靶向表皮生长因子受体的寡肽与力达霉素强化融合蛋白的构建及其抗肿瘤活性

背景与目的：表皮生长因子受体（epidermal growth factor receptor,EGFR）在多种肿瘤细胞表面异常表达。力达霉素是从链霉菌中分离得到的具有强抗肿瘤作用的烯二炔类抗生素。本研究的目的是制备一种特异性靶向EGFR的寡肽配体与力达霉素的强化融合蛋白（Ec—LDP-AE）,并初步探讨其抗肿瘤活性。方法：制备融合蛋白（Ec-LDP）,并分离纯化,用高效液相色谱法检测Ec-LDP的纯度,ELISA、流式细胞技术以及细胞免疫荧光方法分析纯化的Ec-LDP蛋白对不同肿瘤细胞的免疫结合活性。将纯化的Ec-LDP蛋白与力达霉素发色团进行组装,构建Ec-LDP-AE,用高效液相色谱法检测350nm处的吸收峰。MTY法检测Ec—LDP-AE的体外抗肿瘤活性。结果：成功构建并表达了Ec-LDP蛋白,目的蛋白分泌至大肠杆菌周质腔中,产量为每升发酵液18mg活性蛋白,其纯度为95．3％。ELISA和细胞流式检测结果均显示Ec-LDP蛋白对EGFR高表达的肿瘤细胞系A431和MCF-7都有很强的免疫结合活性．而对不表达EGFR的NIH3T3细胞则无结合活性。免疫荧光实验也证实Ec—LDP蛋白可与A431细胞膜受体结合。Ec-LDP-AE蛋白在350nm波长处出现特定吸收峰,表明其构建成功。MTT法检测结果显示Ee-LDP—AE对体外培养的肿瘤细胞有强烈的杀伤作用,对MCF-7和A431细胞的IC。值分别为3．06×10^11mol/L和9．38×10^-13mol／L。结论：本研究制备的强化融合蛋白Ec—LDP—AE对表皮生长因子受体有靶向作用,并且保留了力达霉素的强烈细胞毒作用。     

TGF-α与绿脓杆菌外毒素融合蛋白的表达、纯化及对肿瘤细胞的杀伤作用

目的:表达并纯化转化生长因子α与绿脓杆菌外毒素融合蛋白(TGFα-PEA0),探讨其对EGF受体(EGFR)阳性肿瘤细胞的靶向杀伤作用.方法:用pET28a表达载体构建表达TGFα-PE40蛋白的重组体pV28,IPTG诱导其表达后,提取包涵体蛋白并用Ni柱纯化,MTT法观察复性融合蛋白对肿瘤细胞A431和SK-OV3的杀伤效用.结果:成功构建基因重组质粒pV28,纯化后的包涵体蛋白中TGFα-PFA0蛋白纯度达98%以上,这种活性毒素蛋白对EGFR高表达的A431癌细胞抑制率为50%(IC50)时所需蛋白浓度为(0.86±0.07)μg/ml,低于EGFR低表达的SK-OV3癌细胞的IC50(6.37±2.18μg/ml),差异显著(P＜0.05).结论:融合蛋白TGFα-PE40对肿瘤细胞的毒性与肿瘤细胞表面表达的EGFR数量成正相关,可选择性杀伤肿瘤细胞.     

TGF-α与绿脓杆菌外毒素融合蛋白的表达、纯化及对肿瘤细胞的杀伤作用

目的： 表达并纯化转化生长因子α与绿脓杆菌外毒素融合蛋白（TGFαPE40）,探讨其对EGF受体（EGFR）阳性肿瘤细胞的靶向杀伤作用。方法：用pET28a表达载体构建表达TGFαPE40蛋白的重组体pV28,IPTG诱导其表达后,提取包涵体蛋白并用Ni柱纯化,MTT法观察复性融合蛋白对肿瘤细胞A431和SKOV3的杀伤效用。结果：成功构建基因重组质粒pV28,纯化后的包涵体蛋白中TGFαPE40蛋白纯度达98％以上,这种活性毒素蛋白对EGFR高表达的A431癌细胞抑制率为50％（IC50）时所需蛋白浓度为(0.86±0.07)μg/ml,低于EGFR低表达的SKOV3癌细胞的IC50 (6.37±2.18μg/ml),差异显著（P<0.05）。结论： 融合蛋白 TGFαPE40 对肿瘤细胞的毒性与肿瘤细胞表面表达的EGFR数量成正相关,可选择性杀伤肿瘤细胞。     

