妇女子宫颈感染人乳头瘤病毒高危亚型HPV52的遗传变异性分析

目的了解宁波地区人乳头瘤病毒(human papillomavirus,HPV)高危亚型HPV52的分布和癌相关基因E6、E7区基因变异情况。方法收集宁波市社区妇女的宫颈脱落细胞样本进行HPV分型检测,并对其中HPV52亚型阳性株进行癌相关基因测序和序列分析。结果从778份样本中检出HPV52阳性24例,检出率为3.1%。获得E6、E7基因序列分别为21和19条。E6区有3个位点发生碱基颠换:G350T、G356A和A379G,其中G350T和A379G出现率最高,占95.2%;E7区有3个位点发生碱基颠换:C751T、A801G和A849C,其中C751T和A801G出现率最高,占94.7%。系统进化树分析显示,宁波地区HPV52型存在亚洲型和欧洲型。结论宁波社区人群HPV52感染率较高,多数感染者携带病毒的癌相关基因发生变异,应加强对人群HPV感染状况和病毒株变异情况的监测,及时调整宫颈癌防治策略。     

人乳头瘤病毒16型L1/E7嵌合基因的克隆及其在毕赤酵母中的表达

目的克隆人乳头状瘤病毒16型(HPV16)L1/E7嵌合蛋白基因,并在毕赤酵母中表达。方法PCR扩增HPV16L1/E7基因,并克隆至毕赤酵母表达载体pPIC-3·5K和pPIC-9K中,构建含HPV16L1/E7基因的pPIC3·5K-HPV16L1/E7和pPIC9K-HPV16L1/E7重组表达载体,经测序证实插入的外源基因读码框正确后,分别转入GS115和KM71菌株中,筛选阳性转化菌株,经PCR鉴定,甲醇诱导表达HPV16L1/E7嵌合蛋白,经SDS-PAGE和Westernblot鉴定,电镜观察,并经离心法纯化VLPs。结果HPV16L1/E7基因已成功插入pPIC-3·5K和pPIC-9K载体中,共获得6株KM71HPV16L1/E7-pPIC3·5K、5株GS115HPV16L1/E7-pPIC3·5K、1株KM71HPV16L1/E7-pPIC9K和2株GS115HPV16L1/E7-pPIC9K阳性转化菌株,并可表达相对分子质量约为69000的蛋白,与预期值一致。表达量约为0·35~1·04mg/ml。Westernblot显示该蛋白可与抗-HPV16L1蛋白和抗-HPV16E7蛋白的单克隆抗体特异性结合。在透射电镜下可观察到VLPs的形成,其形态与HPV16天然病毒颗粒相似。经纯化的目的蛋白纯度接近100%。结论已成功构建了pPIC3·5K-HPV16L1/E7和pPIC9K-HPV16L1/E7重组表达载体,阳性转化菌株可表达结构与野生型HPV16一致的VLPs。     

N+离子注入诱变筛选胸腺嘧啶缺陷型大肠杆菌

利用N+离子注入诱变大肠杆菌,经甲氧苄啶的选择性筛选,得到胸腺嘧啶营养缺陷型菌株.通过重组DNA技术将该突变株胸苷酸合成酶(TS)基因连接到载体pMD-18T质粒上,经测序发现TS基因第173个碱基由AT→CG,其相应的氨基酸由谷氨酸(E58)突变为丙氨酸(A58).该突变株的获得为后续研究嘧啶核苷酸的代谢奠定了基础.     

HPV-DNA分型检测联合液基细胞学对宫颈癌筛查的意义及临床价值的研究

目的研究HPV-DNA分型检测联合宫颈液基细胞学检查(TCT)对宫颈癌筛查的意义及临床价值。方法将于我院就诊的564例妇女同时行TCT、PCR荧光检测HPV-DNA和宫颈活检组织学检查,采用膜杂交法检测23个HPV基因型别(低危型:HPV6、11、42、43、45和高危型:HPV16、18、31、33、35、39、45、51、52、53、56、58、59、66、68、73、83、MM4)和液基细胞学(TCT)作为宫颈癌和癌前病变的筛查。比较TCT对宫颈细胞异常的检出率以及TCT检测联合PCR荧光检测HPV-DNA对HPV感染的检出率,以病理学诊断为标准。结果 PCR荧光法检测HPV-DNA总检出率为21.0%,组织学阳性病例检出率70.9%,组织学阴性病例检出率4.5%,均显著高于TCT,呈极其显著性差异,P<0.01。其中单一型HPV感染中,低危型占34%、高危型占60%、二型和二型以上混合感染占6%。结论 HPV基因分型检测是有价值的辅助诊断技术,与细胞学联合,是最佳的宫颈癌筛查的方案,能明显提高诊断的准确性和早发现癌前病变。     

