期保护作用.方法:分离存活的第二代小鼠骨髓有核细胞,直接移植给经致死剂量照射的同系小鼠,以MTX筛选,观察小鼠血象和生存率变化,PCR和Southem印迹杂交分析目的基因在小鼠染色体DNA中的整合与表达情况.结果:在大剂量MTX筛选下,实验组小鼠造血功能逐渐恢复,对照组3周内全部死亡.实验组生存率和生存期明显高于对照组,但较前两代生存率低.PCR和Southern印迹分析结果提示,实验组脾脏和肝脏组织中均检测到前病毒标志基因neo~R和dhfr基因的特异务带.结论:转染了人突变dhfr基因的第二代小鼠骨髓,能有效地重建经致死剂量照射的第三代小鼠造血功能.保护骨髓免遭大剂量MTX所致的严重骨髓抑制,dhfr基因在小鼠基因组DNA中的稳定整合是这种长期保护作用的物质基础.     

人精子携带的HBs和HBc基因在早期胚胎细胞中的蛋白表达

背景与目的:探讨由人精子带入受精卵中的HBs和HBc基因能否在早期胚胎细胞中进行蛋白表达.材料与方法:人精子经重组质粒pIRES2-EGFP-HBV转染后,与金黄地鼠去透明带卵母细胞离体受精,选择带绿色荧光的2-细胞胚,用免疫荧光技术检测HBs和HBc基因在胚胎细胞中的蛋白表达,用ELISA方法分别对HBsAg和HBcAg进行半定量和定性分析.结果:在带绿色荧光的2-细胞胚中免疫荧光检测可见清楚的HBsAg和HBcAg阳性信号;ELISA结果表明,单个2-细胞胚内HBsAg的量小于0.064 ng/ml,对HBcAg的检测得到阳性结果.结论:人精子携带到卵内的HBV基因可在早期胚胎细胞中表达表面抗原和核心抗原.     

水泡口炎病毒M蛋白对小鼠Lewis肺癌LL/2c细胞增殖与凋亡的影响

背景与目的:水泡口炎病毒(vesicular stomatitis virus,VSV)具有明显的抗肿瘤作用,在人的A549肿瘤模型和小鼠的LL/2c模型中已取得了明显的治疗效果.但复制完全型病毒在临床应用中存在着一定的安全隐患,且研究报道VSV抗肿瘤作用与其基质蛋白(matrix protein,M蛋白)有关.本研究旨在探讨VSV-M蛋白对小鼠的LL/2c肿瘤细胞增殖和凋亡的影响.方法:采用分子克隆技术构建VSV-M蛋白重组真核表达质粒pcDNA3.1-M,经酶切、PCR、测序鉴定得到阳性重组子,体外转染LL/2c细胞,RT-PCR、Western blot检测M蛋白的表达.采用噻唑蓝(MTT)法检测了M蛋白质粒对LL/2c肿瘤细胞增殖的影响.DNALadder、Hoechst33528染色检测LL/2c肿瘤细胞凋亡.结果:构建了VSV-M蛋白真核表达质粒pcDNA3.1-M.重组质粒体外转染LL/2c细胞后,检测到M蛋白的表达;倒置显微镜下观察到细胞形态学的改变,且48 h后细胞生长抑制明显,抑制率达41.3%(P＜0.05),而空载体转染组只有6.5%的抑制率(P＞0.05).琼脂糖凝胶电泳可见DNA ladder形成,Hoechst 33528染色检测到LL/2c细胞凋亡,细胞核改变.结论:VSV-M蛋白可抑制小鼠LL/2c细胞增殖,并诱导细胞凋亡.     