靶向EGFR基因的shRNA抑制胰腺癌PANC-1细胞增殖的研究

目的探讨EGFR基因对胰腺癌PANC-1细胞的增殖抑制作用。方法构建针对EGFR序列特异性shRNA的表达载体,用脂质体转染胰腺癌PANC-1细胞。采用RT-PCR、Western blot检测EGFR mRNA和蛋白的表达;流式细胞仪检测细胞周期及凋亡;克隆形成实验检测细胞增殖。结果靶向EGFR的序列特异性shRNA明显抑制EGFR mRNA和蛋白的表达,EGFR mRNA和蛋白的抑制率分别为72.1％和67.6％;G1期细胞增多、S期细胞减少(P<0.05);细胞凋亡增加(P<0.05);克隆形成减少(P<0.05)。结论靶向EGFR的序列特异性shRNA能明显抑制胰腺癌细胞增殖、促进凋亡。     

EGF-Ang融合蛋白的构建、表达及活性研究

目的：构建由人表皮生长因子（epidermal growth factor,EGF）和人血管生成素（angiogenin, Ang）组成的人源化的融合蛋白，并检测其对肿瘤细胞的靶向杀伤能力。方法：利用基因工程技术将EGF和Ang基因连接起来，克隆到高效表达载体pET28a(+)中，构建重组表达质粒pEGFAng，并在大肠杆菌中表达该融合蛋白（EGFAng）。经DEAESepharose FF阴离子交换柱层析纯化后，用MTT法检测复性蛋白的细胞毒性。结果：SDSPAGE和薄层扫描分析表明外源蛋白的表达量占菌体裂解蛋白总量的18.6%。细胞活性检测表明EGFAng重组蛋白能明显地抑制Hep2细胞的生长，而不影响MA104细胞的正常生长。结论：融合蛋白EGFAng在体外对过度表达EGFR的Hep2细胞具有明显的杀伤作用。     

单抗导向超抗原对人大肠癌细胞的抑制作用研究

[目的]研究单抗导向超抗原对人大肠癌细胞的抑制作用。[方法]MTT法观察融合蛋白对Colo205细胞的体外抑制效应;DNA电泳法和电子显微镜法检测融合蛋白处理后肿瘤细胞的凋亡。[结果]在效应细胞介导下,肿瘤细胞的生长抑制率与随融合蛋白作用浓度的增加而逐渐增大;DNA电泳法和电子显微镜法显示被融合蛋白活化的免疫效应细胞攻击后的肿瘤细胞表现出典型的凋亡特征性变化。[结论]在效应细胞介导下,融合蛋白能对表达相应抗原的肿瘤细胞显示出有效的抑制作用;在融合蛋白介导的肿瘤细胞体外生长抑制过程中伴随有靶细胞的凋亡。     

EGF-PE35KDEL嵌合毒素对Hela细胞的毒性作用

目的: 构建由人表皮生长因子和绿脓杆菌外毒素A组成的融合蛋白,并检测其对肿瘤细胞的杀伤能力.方法: 利用基因工程技术连接EGF和PE35KDEL基因,克隆到表达载体pET28a中,将其转化至BL21(DE3),在BL21(DE3)以可溶的形式表达EGF-PE35KDEL嵌合毒素,经DEAE-Sepharose FF阴离子交换等层析后,纯度达到95%.结晶紫检测EGF-PE35KDEL对肿瘤细胞的活性.结果: SDS-PAGE和薄层扫描分析外源蛋白的表达量占菌体蛋白的20%.细胞活性检测证明EGF-PE35KDEL能够抑制Hela细胞的生长,IC50为0.07 μg/ml.结论: EGF-PE35KDEL嵌合毒素对体外表达EGFR的Hela细胞有杀伤作用.     

