HLA-G在卵巢癌组织中的表达及对NK细胞杀伤活性的影响

通过免疫组织化学法(IHC)检测卵巢癌组织中HLA-G的表达;克隆编码全长HLA-G1cDNA,将该基因通过真核表达载体pVITRO2-mcs转染至HLA-G阴性的HO-8910、OVCAR-3细胞株,采用RT-PCR、流式细胞术(FACS)及Westernblot鉴定、分析转染细胞中HLA-G的mRNA及蛋白质表达;LDH释放法检测卵巢癌细胞表达HLA-G后对NK细胞杀伤功能的影响。结果显示,66.7%(22/33)卵巢癌组织表达HLA-G分子,而正常组织未见其表达;基因转染后,HLA-G在HO-8910及OVCAR-3细胞表面获得稳定表达。细胞毒实验结果显示,HLA-G能显著抑制NK-92对卵巢癌细胞的杀伤活性(P<0.01);HLA-G特异性抗体87G阻断后,能明显恢复NK-92细胞对HO-8910-G、OVCAR-3-G的杀伤功能。本研究结果提示,卵巢癌细胞通过表达HLA-G分子抑制NK细胞的杀伤活性,在卵巢癌细胞逃避宿主的免疫监视中起重要作用。     

膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响

目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.     

HCMV感染对人THP-1细胞HLA-G及其受体表达的影响

目的:研究人巨细胞病毒(HCMV)感染对人THP-1细胞人类白细胞抗原G(HLA-G)异构体及其受体表达的影响探讨HLA-G在HCMV逃逸宿主免疫应答中的作用。方法:HCMV Towne株感染THP-1细胞后,采用RT-PCR和Western blot检测HLA-G异构体mRNA和蛋白水平,流式细胞术检测THP-1细胞HLA-G及其表面受体ILT2、ILT4的表达,ELISA检测细胞培养上清中IL-10及可溶性HLA-G(sHLA-G)水平,同时检测细胞存活率。结果:HCMV感染后细胞未出现明显凋亡,细胞存活率高。HCMV感染THP-1细胞1 d后HLA-G1、-G3、-G4和-G5的mRNA表达明显上调,HLA-G1和HLA-G5的蛋白表达明显上调。THP-1细胞HLA-G、ILT2和ILT4的表达在感染1 d后明显上调。sHLA-G水平在感染1 d后显著升高,与对照组比较差异有统计学意义(P0.01)。THP-1细胞培养上清液IL-10水平在感染1 d后明显上调,与对照组比较,差异有统计学意义(P0.05)。结论:HCMV感染THP-1细胞能诱导HLA-G异构体的差异表达,以HLA-G1和HLA-G5为主,且上调其表面受体ILT2/ILT4的表达。同时,HCMV感染能诱导THP-1细胞分泌IL-10。该研究为进一步探讨HCMV逃避机体免疫应答的机制提供实验依据。     

RNA干扰技术抑制JEG-3细胞中HLA-G的表达

目的:以人类白细胞抗原G基因(HLA-G)为靶基因,采用RNA干扰(RNAi)技术特异性地抑制JEG-3细胞中HLA-G的表达。方法:针对HLA-G基因设计一段小干扰RNA(siRNA),由Ambion公司化学合成,用脂质体lipofectamine2000将该siRNA转染JEG-3细胞,采用RT-PCR、流式细胞术和Westernblot分别从mRNA和蛋白质水平检测该siRNA对HLA-G表达的影响。结果:针对HLA-G的siRNA能够明显下调JEG-3细胞中HLA-G的表达水平,并具有良好的特异性。结论:在JEG-3细胞系中采用RNAi技术特异性下调了HLA-G基因的表达,为进一步研究HLA-G在人类的母胎免疫耐受、正常妊娠的维持及助孕技术应用等方面的作用奠定了实验基础。     