人乳头状瘤病毒16亚型E6E7基因重组腺病毒的构建及其在小鼠骨髓源树突状细胞中的表达

目的 构建人乳头状瘤病毒16亚型(Human papillomavirus 16,HPV16)E6E7基因重组腺病毒,并在小鼠骨髓源树突状细胞(Dendritic cell,DC)中表达。方法双酶切质粒pET-32a(+)-E6E7获得E6E7,基因片段,插入腺病毒穿梭质粒pAd-Track-CMV中,转染HEK293细胞,扩增病毒,经同源重组、包装后获得重组腺病毒pAd-E6E7,转染体外培养的小鼠骨髓源DC,激光共聚焦显微镜观察转染的小鼠DC的形态,流式细胞术检测转染前后小鼠DC表面标志物(CD40、CD86、MHCⅡ和CD11C),Western blot检测E6蛋白的表达。结果双酶切及测序证实,重组腺病毒质粒pAd-E6E7构建正确,插入的E6E7基因片段序列正确;转染HEK293细胞48 h,倒置荧光显微镜下可见绿色荧光蛋白的表达,重组腺病毒的滴度为3×107 CCID50/ml;重组腺病毒pAd-E6E7感染后DC的状态与成熟DC表面标志物(CD40、CD86、MHCⅡ和CD11C)相符合,感染后的DC表面有许多树枝状、刺状突起,符合成熟DC的表面形态;感染后的DC中存在E6蛋白的表达。结论已成功构建了HPV16 E6E7基因重组腺病毒表达质粒,其能在小鼠骨髓源DC中表达。     

N^＋离子注入诱变筛选胸腺嘧啶缺陷型大肠杆菌

利用N＋离子注入诱变大肠杆菌,经甲氧苄啶的选择性筛选,得到胸腺嘧啶营养缺陷型菌株。通过重组DNA技术将该突变株胸苷酸合成酶（TS）基因连接到载体pMD-18T质粒上,经测序发现TS基因第173个碱基由AT→CG,其相应的氨基酸由谷氨酸（E58）突变为丙氨酸（A58）。该突变株的获得为后续研究嘧啶核苷酸的代谢奠定了基础。     

adr和adw亚型HBsAg单克隆抗体的制备

目的制备adr和adw亚型乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)单克隆抗体,并用其区分两亚型HBsAg。方法利用杂交瘤细胞融合技术制备adr和adw亚型HBsAg单克隆抗体,采用间接ELISA法进行筛选;优化鉴别试验抗原包被剂量,并对HBsAg亚型鉴别试验进行验证。结果共获得抗两亚型HBsAg的单克隆抗体7株;鉴别试验抗原的最佳包被剂量为10μg;1H6和4D3株单抗检测2份编盲adw亚型HBsAg样品的adwA450/adrA450值均大于2,判定其为adw亚型;1E9株单抗检测2份编盲adr亚型HBsAg样品adrA450/adwA450值均大于2,判定其为adr亚型;3A5株单抗检测4份编盲样品的adrA450/adwA450值和adwA450/adrA450值均小于2,未能区分两亚型HBsAg。结论抗adr亚型单抗1E9株及抗adw亚型单抗1H6株和4D3株可用于adr亚型汉逊酵母乙肝疫苗研制过程中HBsAg亚型的鉴别。     

慢病毒介导的人乳头瘤病毒16型E6基因shRNA对荷瘤裸鼠宫颈癌生长的抑制作用

目的探讨慢病毒介导的人乳头瘤病毒(Human papilloma virus,HPV)16型E6基因shRNA在体内对荷瘤裸鼠宫颈癌生长的抑制作用。方法将BALB/c-nu裸鼠随机分为空白对照组、干扰组和无义干扰组,经皮下接种宫颈癌Caski细胞(2×106个),移植瘤直径达0.5 cm时,空白对照组于瘤体局部注射PBS,干扰组和无义干扰组分别于瘤体局部注射靶向HPV16型E6基因的shRNA-PLL3.7干扰慢病毒和无义干扰慢病毒(3×108TU/ml),2周后检测瘤体大小和重量的变化,采用免疫组化法检测肿瘤组织中HPV16型E6、P53和P21蛋白的表达。结果与无义干扰组和空白对照组比较,干扰组裸鼠体内肿瘤明显缩小,瘤体重量明显降低(P<0.000 1),无义干扰组与空白对照组比较,差异无统计学意义(P>0.05);干扰组肿瘤组织中HPV16型E6蛋白的表达被抑制,P53和P21蛋白的表达水平明显升高。结论慢病毒介导的HPV16型E6基因shRNA能有效抑制动物体内宫颈癌的生长,具有潜在的开发应用前景。     