重组人野生型P53融合蛋白的克隆表达和对肿瘤细胞的作用

目的通过基因重组改善P53蛋白的功能，并研究它对肿瘤细胞的作用。方法构建人野生型P53(wtP53)融合蛋白的原核细胞表达载体PGEX-3X—PTD—P53，诱导表达和亲和层析纯化后，采用MTT法和流式细胞仪检测它对肿瘤细胞的增殖抑制作用和促细胞凋亡。结果成功构建P53融合蛋白的原核细胞表达载体PGEX-3X—PTD—P53，通过诱导表达和纯化获得重组人wtP53融合蛋白。MTT法和细胞流式分析证明该蛋白对几种癌细胞有明显的增殖抑制作用和促细胞凋亡。结论这种原核细胞表达的重组人wtP53融合蛋白保留了wtP53蛋白的生物学性状。     

miR-134-5p 对宫颈癌细胞增殖和凋亡的影响及其分子机制

目的:观察微小核糖核酸-134-5p(miR-134-5p)转染对宫颈癌细胞增殖和凋亡的影响,验证其可能的分子机制.方法:收集湖北医药学院附属人民医院肿瘤中心2016年5月至8月收治的8名宫颈癌患者肿瘤组织和相应癌旁组织.利用lipo-fectamine 2000将miR-134-5pmimics转染至宫颈癌Hela和SiHa细胞.采用MTT法和集落形成实验检测细胞增殖活性;流式细胞术(FCM)检测细胞周期和细胞凋亡;qRT-PCR检测宫颈癌组织和细胞miR-134-5pmRNA表达以及宫颈癌细胞EGFR mRNA表达;Western blotting检测宫颈癌细胞EGFR信号通路相关蛋白的表达.结果:宫颈癌组织miR-134-5pmRNA表达显著低于癌旁组织(P＜0.01).和转染miR-NC的Hela和SiHa细胞比较,转染miR-134-5pmimics的宫颈癌Hela和SiHa细胞miR-134-5pmRNA表达显著升高;细胞增殖能力显著降低(转染第5天,Hela细胞:1.06±0.13 vs 1.32±0.07;SiHa细胞:1.12±0.10 vs 1.42±0.12,均P＜0.05);形成的集落数减少;G0/G1期细胞比例显著上升,S期和G2/M期细胞比例显著下降;细胞凋亡率显著增加[Hela细胞:(26.53±13.48)％ vs(3.25±1.74)％;SiHa细胞:(30.49±12.04)％ vs(5.10±2.86)％,均P＜0.05];EGFR mRNA和EGFR蛋白表达显著下调,其中EGFR mRNA,Hela细胞下调58％(P＜0.01),SiHa细胞下调41％ (P＜0.05);EGFR下游靶蛋白p-AKT、p-ERK1/2和CyclinD1蛋白及pEGFR蛋白表达显著下调.结论:miR-134-5p可显著抑制宫颈癌细胞增殖并促进细胞凋亡,其可能的分子机制是通过抑制EGFR基因的表达,抑制EGFR通路的活化.     