卵巢癌细胞IL-6、IL-8及其受体表达的研究

目的分析比较五种常见的上皮性卵巢癌细胞系IL-6、IL-8及其受体表达的差异。方法IL-6、IL-8的表达分别采用RT-PCR和ELISA法进行检测,IL-6受体（IL-6Rα和gp130）及IL-8受体（IL-8RA和IL-8RB）的表达采用免疫印迹技术进行测定。结果①五种上皮性卵巢癌细胞均组成性表达IL-6和IL-8。IL-6和IL-8在CAOV-3细胞中的表达水平均最高,而在HO-8910PM细胞中的表达水平均最低,IL-6在SKOV-3、HO-8910、OVCAR-3细胞中的表达水平依次降低,IL-8在OVCAR-3、SKOV-3、HO-8910细胞中的表达水平依次降低。②五种上皮性卵巢癌细胞均表达IL-6Rα、gp130及IL-8RA;除CAOV-3细胞外,其它细胞均表达IL-8RB。结论本研究旨在筛选表达IL-6和IL-8及其相应受体的细胞株,为研究IL-6、IL-8与卵巢癌发生、发展关系奠定基础,同时也为今后卵巢癌的免疫治疗提供一个新的思路。     

木犀草素对卵巢癌细胞株转移能力的影响

目的：研究木犀草素对卵巢癌细胞株HO-891 0PM的体外侵袭能力的影响，并探讨其可能的作用机制。方法：HO-8910P M细胞经木犀草素处理后用Boyden小室法检测其体外、侵袭和运动能力。并采用明胶酶 谱法检测HO-8910PM细胞分泌的明胶酶活性。RT-PCR检测TIMP-1、NM23基因mRNA的表达情况，蛋白印迹法检测ERK2的蛋白表达变化。结果：HO-8910PM细胞经木犀草 素处理后，体外侵袭、运动能力呈剂量依赖性下降。HO-8910PM细胞MMP-9的分泌下降。ERK2 蛋白表达明显降低，但TIMP-1、NM23基因mRNA的水平无明显变化。结论:木犀草素在体外剂量依赖性地抑制卵巢癌细胞HO-8910PM的转移能力，这可能与木犀草素 抑制MMP-9的分泌及下调ERK2表达有关。     

人卵巢浆液性囊腺癌高低转移细胞株E-cadherin差异表达

目的探讨上皮性细胞粘附分子(E-cadherin,E-cad)在人卵巢浆液性囊腺癌高低转移细胞株HO-8910PM及HO-8910中的表达及生物学行为的差异。方法采用免疫荧光法及W estern b lotting检测细胞中E-cad蛋白表达的差异,用粘附实验检测细胞与细胞外基质的粘附能力,用Transwell小室法检测细胞侵袭能力及迁移能力。结果E-cad在HO-8910细胞中为高表达,在HO-8910PM细胞中为低表达,HO-8910PM细胞与细胞外基质的粘附能力、侵袭能力及迁移能力均明显高于HO-8910细胞(P<0.05,P<0.01)。结论人卵巢浆液性曩腺癌HO-8910PM细胞的浸润、转移等恶性行为可能与E-cad的表达下降有关。     

针对HLA-G的siRNA的载体构建与表达及效应检测

目的构建针对HLA-G的siRNA的表达载体,研究其对NK细胞杀伤效应的影响。方法构建针对HLA-G的siRNA真核表达质粒pSuppressor-U6-neo-HLA-G,转染JEG-3滋养细胞系。采用RT-PCR、Western blot检测转染的JEG-3细胞中HLA-G的表达水平。将NK-92MI细胞(效应细胞)与滋养细胞(靶细胞)共同培养,以LDH释放法观察不同效靶比例时NK-92MI细胞对靶细胞的杀伤效应。结果NK-92MI细胞对转染针对HLA-G的siRNA的JEG-3细胞的杀伤作用较未转染细胞明显升高(P<0.001)。结论针对HLA-G的SiRNA增加NK细胞对滋养细胞的杀伤,HLA-G具有保护滋养细胞免受NK细胞杀伤的作用。     