多重荧光PCR方法在HPV基因分型中的应用

目的:探讨多重荧光PCR方法在HPV基因分型中的应用。方法:用已构建的18种中高危型HPV基因分型的多重荧光PCR方法检测510例临床样本,并与核酸测序法结果比较以考察该技术在HPV基因分型中应用的可行性。结果:临床研究表明,HPV 16、18、26、31、33、35、39、45、51、52、53、56、58、59、66、68、73、82共18种型别检测与核酸测序法结果几乎完全一致。结论:多重荧光PCR方法具有灵敏性高、特异性强、操作简单、通量高,耗样量少的特点,在中小规模临床样本检测中应用前景广阔。     

山东地区女性人乳头瘤病毒感染亚型、多重感染情况及年龄分布差异

目的了解山东地区女性人乳头瘤病毒(human papillomavirus,HPV)感染状况以及23种亚型分布特征、多重感染情况、在不同年龄组的分布差异。方法利用反向点杂交技术,对2013年1月至2014年11月山东省内13 973份门诊女性宫颈脱落细胞样本进行HPV分型检测,分析HPV感染亚型、多重感染情况及年龄分布差异。结果13 973份样本中,共检测出阳性样本4 105份,总检出率29.38%,23种亚型型别均有感染。单一型别感染2 513份,占阳性样本的61.25%,其中高危型感染1 815份,占阳性样本的44.21%,低危型感染698份,占阳性样本的17.00%;高危型感染最常见型别依次为16、52、58、56、53、66、18、68,低危型感染最常见型别依次为6、11、81、43、42。多重感染为1 592份,占阳性样本的38.75%,高低危混合感染占55.84%,纯高危型感染占39.20%;随着合并感染的型别数增加,比例逐渐下降。小于30岁人群有较高感染率,之后随年龄增长感染率呈下降趋势,而大于50岁年龄组,感染率又有所增加。结论山东地区女性HPV感染以单重感染为主,且高危型感染占优势;高危型感染以16型为主,低危型感染以6型为主。     

Proteomics Analysis of Andrographolide-Induced Apoptosis via the Regulation of Tumor Suppressor p53 Proteolysis in Cervical Cancer-Derived Human Papillomavirus 16-Positive Cell Lines

Regardless of the prophylactic vaccine accessibility, persistent infections of high-risk human papillomaviruses (hr-HPVs), recognized as an etiology of cervical cancers, continues to represent a major health problem for the world population. An overexpression of viral early protein 6 (E6) is linked to carcinogenesis. E6 induces anti-apoptosis by degrading tumor suppressor proteins p53 (p53) via E6-E6-associated protein (E6AP)-mediated polyubiquitination. Thus, the restoration of apoptosis by interfering with the E6 function has been proposed as a selective medicinal strategy. This study aimed to determine the activities of andrographolide (Androg) on the disturbance of E6-mediated p53 degradation in cervical cancer cell lines using a proteomic approach. These results demonstrated that Androg could restore the intracellular p53 level, leading to apoptosis-induced cell death in HPV16-positive cervical cancer cell lines, SiHa and CaSki. Mechanistically, the anti-tumor activity of Androg essentially relied on the reduction in host cell proteins, which are associated with ubiquitin-mediated proteolysis pathways, particularly HERC4 and SMURF2. They are gradually suppressed in Androg-treated HPV16-positive cervical cancer cells. Collectively, the restoration of p53 in HPV16-positive cervical cancer cells might be achieved by disruption of E3 ubiquitin ligase activity by Androg, which could be an alternative treatment for HPV-associated epithelial lesions.     

Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.     

The Dysregulation of MicroRNAs in the Development of Cervical Pre-Cancer—An Update