缺氧诱导因子-1α的表达与胰腺癌细胞增殖和凋亡研究

目的　探讨缺氧诱导因子- 1α(HIF -1α)在胰腺癌组织中表达及其与bcl 2和PCNA的关系。方法　应用免疫组织化学PicTureTM 检测47例胰腺癌组织中HIF- 1α、PCNA和bcl 2的表达。结果　47例胰腺癌组织HIF 1α阳性表达率为5 5 .3 % (2 6/4 7) ,有周围脏器浸润者与无浸润者的HIF- 1α阳性表达率分别为66.7% (2 0 /3 0 ) ,3 5 .3 % (6/17) ,两者有显著性差异(χ2 =4.3 2 ,P<0 .0 5 ) ;在47例胰腺癌组织中高增殖活性2 8例(Ⅲ/Ⅳ)、低增殖活性19例(Ⅰ/Ⅱ)。bcl 2在47例胰腺癌组织中阳性率为2 9.8%(14 /4 7)。HIF- 1α表达与胰腺癌细胞增殖程度呈正相关性(γ=0 .5 86,χ2 =15 .15 ,P <0 .0 1) ,与Bcl 2蛋白表达无相关性(γ=-0 .2 11,χ2 =15 .15 ,P >0 .0 5 )。结论　HIF -1α表达与胰腺癌的增殖、浸润和转移密切相关,与肿瘤细胞凋亡无关。     

Urinary human polyomavirus and papillomavirus infection and bladder cancer risk

Background:

The association of transitional cell carcinomas of the bladder (TCB) with Schistosoma haematobium suggested a possible role of infections in the aetiology of TCB.

Methods:

In all, 114 TCB cases and 140 hospital controls from Pordenone Province were enrolled within an Italian multi-centric case–control study. Urine samples were screened for DNA from five human polyomaviruses (HPyV) (JCV, BKV, MCV, WUV, and KIV); SV40; and 22 mucosal human papillomaviruses (HPV) using highly sensitive PCR assays. Odds ratios (ORs) and corresponding confidence intervals (CIs) were computed for risk of TCB by HPyV- or HPV-positivity using unconditional logistic regression.

Results:

Human polyomavirus prevalence was similar in TCB cases (71.7%) and controls (77.7%) (OR for TCB=0.85; 95% CI: 0.45–1.61). JCV was the most frequently detected HPyV type. No individual HPyV showed a significant association. Among cases, HPyV-positivity was not associated with tumour characteristics, but it was significantly lower in women than men and among current and former smokers than never smokers. Human papillomavirus was detected in seven cases and five controls (OR=1.52; 95% CI: 0.42–5.45).

Conclusion:

The present small study does not support an involvement of HPyV or HPV infection in TCB aetiology in immunocompetent individuals. Differences in HPyV-positivity by sex and smoking may derive from differences in either acquisition or persistence of the infection.     

Against which human papillomavirus types shall we vaccinate and screen? the international perspective

At least 15 types of HPV have been associated with cervical cancer, but current HPV vaccines confer only type-specific immunity. To determine geographic variations in the HPV type distribution in cervical cancer, we carried out a pooled analysis of data from an international survey of HPV types in cervical cancer and from a multicenter case-control study, both co-coordinated by the IARC. Study cases were 3,607 women with incident, histologically confirmed cervical cancer recruited in 25 countries. HPV DNA detection and typing in cervical cells or biopsies were centrally done using PCR assays. Estimates of the potential number of cases prevented by HPV type-specific vaccines and changes in the validity of different HPV screening cocktails were calculated. HPV DNA was detected in 96% of specimens, and 30 different types were detected. The 15 most common types were, in descending order of frequency, 16, 18, 45, 31, 33, 52, 58, 35, 59, 56, 39, 51, 73, 68 and 66. Higher than average proportions of type 16 were found in northern Africa, of type 18 in south Asia, of type 45 in sub-Saharan Africa and of type 31 in Central/South America. A vaccine including types 16 and 18 could potentially prevent 71% of cervical cancers worldwide, but its impact with regard to the percentage of cases potentially prevented would be higher in Asia and Europe/North America. In contrast, a vaccine containing the 7 most common HPV types would prevent about 87% of cervical cancers worldwide, with little regional variation. The impact of modifying the number of types in the screening cocktail tests would be small and probably irrelevant for screening programs.     