人类白细胞抗原-G突变体cDNA克隆及在K562细胞上的表达

目的：克隆人类白细胞抗原-G(Human leukocyte antligen-G，HLA-G)突变体cDNA，并使其在HLA-I类阴性的K562细胞上获得稳定表达，为研究配-受体之间的识别机制奠定基础。方法：用RT-PCR方法从人子宫蜕膜组织扩增出HLA-GcDNA，得到全长HLA-GPCR产物后，用桥式PCR方法进行定点点突变，将突变的目的基因亚克隆于逆转录，将突变的目的基因亚克隆于逆转录mG-pLNCX表达载体，采用感染的方法将重组质粒转入K562细胞，最后经G418筛选及有限稀释，利用单克隆抗体W6/32进行FACS及mRNA检测，观察HLA-G突变体在靶细胞表面的表达。结果：HLA-G突变体分子在经mG-pLNCX转染的靶细胞表面获得稳定高表达。结论：成功构建了mG-pLNCX表达载体，并使HLA-G突变体分子在HLA-I类阴性的靶细胞K562细胞上获得稳定表达。     

HLA-G在肿瘤中的表达

自1987 年Geraghty等[1]同事第一次报道发现HLA-G 以来, 至今已有十余年, 人们对这个非经典HLA -I 类分子的了解逐渐深入.HLA-G(human leucocyte antigen-G) 即人类白细胞抗原系统-G 是一种非经典的HLA- I类抗原,它定位于6 号染色体短臂HLA 远端300bp以内, 并选择性的在胎盘表达,参与保护胚胎免受母体淋巴系统的攻击.1900年Kovats[2]发现它在胎盘绒毛远端的细胞滋养层特异表达.1995 年WHO 组织的HLA 命名大会上Bodmer等[3]报道,HLA-G 有17个等位基因.随着检测水平的发展, 在人类胚胎的很多器官都检测到HLA-G的表达.最早发现是在母胎界面的绒毛膜滋养层细胞上分布,参与保护胚胎免受母体淋巴系统的攻击.     

Multiple steps of HLA-G in ovarian carcinoma metastasis: Alter NK cytotoxicity and induce matrix metalloproteinase-15 (MMP-15) expression

Previous study showed that aberrant HLA-G expression in cancer cells plays important roles in disease progression and it was associated with tumor metastasis and with poor survival in an animal model with ovarian cancer; however, the mechanisms remain to be explored. In this study, we found that HLA-G expression could dramatically decreased the NK cytotoxicity against the ovarian carcinoma cell line (HO-8910) engineered to express HLA-G (HO-8910-G), and matrix metalloproteinase-15 (MMP-15) expression was up-regulated in HO-8910-G cells. Consistent with this, a strong correlation between HLA-G and MMP-15 expression were observed in a cohort of ovarian cancer samples. Knockdown the HLA-G induced MMP-15 expression by small interfere RNA (siRNA) significantly decreased the HO-8910-G cell migration potential and tumor metastasis. Collectively, our study indicated that HLA-G involved in tumor invasiveness or metastasis may rely on the NK cytotoxicity inhibition and induction of MMP-15 expression in ovarian cancer.     

HLA-G3蛋白能够抑制多克隆NK细胞的杀伤功能

目的 研究HLA－G3蛋白对NK细胞杀伤功能的抑制作用。方法 用RT－PCR方法扩增HLA－G3和HLA－G1的cDNA，并将其克隆到真核表达载体pcDNA3中，采用脂质体转染法将其转入K562细胞，用免疫荧光法确定其在细胞表面表达，细胞毒试验研究其对NK细胞杀伤功能的抑制作用。结果 HLA－G3蛋白和HLA－G1表达在细胞表面，以PBL细胞为效应细胞的细胞毒实验表明K562－G1细胞和K562－G3细胞的细胞裂解率都比K562细胞裂解率低。结论 HLA－G3蛋白能够抑制多克隆NK细胞的杀伤功能。     

