重组人IL-2抑制乳腺癌细胞增殖及其机制的研究

目的:观察重组人IL-2对体外培养乳腺癌细胞生长的影响,及对洛铂体外抑瘤效果的影响。方法:选择乳腺癌细胞株MCF-7和MDA-MB-231作为实验对象。利用MTT实验检测重组人IL-2和对照药物洛铂对肿瘤细胞生长的抑制率,利用RT-PCR方法分析MCF-7细胞中Syk基因在不同处理因素作用下表达情况。结果:洛铂能明显抑制两种乳腺癌细胞体外生长,重组人IL-2对两种癌细胞有一定的抑制作用。重组人IL-2和洛铂同时处理时,对肿瘤细胞的抑制率比单独应用洛铂时明显升高,并且同时处理时MCF-7细胞中Syk mRNA表达水平比洛泊单独处理时高。结论:重组人IL-2能增强洛铂对乳腺癌细胞生长抑制效果。重组人IL-2单独作用乳腺癌细胞时也有一定的抑制效果,针对MCF-7细胞,重组人IL-2刺激信号传递可能有Syk参与。提示在体内时IL-2不仅对机体免疫细胞起重要的刺激激活作用,还可能对瘤细胞本身也有抑制作用。     

三羟异黄酮对人乳腺癌MCF-7/ADM细胞体外抑瘤效应、细胞周期及凋亡的影响

 目的探讨三羟异黄酮（Genistein GEN）对体外培养人乳腺癌耐药细胞MCF-7/ADM抑瘤作用、细胞周期及细胞凋亡的影响。方法采用MTT法检测GEN单独及联合阿霉素对体外培养人乳腺癌MCF-7/ADM细胞的抑制作用;荧光分光光度法检测GEN对阿霉素在MCF-7/ADM细胞的蓄积作用;用流式细胞仪（FCM）检测细胞周期及凋亡率的变化。结果GEN单独及联合阿霉素对体外培养MCF-7/ADM细胞均有明显生长抑制作用,GEN单独作用48 h后表现为随时间的延长抑制作用明显增强,当GEN浓度达到60 μg/ml时,其抑制作用急剧上升（P<0.01）。联合阿霉素后与对照组相比抑制率明显上升（P<0.01）,并随GEN浓度的加大其抑制作用增强,细胞内阿霉素的浓度也随之升高。与对照组相比,细胞周期均有G2/M期阻滞作用,细胞凋亡百分比以联合组最高（P<0.01）,G₁期前出现典型的亚二倍体凋亡峰。结论GEN单独及联合阿霉素对体外培养人乳腺癌MCF-7/ADM细胞具有抑瘤增效作用,可以提高阿霉素在MCF-7/ADM细胞内的蓄积、对细胞周期具有G₂/M期阻滞作用,显著诱导MCF-7/ADM细胞凋亡,可能是其发挥逆转多药耐药分子生物学机制之一。     

氧化苦参碱对人乳腺癌 MCF -7 细胞的生长抑制作用及其机制研究

目的：了解中药苦参的有效成分氧化苦参碱对人乳腺癌细胞系MCF-7肿瘤细胞的增殖及其相关的调控机制的干预作用.方法：以乳腺癌细胞株（MCF-7）为研究对象,利用MTT法检测氧化苦参碱的体外杀伤活性;RT-PCR法、Western blot法、流式细胞技术、细胞免疫荧光计数检测基因、蛋白表达水平,对其相关调控基因的表达水平进行检测.结果：氧化苦参碱对体外培养的MCF-7细胞的生长有明显的抑制作用;可以诱导乳腺癌 MCF-7细胞凋亡,而且这种生长抑制及促进凋亡的作用可能是通过抑制Wnt/β-catenin信号转导通路的活性来实现.结论：氧化苦参碱可以通过抑制Wnt/β-catenin信号转导通路的活性来抑制人乳腺癌MCF-7细胞系的增殖,促进其凋亡.     