Efficacy of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine in women aged 15-25 years with and without serological evidence of previous exposure to HPV-16/18

In the Phase III PATRICIA study (NCT00122681), the human papillomavirus (HPV)‐16/18 AS04‐adjuvanted vaccine (Cervarix^®, GlaxoSmithKline Biologicals) was highly efficacious against HPV‐16/18 infections and precancerous lesions in women HPV‐16/18 deoxyribose nucleic acid (DNA) negative and seronegative at baseline. We present further data on vaccine efficacy (VE) against HPV‐16/18 in the total vaccinated cohort including women who may have been exposed to HPV‐16/18 infection before vaccination. In women with no evidence of current or previous HPV‐16/18 infection (DNA negative and seronegative), VE was 90.3% (96.1% confidence interval: 87.3–92.6) against 6‐month persistent infection (PI), 91.9% (84.6–96.2) against cervical intraepithelial neoplasia (CIN)1+ and 94.6% (86.3–98.4) against CIN2+ [97.7% (91.1–99.8) when using the HPV type assignment algorithm (TAA)]. In women HPV‐16/18 DNA negative but with serological evidence of previous HPV‐16/18 infection (seropositive), VE was 72.3% (53.0–84.5) against 6‐month PI, 67.2% (10.9–89.9) against CIN1+, and 68.8% (?28.3–95.0) against CIN2+ [88.5% (10.8–99.8) when using TAA]. In women with no evidence of current HPV‐16/18 infection (DNA negative), regardless of their baseline HPV‐16/18 serological status, VE was 88.7% (85.7–91.1) against 6‐month PI, 89.1% (81.6–94.0) against CIN1+ and 92.4% (84.0–97.0) against CIN2+ [97.0% (90.6–99.5) when using TAA]. In women who were DNA positive for one vaccine type, the vaccine was efficacious against the other vaccine type. The vaccine did not impact the outcome of HPV‐16/18 infections present at the time of vaccination. Vaccination was generally well tolerated regardless of the woman's HPV‐16/18 DNA or serological status at entry.     

Cis-Diamminedichloroplatinum(II) Toxicity in Human Melanoma Cells and Lymphocytes as Related to Cellular Platinum Accumulation, DNA Cross-Linking and Inhibition of DNA Synthesis

The cytotoxic effect of cis-diamminedichloroplatinum(II) (cis-DDP), as measured by a dye exclusion assay was much more pronounced in bone marrow cells and phytohemagglutinin (PHA)-stimulated lymphocytes than in a human melanoma cell line. DNA synthesis measured by incorporation of ³H-thymidine was much more sensitive to cis-DDP in PHA-stimulated lymphocytes than in melanoma cells. These differences were not caused by a difference in drug accumulation since measurements of cellular platinum content gave similar results in both cell types. The total amount of DNA cross-links and DNA interstrand crosslinks induced by cis-DDP was measured with alkaline elution of DNA. In both PHA-stimulated lymphocytes and melanoma cells low total levels of DNA cross-links and DNA interstrand crosslinks were found immediately after drug exposure, followed by a protracted increase in DNA cross-linking for 6-12 hours during further incubation after removal of cis-DDP. The relationship between the concentration of cis-DDP and peak levels of total DNA cross-links as well as DNA interstrand cross-links was linear in both cell types. Cis-DDP was found to induce 5.6 times higher total levels of DNA cross-links and 6.1 times higher levels of DNA interstrand cross-links in PHA-stimulated lymphocytes than in melanoma cells. The incorporation of ³H-thymidine was much more reduced in PHA-stimulated lymphocytes than in melanoma cells at similar levels of DNA cross-linking. Thus, both reduced DNA cross-linking and lower effect of DNA cross-links on the DNA synthesis may contribute to the greater resistance of melanoma cells to cis-DDP.     

