卷曲乳酸杆菌对Hela细胞的粘附研究

目的研究不同时期的生殖道卷曲乳酸杆菌A7对Hela细胞的粘附性。方法将对数期及稳定期的卷曲乳酸杆菌A7分别与Hela细胞进行粘附试验,经革兰染色后,通过显微计数法计算粘附数。结果处于对数期的卷曲乳酸杆菌A7在粘附时间为3 h时,其效果最佳。结论阴道卷曲乳酸杆菌A7对Hela细胞具有较强的粘附性。     

乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的抑制作用

目的观察乳酸杆菌DM9811发酵滤液及其主要成分对宫颈癌细胞株Hela细胞的体外增殖的影响,探索乳酸杆菌发酵滤液对宫颈癌细胞是否有抑制作用及解析作用的有效成分。方法用MTr法研究不同浓度乳酸杆菌DM9811发酵滤液在不同时间对Hela细胞的抑制作用,在此基础上研究脂肪酸、菌体核酸在不同时间对Hela细胞的抑制作用。结果不同浓度乳酸杆菌DM9811发酵滤液及相关物质在不同时间对Hela细胞的抑制作用显示：（1）乳酸杆菌DM9811发酵滤液各浓度组对Hela细胞的生长均有抑制作用,且这种抑制作用呈剂量-时间依赖方式。24、48、72h达到半数抑制率的发酵滤液浓度分别为8．9％、5．3％、3．8％。（2）乳酸杆菌DM9811发酵滤液脂肪酸对Hela细胞的生长有一定抑制作用,抑制率在7．0％～34．0％。（3）乳酸杆菌DM9811菌体核酸对Hela细胞的生长有抑制作用,抑制率为9．7％-53．4％,呈剂量一时间依赖方式。72h达到半数抑制率核酸的浓度为5．5μg/ml。结论乳酸杆菌DM9811发酵滤液对Hela细胞的生长具有显著的抑制作用,其中脂肪酸组分是有效成分之一。     

乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的细胞周期和细胞凋亡的影响

目的观察乳酸杆菌DM9811发酵滤液对宫颈癌细胞株Hela细胞的细胞周期和细胞凋亡的影响,探索乳酸杆菌发酵滤液对宫颈癌细胞作用的可能机制。方法用光镜、电镜和流式细胞仪分析不同浓度乳酸杆菌DM9811发酵滤液对Hela细胞凋亡的诱导效果;用流式细胞仪分析不同浓度乳酸杆菌DM9811发酵滤液对Hela细胞细胞周期的影响。结果（1）乳酸杆菌DM9811发酵滤液可诱导宫颈癌Hela细胞凋亡。形态学观察处理后的Hela细胞,可见细胞变形,细胞皱缩,体积变小,细胞间隙增大,细胞核固缩。流式细胞仪分析,1%、2%的乳酸杆菌DM9811发酵滤液在48、72h可诱导Hela细胞凋亡;5%的乳酸杆菌发酵滤液在24、48和72h均可诱导Hela细胞凋亡。（2）乳酸杆菌DM9811发酵滤液阻滞宫颈癌Hela细胞于S期,不同浓度的乳酸杆菌发酵滤液作用24、48和72h均可使S期细胞比阴性对照组增多。结论乳酸杆菌DM9811发酵滤液可诱导部分Hela细胞凋亡,其对Hela细胞的生长抑制作用可能通过S期阻滞实现。     

植物乳杆菌黏附大鼠小肠黏液及机制的研究

分析了6种植物乳杆菌黏附大鼠小肠粘液的能力,并分析了介导黏附性的主要因素。结果表明,植物乳酸杆菌向大鼠小肠粘液的黏附具有菌种特异性,其黏附作用是甘露糖特异性的,细胞外表蛋白质、碳水化合物和(脂)磷壁酸可能参与了黏附过程。     