TRAIL真核表达治疗肝细胞癌作用的研究

目的：构建鼠源TRAIL（mTRAIL）真核表达质粒。研究其体内、外表达对肝癌细胞的诱导凋亡作用，抑制肝癌生长作用及与重组人FN多肽真核表达质粒pCH510协同抑制肝癌生长作用。方法：采用RTPCR及重组DNA技术构建mTRAIL真核表达质粒；体内、外进行基因转染；用流式细胞仪检测肝癌细胞凋亡率，用TdTmediated dUTP nick end labeling (TUNEL)法及免疫组织化学技术检测肝癌细胞凋亡；小鼠实体瘤块模型研究基因转染抑制肝癌生长作用。结果：用RTPCR方法自小鼠脾细胞RNA扩增出TRAIL基因的全长cDNA，并克隆至真核表达载体pcDNA3.1中获重组质粒pX1；以pX1转染BHK细胞株后攻击肝癌细胞株H22细胞，可检测到肝癌细胞凋亡；肌肉内注射转染质粒DNA pX1，通过诱导肿瘤细胞凋亡抑制肝癌生长；pX1与FN多肽真核表达质粒pCH510有协同抑制肝癌生长作用。结论：质粒pX1可在细胞及小鼠体内表达；体内、外表达可诱导肝癌细胞凋亡并可通过该机制抑制肝癌生长；与FN多肽真核表达质粒pCH510有协同抑制肝癌生长作用。     

Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction

Summary Oncogenic human papillomavirus (HPV) types 16 and 18 commonly associated with cervical cancer are found in many epithelial malignancies at extra-genital sites including breast. The transforming gene products of HPV have also been shown to immortalize breast epithelial cellsin vitro. But the findings of HPV DNA in breast carcinoma are found to be contradictory. In the present study fine needle aspirate cell (FNAC) samples from 26 breast cancer patients and four breast tumour biopsies were analysed for the presence of HPV 16 and 18 DNA sequences by both polymerase chain reaction (PCR) and Southern blot hybridization. Of 26 fine needle aspi rate cell samples and four breast cancer biopsies, not a single sample was found to be positive by either PCR or Southern blot hybridization. The observation of complete absence of HPV DNA sequences in breast cancer refute the possibility of any role for oncogenic genital HPV types 16 and 18 in the pathogenesis of breast cancer.     

酵母双杂合体系筛选乙型肝炎病毒X蛋白结合蛋白

乙型肝炎病毒(hepatitis B virus,HBV)X 蛋白是一个广义反式作用因子,同原发性肝癌的发生发展密切相关,由于其功能同蛋白-蛋白间相互作用有关,故本研究利用酵母双杂合体系筛选并鉴定HBV X 蛋白结合蛋白。     

顺铂、卡铂和双环铂对pBR322质粒DNA的断裂作用

目的：研究铂类抗癌药顺铂、卡铂和双环铂对pBR322质粒DNA的致断性。 方法：用琼脂糖凝胶电泳和凝胶成像分析方法 结果：顺铂、卡铂、双环铂均可诱发pBR322质粒DNA断裂,对质粒DNA的半数致断剂量分别为33.71 μmol/L、3.31 mmol/L和1.61 mmol/L。 结论：对pBR322质粒DNA的致断性强弱依次为：顺铂>双环铂>卡铂。     

Integrated state of subgenomic fragments of hepatitis B virus DNA in hepatocellular carcinoma from mainland China