整合NGR肽抗EGFR单链抗体制备及其抗肿瘤活性研究

目的：利用大肠埃希菌表达基于抗表皮生长因子受体（epidermalgrowthfactorreceptor,EGFR）单链抗体和靶向CD13环状NGR肽的双靶点重组融合蛋白ER（Fv）-NGR,研究其对肿瘤细胞的亲和能力和体内外抑制作用。方法：用基因工程的方法构建重组质粒pET-scfv-ngr,转入大肠埃希菌BL21,IPTG诱导表达带有His-tag标签肽的重组蛋白ER（Fv）-NGR,用Ni离子亲和柱进行纯化。运用细胞免疫荧光检测ER（Fv）-NGR与肿瘤细胞的结合;流式细胞术分析重组蛋白与肿瘤细胞的亲和力;克隆形成实验测定重组蛋白对肿瘤细胞体外增殖的抑制作用;利用人乳腺癌MCF-7裸鼠移植瘤模型对ER（Fv）-NGR的体内抗肿瘤活性进行研究。结果：重组蛋白ER（Fv）-NGR由抗EGFR单链抗体和3个连续的环状NGR肽构成,相对分子质量约为27×10^4,以包涵体形式在大肠埃希菌内表达,表达量约为5mg／L。细胞免疫荧光结果表明,重组蛋白与EGFR和CD13皆高表达MCF-7细胞有较强的结合能力,而与正常HEK293细胞结合较弱。ER（Fv）-NGR与MCF-7细胞结合的解离常数为8．4×10-7nmol／L。ER（Fv）-NGR在体外对肿瘤细胞的增殖有抑制作用,作用于MCF-7和A549细胞的Ic50值分别为1．2×10^-6和4．7×10-mol／L。在体内对人乳腺癌MCF-7裸鼠移植瘤有生长抑制作用,在10mg／kg药物剂量下28d对照组与实验组瘤体积分别为（1．06±0．17）和（0．63±0．11）cm2,F-18．7,P=0．003;瘤质量分别为（0．95±0．12）和（0．52±0．07）g,F=42．7,P〈0．001;抑瘤率为45．4％。结论：成功制备了基于单链抗体和靶向肽的双靶点融合蛋白ER（Fv）-NGR,其对肿瘤细胞具有良好的亲和力和体外增殖抑制作用,能够有效抑制裸鼠移植瘤的生长,为研制新型双靶点抗肿瘤药物提供依据。     

咖啡因对乳腺癌化疗增效作用的体外实验研究

目的：体外研究咖啡因（caffeine，CF）联合多柔比星（adriamycin，ADM）对乳腺癌细胞株MCF-7的抑制效应。方法：用MTT法评估瘤细胞的生长抑制情况，通过电镜检测瘤细胞的凋亡，并用流失式细胞仪确定瘤细胞的凋亡率。结果：在0～8．0mg／mL浓度范围内，CF对MCF-7的抑制作用和其浓度呈正相关，且CF联合ADM（0．5～2．5μg／mL）对MCF-7的生长抑制显著大于ADM的单独作用，P〈0．05；先加入CF或CF与ADM同时作用，对MCF-7的生长抑制高于后加入CF组或ADM单独使用组，P〈0．05；CF和ADM联合作用对MCF-7的凋亡诱导，和CF或ADM单独作用比，凋亡率未有明显改变。结论：CF对MCF-7的生长有抑制作用，与ADM联合应用的抑制作用明显大于单独使用ADM，先加入CF或CF与ADM同时作用的给药顺序优于后加入CF；ADM和CF抑制MCF-7生长的机制之一是诱导凋亡，两者共同作用并未显著增加MCF-7的凋亡率，但有协同杀伤MCF-7的作用，其中化学性杀伤仍起主要作用。     

人端粒酶逆转录亚单位(hTERT)基因特异性siRNA对MCF-7细胞生长抑制作用的研究

目的 研究人端粒酶逆转录亚单位（hTERT）特异性siRNA对MCF-7细胞体外生长及体内肿瘤形成能力的抑制作用。方法 设计并化学合成针对hTERT的siRNA，用脂质体转染法将其导入MCF-7细胞内，通过软琼脂克隆形成试验及接种裸鼠的肿瘤形成实验，观察hTERT-siRNA对人乳腺癌MCF-7细胞体外及体内的生长抑制作用。结果 软琼脂克隆形成试验显示，hTERT-siRNA转染的MCF-7细胞克隆形成数量明显少于对照组，抑制率达到84．1％。裸鼠体内实验结果显示，hTERT-siRNA转染组肿瘤生长速度也明显慢于对照组。结论 hTERT-siRNA在体内外均显示可以有效、特异地抑制肿瘤细胞的生长。     