阴道卷曲乳酸杆菌对阴道致病菌的体外生物拮抗作用

目的研究阴道卷曲乳酸杆菌对阴道致病菌的体外生物拮抗作用。方法将阴道卷曲乳酸杆菌分别与3株阴道致病菌（金黄色葡萄球菌、大肠埃希菌、粪肠球菌）混合接种于GAM液体培养基中进行厌氧培养,通过平板菌落计数法计算阴道致病菌的菌数。结果经混合培养后阴道致病菌的菌数明显下降。结论阴道卷曲乳酸杆菌A7在体外能明显抑制3株肠道致病菌的生长繁殖。     

卷曲乳杆菌对三种结肠上皮细胞的粘附性

目的研究卷曲乳杆菌对3种结肠上皮细胞(SW480细胞、SW620细胞、LOVO细胞)的粘附性。方法将处于对数生长期的卷曲乳杆菌A7分别与SW480细胞、SW620细胞、LOVO细胞进行体外粘附试验,革兰染色后显微镜观察卷曲乳杆菌A7对3种结肠上皮细胞的粘附结果并计数。结果卷曲乳杆菌A7对3种结肠上皮细胞的粘附均具有显著性,其中对于SW480细胞和LOVO细胞的粘附性明显高于SW620细胞。结论卷曲乳杆菌A7对SW480细胞、SW620细胞、LOVO细胞均具有较强的粘附性,提示该菌株有望成为肠道益生菌的新成员。     

植物乳酸杆菌CGMCC NO．1258表层黏附蛋白同致病性大肠埃希菌竞争黏附肠上皮细胞的研究

目的了解植物乳酸杆菌CGMCC NO．1258表层黏附蛋白（IMP2）在同肠上皮细胞（Caco-2）黏附作用中的机制。方法在制备和鉴定IMP2的多克隆抗体的基础上,同EPEC进行竞争黏附肠上皮细胞中,采用体外抗体阻断的方法检测IMP2对植物乳酸菌CGMCC NO．1258黏附作用的影响。结果多克隆抗体能够有效抑制植物乳酸菌CGMCC NO．1258对肠上皮细胞的黏附,并且导致EPEC对Caco-2细胞黏附的增加。结论IMP2在植物乳酸杆菌CGMCC NO．1258同肠上皮细胞黏附中发挥着重要的作用。     

乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的拮抗作用

【目的】对嗜酸乳杆菌的S-层蛋白(S-layer protein)进行提纯,研究嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附和入侵的拮抗作用。【方法】应用阴离子交换柱(DE52)对嗜酸乳酸杆菌的S-层蛋白进行提纯,然后分别研究了嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的作用。【结果】S-层蛋白能显著地抑制鼠伤寒沙门氏菌的黏附及入侵;在竞争、排斥、置换3种黏附试验中,S-层蛋白可显著降低鼠伤寒沙门氏菌的黏附,其相对黏附力分别为1.17%±5.97%、8.71%±1.36%、10.56%±0.92%,差异极显著(p0.01),其中竞争试验效果最好;并且S-层蛋白对鼠伤寒沙门氏菌黏附抑制作用极显著高于嗜酸乳酸杆菌(p0.01);此外,S-层蛋白也能显著抑制鼠伤寒沙门氏菌入侵。【结论】乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌可产生显著的拮抗作用,这可能与S-层蛋白和鼠伤寒沙门氏菌的宿主黏附受体存在竞争作用有关;提示乳酸杆菌S-层蛋白可用于预防和治疗鼠伤寒沙门氏菌感染,并有望成为抗生素的替代品。     