Expression of HLA-G in the human placenta

The unique structure of the human placenta provides a separation between maternal and fetal tissues and circulations. Although the maternal–fetal proximity is very close, the placenta and the fetus are not rejected by the maternal immune system. Among the various factors implicated in the mother's tolerance of the fetus, a key factor has been attributed to HLA-G molecules, which are expressed by specialized trophoblast cells. By the alternative splicing of its primary mRNA, HLA-G, belonging to Class I MHC molecules, results in four membrane bound (HLA-G1, -G2, -G3, -G4) and three soluble proteins (HLA-G5, -G6, -G7). Which of these HLA-G isoforms are expressed on the cell surface and which are secreted was the aim of our study.
Whereas the generally agreed upon opinion is that the full length HLA-G1 isoform is expressed by extravillous cytotrophoblast cells, the source of soluble HLA-G5 isoform and the expression of all other isoforms in the human placenta is still under debate. However, soluble HLA-G products, which might be present in body fluids, could also come from membrane bound HLA-G1 by shedding; a process, that has been described for classical HLA molecules. HLA-G expression in organs, other than placenta and certain tumors, has not yet been explained convincingly.
Since immunological detection methods crucially depend on the antibodies and preparation techniques used, we investigated HLA-G specific antibodies for their specificity and binding properties. We compared the binding ability of some antibodies on HLA-G1, G2, -G5 transfected cell lines with naturally expressed placental HLA-G molecules. A broad range of methods, from immunolocalization to protein-biochemical and Elisa techniques, were used to clarify which HLA-G isoform is actually expressed in the normal placenta.     

The expression of human leukocyte antigen-G on trophoblasts abolishes the growth-suppressing effect of interleukin-2 towards them

PROBLEM: We have shown the attenuated human leukocyte antigen (HLA)-G expression on trophoblasts and an aberrant expression of interleukin (IL)-2, a cytotoxic cytokine, in decidual tissue in preeclampsia, where deteriorated trophoblastic invasion into decidual layers may constitute a crucial pathogenesis. We hypothesized that the absence of HLA-G might make trophoblasts susceptible to compromise by IL-2. METHOD OF STUDY: We analyzed the growth of HLA-G-negative and positive cell lines, all of which possessed IL-2 receptors, in the culture with or without IL-2 supplementation. RESULTS: The proliferation of HLA-G positive trophoblastic cell lines (BeWo and JEG-3) was not influenced by the addition of IL-2, whereas a HLA-G-negative trophoblastic cell line (JAR) exhibited significantly decreased proliferation when cultured with IL-2. Interestingly, the transfection of JAR cells with HLA-G completely eliminates the growth-inhibitory effect of IL-2. CONCLUSION: The expression of HLA-G may commit trophoblasts to evade cell damage by IL-2, which may be relevant to maternal tolerance of the fetus during pregnancy and its derangement as exemplified by preeclampsia.     

Reassessment of HLA-G isoform specificity of MEM-G/9 and 4H84 monoclonal antibodies

Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule thought to play a key role in maternal-fetal tolerance. Although initial studies suggested that HLA-G expression is restricted to extravillous cytotrophoblasts, expression was subsequently reported in a wide variety of other human tissues and tumor cells. However, consensus as to the validity of these collective findings has proven difficult because the antibodies used to define the temporal and spatial expression patterns of HLA-G remain incompletely characterized. The aim of our study was to reassess two of the most widely used HLA-G antibodies (MEM-G/9 and 4H84) in HLA-G-positive (JEG-3 and HLA-G transduced) and -negative (dermal fibroblast, mesenchymal stem cell, K562, and Jurkat) lines using flow cytometry, immunofluorescence, and western blotting. We found that MEM-G/9 recognized HLA-G3 by flow cytometry, indicating that its epitope is present on the α1 domain of HLA-G. Although 4H84 preferably recognized unfolded HLA-G-free chains, it showed strong non-specificity under certain methodological conditions.