重组人NDRG2蛋白对肿瘤细胞抑制作用研究

目的:考察原核表达的重组人NDRG2蛋白对肿瘤细胞的作用.方法:通过设置不同浓度的蛋白剂量并设置对照组,采用细胞MTT比色法和细胞计数检测NDRG2蛋白对7901、HHCC细胞的抑制效果,流式细胞仪检测细胞周期的变化,体内实验采用裸鼠抑瘤试验.结果:重组人NDRG2对肿瘤细胞生长有较强的抑制作用,约50μg/ml浓度时对肿瘤细胞生长的抑制作用明显,流式细胞术结果显示肿瘤细胞G1期变化,体外能抑制裸鼠肿瘤的生成.结论:重组人NDRG2蛋白在体内体外具有一定的抑制肿瘤细胞生长作用,在肿瘤治疗方面将具有一定的意义.     

基因转染LL-37/hCAP-18对人乳腺癌细胞凋亡的影响

目的：探讨基因转染人源抗菌肽LL-37/hCAP-18对人乳腺癌细胞MCF-7增殖、凋亡的影响。方法：将含人LL-37/hCAP-18的真核表达质粒瞬时转染人MCF-7细胞,48h后,MTT比色检测MCF-7细胞的增殖,流式细胞术检测MCF-7细胞周期及凋亡,细胞免疫组化染色检测MCF-7细胞中Bax、Bcl-2的表达。结果：重组基因瞬时转染MCF-7细胞48h后,与各对照组相比,肿瘤细胞的生长增殖受到显著抑制（P〈0.05）;转染重组基因组肿瘤细胞凋亡率显著高于各对照组（P〈0.05）,但细胞周期无明显变化;与对照组相比,转染重组基因pEGFP-c1-LL-37的MCF-7细胞中Bcl-2表达显著下降（P〈0.05）,Bax由阴性表达转为表达明显升高。结论：LL-37/hCAP-18可以通过上调促凋亡因子Bax、下调抗凋亡因子Bcl-2诱导肿瘤细胞凋亡,从而抑制乳腺癌细胞MCF-7的增殖。     

咖啡因对乳腺癌化疗增效作用的体外实验研究

目的:体外研究咖啡因(caffe-ine,CF)联合多柔比星(adriamycin,ADM)对乳腺癌细胞株MCF-7的抑制效应。方法:用MTT法评估瘤细胞的生长抑制情况,通过电镜检测瘤细胞的凋亡,并用流失式细胞仪确定瘤细胞的凋亡率。结果:在0~8·0mg/mL浓度范围内,CF对MCF-7的抑制作用和其浓度呈正相关,且CF联合ADM(0·5~2·5μg/mL)对MCF-7的生长抑制显著大于ADM的单独作用,P<0·05;先加入CF或CF与ADM同时作用,对MCF-7的生长抑制高于后加入CF组或ADM单独使用组,P<0·05;CF和ADM联合作用对MCF-7的凋亡诱导,和CF或ADM单独作用比,凋亡率未有明显改变。结论:CF对MCF-7的生长有抑制作用,与ADM联合应用的抑制作用明显大于单独使用ADM,先加入CF或CF与ADM同时作用的给药顺序优于后加入CF;ADM和CF抑制MCF-7生长的机制之一是诱导凋亡,两者共同作用并未显著增加MCF-7的凋亡率,但有协同杀伤MCF-7的作用,其中化学性杀伤仍起主要作用。     

Interleukin-2 differentially affects the proliferation of a hormone-dependent and a hormone-independent human breast cancer cell line in vitro and in vivo

Direct in vitro and in vivo effects of the lymphokine, interleukin-2 (IL-2), on hormone-dependent (MCF-7) and- independent (MDA-231) human breast cancer cell proliferation were investigated. In vitro, picomolar concentrations of IL-2 directly inhibited MCF-7 cell proliferation after 12 days of culture, while nanomolar doses of IL-2 significantly stimulated MCF-7 cell growth over the same time period. In addition, micromolar concentrations of IL-2 had virtually no effect on the in vitro proliferation of MCF-7 cells. In parallel in vitro growth experiments, the hormone-independent cells, MDA-231, were not affected by IL-2 regardless of concentration. IL-2 treatment of overiectomized, estrogen-treated nude mice, burdened with MCF-7 or MDA-231 tumors, inhibited MCF-7 tumor growth, but had no effect on MDA-231 tumors. Examination of T, B and natural killer (NK) cell function in these animals indicated that the interleukin-2-mediated effect on MCF-7 cell growth in vivo is independent of the proliferative abilities of these lymphoid cells, suggesting that IL-2 may directly affect the growth of these hormone-dependent human breast cancer cells.     