重组七鳃鳗细胞毒素蛋白rLj-RGD3表达及对Hela细胞活性的影响

为研究重组七鳃鳗细胞毒素蛋白rLj-RGD3的抗肿瘤活性,确定其生物学地位及意义,本研究提取成体七鳃鳗(Lampetra japonica)口腔腺组织总RNA,经RT-PCR获得cDNA序列长度为357bp的目的基因片段,将其克隆于pET23b载体,获得带有组氨酸标签的分子量为15kD的基因重组蛋白rLj-RGD3的高效可溶性表达,通过组氨酸亲和层析纯化蛋白。采用MTT法检测不同浓度rLj-RGD3对bFGF诱导下的Hela细胞增殖的抑制作用,结果表明rLj-RGD3能显著抑制Hela细胞的增殖,IC50为2.6μmol/L。rLj-RGD3作用后的Hela细胞经Hoechst染色及DNAladder检测结果显示,细胞均发生凋亡。rLj-RGD3对Hela细胞黏附玻连蛋白(VN)作用的实验结果为有效抑制。采用Transwell细胞培养板对bFGF诱导下的Hela细胞迁移实验表明,rLj-RGD3能够抑制Hela细胞的迁移,且抑制率达60%。采用人工基质膜基质Matrigel及Transwell模仿体内环境研究rLj-RGD3对Hela细胞浸润行为实验显示,以bFGF为趋化剂的Hela细胞穿透Matrigel的...     

Egr-1基因与肿瘤腺癌放射敏感性关系的研究

目的：研究Egr-1基因在2种腺癌细胞A549和Hela放射前后的表达变化及对放射敏感性的影响。方法：培养肺腺癌A549细胞和宫颈腺癌Hela细胞,分别提取4Gyx射线照射前及照射后不同时间点的细胞的总RNA行荧光定量PCR（FQ-PCR）检测Egr-1表达水平;收获4GyX射线照射前及照射后不同时间点的细胞处理行流式细胞术检测其凋亡;对照射不同剂量的细胞继续培养10-14天,进行克隆形成计数,计算克隆形成率及存活分数。结果：FQ-PCR结果显示,放射后Egr．1基因表达水平在2种细胞中均明显升高且于放射后1h达到峰值,A549细胞的峰值明显高于Hela细胞;流式细胞术检测结果显示,A549细胞凋亡明显高于Hela细胞;克隆形成实验结果显示,A549细胞存活分数明显低于Hela细胞。结论：Egr-1基因在不同腺癌细胞表达水平不同并影响其放射敏感性。     

顺铂诱导入宫颈癌细胞系P27表达及其超微弱发光测定的意义

目的：探讨化疗药物对肿瘤增殖活性的影响。方法：选择人宫颈癌细胞系Hela分为两组,分别采用MTT比色法分析测定顺铂处理Hela细胞的浓度;免疫组化SP法分别检测Hela细胞中P27蛋白表达;流式细胞仪分析加药前后细胞周期变化及凋亡情况;用IFFM-D型流动式化学发光仪检测细胞的超弱发光强度。结果：顺铂处理Hela细胞48h的IC50值为3mg／L,当DDP浓度在3mg／L以下时,对Hela细胞无明显毒性作用,超过此浓度时,其毒性呈剂量效应关系（P〈0．001）;流式细胞仪分析细胞周期可见与Hela细胞相比较,Hela＋DDP细胞的G2期细胞数增多,而Gl、S期的细胞数明显减少（P〈0．01）;从细胞凋亡检测显示Hela与Hela＋DDP相比,细胞凋亡率在不同时间点明显升高,在24h、48h、72h结果分别为（11．4±5．8、21．8±7．9、32．5±11．6）％。免疫组化结果显示Hela＋DDP与Hela细胞相比细胞膜上P27蛋白高表达;在10—4mol／L鲁米诺及0.3％的双氧水（H202）条件下Hela细胞超弱发光强度高于用Hela＋DDP细胞（P〈0．001）。结论：超弱发光能够快速、准确、有效地反映肿瘤细胞氧化代谢特点和增殖活动,也可用于筛选敏感的化疗药物。     

NDV CN株对几种人肿瘤细胞的杀伤作用及其机理的分析

本文研究了NDV-CN株对5株不同的人肿瘤细胞的体外杀伤作用。结果表明5株肿瘤细胞对NDV-CN敏感,于感染早期出现细胞收缩变圆,失去贴壁性,感染第5天时细胞存活率低于10％。其中尤以HEP3B细胞最敏感。但NDV-CN株对人二倍体细胞2BS有弱杀伤性。病毒感染早期可检测到感染细胞中有病毒核酸的复制、感染细胞表面有病毒蛋白的表达,脑浆内有病毒粒子存在。NDV-CN株主要诱导HEP3B及T24细胞产生凋亡,主要诱导Hep2、Hela及A549细胞产生坏死。     