Hepatocellular carcinoma (HCC) samples from mainland China were examined for the presence and state of hepatitis B virus (HBV) DNA sequences. HBV DNA was detected by dot-blot hybridization in 13 of 17 cases of HCC from the Shanghai area and in three of six samples from Hangzhou. The HCC cases from Shanghai were then analyzed in more detail. Fifteen of the 17 patients had serologic evidence of past or present infection with HBV (with inadequate information available for the other two), and the 13 HCC samples positive for HBV DNA all came from serologically positive patients. Southern blot analysis showed that the HBV DNA sequences were always integrated in the HCC high-molecular-weight DNA; only one or two viral copies were present per tumor cell, and no common integration site was evident. Hybridization analyses using subgenomic probes of HBV DNA revealed that the tumors seldom retained an entire HBV genome. HBV S-region sequences were always present, X-region sequences were usually represented, and C-region sequences were rarely detectable in virus-positive tumors. A fragment within the HBV DNA X-region, between nucleotides 1441 and 1526, was found to hybridize nonspecifically with cellular DNA; reported sequence data indicated that this fragment would contain approximately 70% guanine + cytosine. Histologic sections were prepared from some of the frozen tissue specimens and stained by an indirect immunoperoxidase technique for hepatitis B surface antigen (HBsAg). Only 1 of 10 HBV DNA-positive samples contained HBsAg in the cytoplasm of tumor cells, although abundant HBsAg was present in adjacent normal cells in all 10 cases. There were no significant differences in histology between HCC that contained HBV DNA sequences and those that were virus negative. These data support the premise that HBV represents a major etiologic factor in the development of HCC in the Shanghai area of China, although the molecular basis of viral involvement remains obscure.     

Detection of human papillomavirus in primary hepatocellular carcinoma.

Human papillomaviruses (HPV) have been linked causally to some human cancers such as cervical carcinoma. To determine whether any additional type of human malignancy contained HPV DNA, we examined 16 hepatocellular carcinoma (HCC) specimens by Southern blot technique and by polymerase chain reaction (PCR). One HCC contained HPV 16 DNA as demonstrated by both Southern blot and PCR. Two other HCC samples contained HPV 18-related nucleotide sequences by PCR but were negative by Southern blot of genomic DNA. HPV could have been carried via blood to the liver, thus indirectly supporting the presence of an HPV viremia. Our findings suggest that oncogenic HPV might constitute a cofactor acting synergistically with hepatitis B virus (HBV) in the development of the HCC in these patients. Alternatively, the presence of HPV in the tumor tissue might be the result of an opportunistic infection.     

Validation of hybridization assays: correlation of filter in situ, dot blot and PCR with Southern blot.

A number of validation experiments have compared the most commonly used HPV hybridization methods with the accepted gold standard, Southern blot hybridization. The methods discussed are filter in situ hybridization (FISH), dot blot hybridization (ViraPap/ViraType), and polymerase chain reaction (PCR). FISH now appears to be too inaccurate to be recommended for future epidemiological studies. ViraPap/ViraType compares well to Southern blot, but is limited to the detection of seven genital HPV types. PCR-based methods may be more sensitive than Southern blot and are likewise capable of detecting most known genital HPV types. Direct comparisons of the several available PCR methods should be performed. Currently, there is no perfect method for HPV testing, because Southern blot itself is prone to some errors in performance and interpretation. Given that the scientific and clinical usefulness of HPV tests depends on the repeatability and accuracy of the assays, more intra- and inter-assay comparisons should be done to establish reference standards applicable to this area of molecular diagnostics.     

Expression of the human beta globin gene and 5'-deletion mutants in erythroleukemic mouse cells studied by DNA mediated gene transfer

A recombinant plasmid pTKH beta GHp-3 carrying genomic human DNA sequences coding for the beta globin gene covalently linked to the thymidine kinase gene of HSV-1 and the ampicillin resistance region of a bacterial plasmid has been constructed. Using Bal 31 exonuclease, deletion mutants have been obtained from a single HpaI site located 800 bp upstream the 5' end of the beta globin gene and towards the cap site. Recipient mouse erythroleukemic Friend TK- cells were transformed with these molecules. Analysis of donor DNA sequences in transformed cells by Southern blotting and filter hybridization has demonstrated the presence of full length multiple copies. RNA isolated from transformed non-induced Friend cells, carrying one or the other of the donor human beta globin deletion mutants, has been analysed by Northern blot and spot hybridization analyses. Evidence has been obtained which suggests that DNA sequences located upstream the CCAAT box (at -76 bp from the cap site) are required for optimal levels of human beta globin specific RNA in transformed cells.