IGF-IR反义基因抑制乳腺癌细胞内源性IGF-IR表达的研究

目的探讨重组的人胰岛素样生长因子I型受体(IGFIR)的反义基因,对乳腺癌细胞内源性IGFIR表达的抑制作用。方法体外构建人IGFIR的反义基因真核表达载体pIGFIR/As,并转染人MCF7乳腺癌细胞,经G418筛选而获得稳定表达外源性反义基因的新细胞株MCF7/As,在基因水平和蛋白质水平检测内源性IGFIR的表达情况。结果人IGFIR的反义基因真核表达载体pIGFIR/As构建成功。RTPCR显示MCF7转染前后IGFIRmRNA的表达水平分别为(0.71±0.04)和(0.43±0.06),表达明显下降(P<0.05);Westernblot显示其蛋白质的表达水平呈相同的变化趋势(P<0.01)。结论本实验构建的载体pIGFIR/As能够稳定转染MCF7细胞,所表达的IGFIR的反义基因能够在基因和蛋白质水平显著抑制内源性IGFIR的表达。     

Interleukin-3: A putative protective factor against breast cancer which is secreted by male but not female breast fibroblasts

The enzyme 17β-hydroxysteroid dehydrogenase (17-HSD) is a key regulator of intracellular 17β-estradiol (E2), which is associated with breast cancer and is influenced by paracrine factors released by breast-cancer fibroblasts. Since the incidence of breast cancer is much higher in females than in males, we have used an in vitro ceil culture system to investigate whether male fibroblasts may inhibit breast-cancer genesis by restricting the intracellular accumulation of E2. Fibroblasts were obtained from normal males and females undergoing reduction mammoplasty, and from females with benign or malignant breast lesions. Fibroblast-conditioned medium (CM) was incubated with the established breast-cancer cell line, MCF-7, and its effects on 17-HSD activity were assessed. CM (25% v/v) from male breast fibroblasts had a significant inhibitory effect on reductive i 7-HSD, decreasing E2 production. This was in direct contrast to the effects of CM from female breast fibroblasts, which had a powerful stimulatory effect on reductive 17-HSD. RT-PCR allowing simultaneous detection of a range of cytokines was performed on each type of fibroblast. IL-3 mRNA was consistently detected in fibroblasts from male but not female breast tissue. Addition of rhIL-3 to cultures of MCF-7 caused a reduction in 17-HSD activity and addition of a polyclonal antibody directed against IL-3 to male CM completely reversed the inhibitory effects of CM. Thus, male breast fibroblasts may be responsible for secreting IL-3-like factors which, given the considerably lower incidence rates of breast cancer in men, may have a protective effect against breast cancer. © 1995 Wiley-Less, Inc.     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的：研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法：采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果：甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达（68.40±4.87）%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论：甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

槲皮素增强乳腺癌细胞化疗敏感度的研究

目的探讨槲皮素(quercetin,Que)对乳腺癌细胞化疗药物敏感度的影响及其可能的机制。方法MTT 法检测不同浓度 Que 对人乳腺癌 MCF-7 细胞生长的影响,单用阿霉素(adriamycin,ADR）或 ADR 联合无毒剂量 Que 作用于人乳腺癌 MCF-7 细胞,MTT 法检测药物对细胞的增殖抑制作用,流式细胞法检测细胞凋亡率;Transwell小室法检测细胞侵袭能力的变化;RT-PCR法及Western blot法分别检测缺氧诱导因子-1α (HIF-1α) mRNA水平和蛋白水平的表达变化。结果不高于 0.7 μM Que对细胞生长无影响。与单用ADR组相比较,0.7 μM Que与ADR联用可提高ADR对人乳腺癌MCF-7细胞的增殖抑制率、诱导其细胞凋亡、降低其体外侵袭和转移能力、下调HIF-1αmRNA和蛋白的表达。结论Que能够增强人乳腺癌MCF-7细胞化疗敏感度,其机制可能与下调该细胞HIF-1α的mRNA和蛋白表达有关。     

Overexpression of macrophage migration inhibitory factor induces angiogenesis in human breast cancer