Globally in 2020, an estimated ~600,000 women were diagnosed with and 340,000 women died from cervical cancer. Compared to 2012, the number of cases increased by 7.5% and the number of deaths increased by 17%. MiRNAs are involved in multiple processes in the pathogenesis of cervical cancer. Dysregulation of miRNAs in the pre-stage of cervical cancer is the focus of this review. Here we summarize the dysregulated miRNAs in clinical samples from cervical pre-cancer patients and relate them to the early transformation process owing to human papillomavirus (HPV) infection in the cervical cells. When HPV infects the normal cervical cells, the DNA damage response is initiated with the involvement of HPV’s E1 and E2 proteins. Later, cell proliferation and cell death are affected by the E6 and E7 proteins. We find that the expressions of miRNAs in cervical pre-cancerous tissue revealed by different studies seldom agreed with each other. The discrepancy in sample types, samples’ HPV status, expression measurement, and methods for analysis contributed to the non-aligned results across studies. However, several miRNAs (miR-34a, miR-9, miR-21, miR-145, and miR-375) were found to be dysregulated across multiple studies. In addition, there are hints that the DNA damage response and cell growth response induced by HPV during the early transformation of the cervical cells are related to these miRNAs. Currently, no review articles analyse the relationship between the dysregulated miRNAs in cervical pre-cancerous tissue and their possible roles in the early processes involving HPV’s protein encoded by the early genes and DNA damage response during normal cell transformation. Our review provides insight on spotting miRNAs involved in the early pathogenic processes and pointing out their potential as biomarker targets of cervical pre-cancer.     

人乳头瘤病毒16型L1蛋白VLP的原核表达及纯化

目的在大肠杆菌中表达人乳头瘤病毒16型(HPV16)L1蛋白病毒样颗粒(VLP),并进行纯化,为研制宫颈癌疫苗提供新的思路。方法以HPV16基因组DNA为模板,PCR扩增HPV16L1基因的编码区,定向克隆至原核表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)编码区下游,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot鉴定后,采用GST纯化柱纯化重组融合蛋白,并用凝血酶切去GST标签,再经S100分子筛纯化,浓缩后,电镜观察纯化蛋白的VLP形态。结果重组表达质粒经双酶切和测序,证明构建正确。表达产物的相对分子质量约为80000,主要以可溶性形式存在。Western blot显示,目的蛋白可与小鼠抗HPV16L1抗体发生特异性反应。纯化蛋白的纯度约为95%,在电镜下可见直径约为50～60nm的VLP。结论已成功地在大肠杆菌中表达了可溶性的HPV16L1蛋白VLP,并进行了纯化,为宫颈癌疫苗及血清学诊断试剂的研制奠定了基础。     

KRAS Mutation Detection in Paired Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Colorectal Cancer Tissues

KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping.     

狂犬病病毒核蛋白的原核表达及其免疫原性

目的原核表达、纯化狂犬病病毒(Rabies virus,RV)核蛋白(Nucleoprotein,NP),并检测其免疫原性。方法 RT-PCR扩增RV CVS-11株NP全长基因序列,克隆至原核表达载体pET-30a(+),构建重组原核表达质粒pET-30a-N,转化E.coliBL21(DE3),IPTG诱导表达,表达的重组蛋白经Ni2+柱亲和层析纯化后,进行Western blot分析,并分别以A(lOH)3和CpG1826作为佐剂经腹腔免疫BALB/c小鼠,ELISA法检测小鼠的血清特异性抗体水平。结果重组原核表达质粒pET-30a-N经双酶切和测序证实构建正确;表达的重组蛋白相对分子质量约51 800,表达量约占菌体总蛋白的35%,主要以包涵体形式存在;纯化的重组蛋白纯度达90%以上,可与抗RV小鼠血清特异性结合,分别以A(lOH)3和CpG1826作为佐剂免疫小鼠的血清特异性抗体滴度对数的GMT值(3.81±0.22和3.68±0.20)明显高于阴性对照组(1.25±0.22),且差异均有统计学意义(P<0.01)。结论已成功表达并纯化了RV NP,其免疫原性良好,为进一步研究其功能奠定了基础。     

58型人乳头瘤病毒L2蛋白的原核表达、纯化及单克隆抗体的制备

目的原核表达并纯化我国高发的致宫颈癌58型人乳头瘤病毒(Human papillomavirus,HPV)次要衣壳蛋白L2,并制备其单克隆抗体。方法从含有HPV-58基因组的质粒pHPV58中扩增L2基因,插入改构的原核表达载体pGEX-KGV中,转化大肠杆菌BL21(DE3),乳糖诱导表达,透析复性并亲和层析纯化重组蛋白,采用杂交瘤技术制备单克隆抗体,并对其进行鉴定。结果重组表达质粒pGEX-KGV-HPV58 L2经双酶切和测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量可占菌体总蛋白的15.5%;纯化的重组蛋白纯度可达95%;共获得10株抗HPV-58 L2的高效价单克隆抗体。结论已成功原核表达、纯化了HPV-58 L2重组蛋白,并制备了单克隆抗体,为下一步HPV-58 L2蛋白抗原表位的研究以及相关预防性抗体药物和广谱重组疫苗的开发奠定了基础。