人免疫缺陷病毒-1 gag基因在人成熟精子中的表达

目的：探讨人免疫缺陷病毒-1（HIV-1）gag基因在人成熟精子中实现mRNA水平表达的可能性。方法：构建含HIV-1gag基因DNA的质粒,转染入人成熟精子,培养24 h后用RT-PCR法检测精子gag mRNA的表达。结果：在转染质粒后的人成熟精子中检测到HIV-1 gag mRNA的表达。结论：HIV基因可在人成熟精子中实现mRNA水平的表达。     

Human papillomavirus type 16 E6 variants and HLA class II alleles among Japanese women with cervical cancer

The enhanced oncogenicity of particular human papillomavirus type 16 (HPV16) E6 variants is population-dependent, implying the involvement of additional genetic cofactors. This study was designed to investigate the association between E6 variants and human leukocyte antigen (HLA) polymorphism within a Japanese population. Fifty-seven women with HPV16-positive cervical cancer were analyzed for E6 sequence variation and its relationship to HLA class II alleles. Compared with local controls (n = 138) and published controls (n = 916), DRB1*1501 and DQB1*0602 frequencies were significantly increased among patients with HPV16 E6 prototype (n = 11). Additionally, DRB1*1502 was positively associated with a particular E6 variant designated D25E (n = 25), although we could not find a significant association between HLA class II alleles and L83V variants (n = 16). Our observations suggest that a specific match between E6 variant proteins and HLA types may contribute to HPV16-related cervical carcinogenesis.     

Human non-malignant and malignant brain tumor derived cell cultures: proliferation and sensitivity to natural human fibroblast (beta) interferon

Twenty-one human brain tumor biopsies were processed by mechanical and enzymatic methods to produce mixed cell suspensions. Cultures were prepared in small plastic flasks, and primary outgrowth occurred in 16/21 cultures. The period required for primary outgrowth ranged from 3 days to 14 days. We established serial propagation with 15/16 of the primary cultures. Sensitivity to HuIFN- was determined between passages 3 to 12, using a microassay based on cell viability (uptake of a supravital stain, netural red). Extracted dye was quantified in acidic-methanol using the MR580 Microelisa Autoreader (Dynatech). We observed a broad range of responsiveness to the drug among the 12 cell-strains tested. Thus, 4 cell strains were relatively sensitive; 4 were resistant to 10⁴ IRU/ml of purified HuIFN-. Four cell strains exhibited a level of responsiveness that was intermediate to that of these two groups.During propagation of these biopsies, cytopathology suggestive of paramyoxvirus-infection appeared in 4 of the cell-strains. This characteristic was not uniformly associated with high sensitivity to human beta interferon which is a very potent, naturally occurring antiviral substance.Our results support the concept that information concerning sensitivity to HuIFN- and other cytostatic agents may be rapidly obtained using microcultures of brain tumor cultures in conjunction with supravital stain uptake studies. Additionally, these results suggest that further clinical studies with interferon should be undertaken to define the parameters which determine successful in vivo application.     

Is human cytomegalovirus a target in cancer therapy?

Human cytomegalovirus (HCMV) is a herpesvirus that is prevalent in the human population. HCMV has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumour cell immune evasion. Moreover, antiviral treatment against HCMV has been shown to inhibit tumour growth in preclinical models. Here we describe the possible involvement of HCMV in cancer and discuss the potential molecular impact expression of HCMV proteins have on tumour cells and the surrounding tumour microenvironment.     

人乳头瘤病毒检测阴性子宫颈癌的研究进展

子宫颈癌是妇科最常见的恶性肿瘤，研究证实99.7%的宫颈癌是因感染人乳头瘤病毒（human papillomavirus，HPV）造成的，但几乎所有的研究中都发现有HPV检测阴性的子宫颈癌存在。HPV检测阴性的子宫颈癌可以概括为假阴性和真阴性两种情况，造成假阴性的原因有病变小、取材有限、病毒载量低、非高危人乳头瘤病毒基因型、技术错误或检测灵敏度不足等。现有的筛查方式具有一定局限性，一些具有高灵敏度和特异性的新型分子标记物如微小核糖核酸、FAM19A4基因甲基化等已被证明有望作为子宫颈癌早期检测和诊断的指标。近年来关于HPV阴性子宫颈癌的研究越来越多，但对HPV阴性子宫颈癌患者临床特点的分析存在差异。本文主要对HPV阴性子宫颈癌假阴性的原因、筛查诊断及临床特点方面进行综述。