Macrophage migration inhibitory factor (MIF) is known to be an important contributor to tumor progression. Overexpression of MIF has been reported in different types of tumors. However, the correlation between MIF expression and tumor pathologic features in patients with breast cancer has not been elucidated. In this study, we examined the expression of MIF, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in human tissues with or without tumor. In addition, we investigated the expression of MIF in MDA-MB-231, MCF-7 (breast cancer cell lines) and MCF-10A (epithelial cell line) cells, and its effect on VEGF and IL-8. We found that MIF was overexpressed in breast cancer tissues compared with normal ones. The level of MIF showed the positive correlation between the expression of IL-8 and tumor microvessel density (MVD). The patients with positive MIF expression in tumor tissues showed a significantly worse disease-free survival compared with negative ones. Increased MIF serum levels were also found to correlate with higher levels of IL-8 in the sera of the patients with breast cancer. In vitro experiments successfully detected MIF in breast cell lines. However, the expression level of it by normal epithelial cells was much less than that of cancer cells. Exogenous MIF did not cause endothelial tube formation and migration but induced a dose dependent increase in VEGF and IL-8 secretion in breast cancer cell lines. In summary, our studies show that human breast cancer tissue expresses MIF. Its in vitro effect on VEGF and IL-8 indicates that MIF may contribute to tumor in angiogenesis and thus play an important role in the pathogenesis of breast cancer.     

Regulation by protein kinase C of TGF-beta 1 expression in cultured cells of breast adenocarcinoma]

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We investigated the involvement of TGF-beta 1 in the PCK-mediated inhibition of breast cancer cell proliferation. Using an RNase protection assay, we showed that TPA induced a dose-dependent increase in levels of TGF-beta 1 mRNA that paralleled the inhibitory effect on MCF-7 proliferation. Similar results were obtained with another TPA-sensitive breast cancer cell line (BT-20). TPA did not increase TGF-beta 1 mRNA levels in the MCF-7:RPh-4 and T47D cell lines, which are both insensitive to the growth inhibitory effects of phorbol esters. In addition, the increase in TGF-beta 1 mRNA level was not observed after treatment of the MCF-7 cell with other inducers of cell differentiation such as forskolin, DMF, HMBA and sodium butyrate. The induction of TGF-beta 1 mRNA by TPA along with its inhibitory effect on cell proliferation suggests that TGF-beta 1 mediates, at least in part, the inhibitory effect of PKC activation.     

Down-regulation of expression of interleukin-6 and its receptor results in growth inhibition of MCF-7 breast cancer cells

Interleukin-6 (IL-6) plays an important role in the neoplastic process through its action on cancer cell adhesion, motility, proliferation, tumor-specific antigen expression, and thrombopoiesis. IL-6 exerts its activity by binding to a high affinity receptor complex consisting of two membrane glycoproteins: the 80 kDa IL-6 a-receptor subunit (IL-6R) and the 130 kDa signal-transducing protein (GP130). In the present study, MCF-7 breast cancer cells were cultured with human IL-6 and IL-6 soluble receptor (sIL-6R). MCF-7 cells were also treated with either antibodies specific to human IL-6 and IL-6R, or synthetic antisense oligodeoxynucleotides (ODNs) targeted to IL-6 and IL-6R genes. Cell growth was measured, and it was found that human IL-6 and sIL-6R did not significantly increase the proliferation of MCF-7 cells. When IL-6 produced by the MCF-7 cells was bound by rabbit anti-human IL-6 antibody, there was a significant dose-dependent inhibition of cell proliferation. IL-6 and IL-6R antisense ODNs caused a marked and specific decrease in IL-6 and IL-6R mRNA and proteins, respectively. Both IL-6 and IL-6R antisense ODNs significantly inhibited the proliferation of MCF-7 cells, but the inhibitory effect of IL-6R antisense ODN was greater than that of IL-6 antisense ODN (IC??: IL-6R: 1 μM; IL-6: 5 μM, 72-hour incubation). Addition of exogenous IL-6 partially reversed the growth inhibition caused by IL-6 antisense ODN but not the growth inhibition caused by IL-6R antisense ODN. In conclusion, IL-6 plays an important role in maintaining the growth of MCF-7 breast cancer cells. These results suggest careful modulation of IL-6 and IL-6R expression of cells as a potential approach for breast cancer therapy.