胡桃楸提取液对肿瘤细胞细胞周期的影响

目的考察胡桃楸提取液对Hela和K562细胞周期的影响。方法用流式细胞仪分析胡桃楸提取液对Hela、K562细胞细胞周期的影响。结果胡桃楸提取物体外作用于Hela细胞可引起细胞周期在S期的停滞,这种效果随药物浓度和作用时间的增加而增加,胡桃楸提取物体外作用于K562细胞,可引起细胞周期在G1期的停滞,随药物浓度的增加而增加。结论胡桃楸提取液对Hela细胞的生长抑制作用可能通过S期阻滞实现,对K562细胞抑制作用可能通过G1期阻滞实现。     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.     

Expression and activity of mTOR and its substrates in different cell cycle phases and in oral squamous cell carcinomas of different malignant grade

In order to study the relationship between mTOR (mammalian target of rapamycin) and tumorigenesis, we investigated the expression and activity of mTOR, and its substrates, alpha1, alpha2, beta1 and beta2 isoforms of p70S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein-1 (4EBP-1) in oral squamous carcinoma and Hela cells using RT-PCR, immunohistochemistry, statistical analysis and Western blotting. The result of Western blots showed that in poorly differentiated oral squamous carcinoma, the expression level of mTOR and p70S6k increased in M phase, while that of 4EBP-1 decreased. The results of RT-PCR and immunohistochemistry assay are the same as that of Western blot. In Hela cells, the RT-PCR results showed that the level of mTOR mRNA did not change during the cell cycle. In M phase, the expression of alpha1, alpha2, beta1 and beta2 isoforms of p70S6K increased noticeably, while the expression of 4EBP-1 decreased. The immunoblot results in Hela cells were consistent with the RT-PCR results. Furthermore, the activity assays in Hela cells suggested that,in phase G2 and M, the activity of mTOR was maintained at a higher level than in any other phase, while 4EBP-1 decreased in phase M. These results may help in further investigations of the important role of mTOR in cell cycle and tumorigenesis.     

MMP-7基因真核表达载体的构建及其在HeLa细胞中的表达与鉴定

目的：构建MMP-7基因真核重组质粒,检测并鉴定MMP-7在人宫颈癌HeLa细胞中的表达。方法：提取宫颈癌组织总RNA,通过基因克隆构建MMP-7基因真核表达重组质粒pcDNA3.1（＋）/MMP-7,酶切、PCR及基因测序鉴定,用阳离子脂质体介导采用基因转染技术转染人宫颈癌Hela细胞,RT-PCR检测外源基因的表达、间接免疫荧光法检测对表达产物进行鉴定。结果：成功构建了重组表达质粒pcDNA3.1（＋）/MMP-7并转染了人宫颈癌Hela细胞,通过RT-PCR可以检测到MMP-7 mRNA在Hela细胞中的表达,经间接免疫荧光反应可检测到明显的阳性反应,而转染空载体组表达阴性。结论：构建的重组质粒pcDNA3.1（＋）/MMP-7能在Hela细胞中表达,为该蛋白在人子宫癌后续的功能研究奠定了基础。     

Interferon-β Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.     

Up-regulation of MELK by E2F1 promotes the proliferation in cervical cancer cells

Cervical cancer is a common gynecologic cancer and a frequent cause of death. In this study, we investigated the role of MELK (maternal embryonic leucine zipper kinase) in cervical cancer. We found that HPV 18 E6/E7 promoted MELK expression by activating E2F1. MELK knockdown blocked cancer cells growth. Furthermore, we used MELK-8A to inhibit the kinase activity of MELK and caused the G2/M phase arrest of cancer cells. Under the treatment of inhibitors, Hela cells formed multipolar spindles and eventually underwent apoptosis. We also found that MELK is involved in protein translation and folding during cell division through the MELK interactome and the temporal proteomic analysis under inhibition with MELK-8A. Altogether, these results suggest that MELK may play a vital role in cancer cell proliferation and indicate a potential therapeutic target for cervical cancer.