培养的血管平滑肌细胞ERα和ERβ的表达与细胞表型转变及增殖时相有关

目的探讨雌激素对血管平滑肌细胞（VSMC）增殖的双重效应机制。方法采用Westernblot、电镜形态定量及细胞计数的方法,动态检测原代培养大鼠VSMC在有或无10^-8mol/L17β-雌二醇（E2）存在下,雌激素受体（ER）α和β表达变化与细胞表型转变及增殖时相的关系。结果无E2存在时,VSMC在从收缩型向合成型转变（原代培养第0到5天）及活跃增殖（第5到12天）过程中,ERβ表达无明显变化,但ERα表达明显上升,导致ERα/ERβ比值升高。这种变化并不随VSMC表型的恢复及增殖停止而逆转。有E2存在时,ERα/ERβ比值在第5天时低于对照组,而第9天后各时点均高于对照组;这种影响与E2对不同状态VSMC的不同作用基本对应,即延长原代收缩型SMC的增殖潜伏期,但促进已发生表型转变的VSMC增殖。结论雌激素对不同表型VSMC的双重效应与表型转变前后ERα/ERβ比值变化有关。     

无蹼壁虎心脏雌激素受体免疫组织化学研究

目的：观察雌激素受体（ERα和ERβ）在非繁殖期成年无蹼壁虎（Gekko swinhonis）心脏的表达并比较性别差异。方法：应用显示雌激素受体的免疫组织化学方法。结果：ERα和ERβ阳性反应均见于无蹼壁虎心肌细胞和成纤维细胞,且受体的表达无性别差异;ERα表达存在明显的心房（11．56±1．67）心室（6．68±1．88）差异（P〈0．01）。结论：雌激素可能是通过ERα主要作用于心房,通过ERβ调节整个心脏的机能;雌激素受体含量与性别无关,可能与生理条件下受体的活性及功能状态有关。     

人、猪、鼠血管内皮及平滑肌细胞培养与纯化方法的改进

对培养和纯化猪主动脉内皮细胞,人脐静脉内皮细胞,猪和鼠主动脉平滑肌细胞的方法进行改进。对其鉴定方法进行了讨论,用胶原酶或胰酶消化法,或机械法分别从猪主动脉,人脐静脉,鼠主动脉获得内皮细胞和平滑肌细胞进行培养,用光学相差显微镜,免疫组化的方法进行鉴定,结果显示,相差显微镜观察,人脐静脉内皮细胞接种后2-3小时贴壁,继而铺开生长,在视野内形成散在细胞团,细胞呈铺路石样排列的单层,随着传代次数增多,可见核分裂,双核及多核,猪血管内皮细胞呈鹅卵石样,多角形,生长分裂旺盛时可见两个或多个核,猪和鼠的平滑肌细胞在相差显微镜下外观为长梭形,细胞生长致密时排列成束,相互平行,并且重叠生长,表现为典型的“波峰”与“浪谷”状。猪血管内皮细胞DiI-Ac-LDL染色,在荧光显微镜下显示特征性的黄绿色荧光,人Ⅷ因子抗原免疫组化检测人脐静脉内皮细胞,显微镜下可见核周胞浆呈现阳性反应,免疫组织化学法进行平滑肌细胞α-肌动蛋白染色,显微镜下见细胞浆内着色,本介绍的培养及纯化猪血管内皮细胞,猪平滑肌细胞,鼠平滑肌细胞的方法简便易行。     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

血管内膜增生过程中核酸代谢相关酶活性变化的研究

目的和方法：应用血管内皮剥脱后再狭窄模型,动态观察胸腹主动脉壁核膜核苷三磷酸酶及核酸代谢和糖代谢相关酶5’—核苷酸酶、腺苷脱氨酶和琥珀酸脱氢酶活性的变化。探讨其与血管平滑肌细胞增生和新生内膜形成的关系。结果：血管内皮剥脱后,胸腹主动脉壁核膜核苷三磷酸酶活性持续升高,与血管内膜增厚程度相平行;血管平滑肌细胞收缩型标志蛋白α-肌动蛋白表达降低及合成型标志蛋白骨桥蛋白表达上调,说明血管平滑肌细胞发生了表型转化,由分化型转变成为去分化型;5’核苷酸酶、腺苷脱氨酶和琥珀酸脱氢酶活性表现为先升后降,三种酶活性均于血管平滑肌细胞增殖旺盛期(术后3～7d)达峰值。结论：细胞内参与mRNA转运及糖、核酸代谢的一系列酶活性的变化是新生内膜形成的生化基础。     

α-平滑肌肌动蛋白在人胸主动脉夹层中的表达

目的:探讨胸主动脉壁中α-平滑肌肌动蛋白(α-SMA)的表达与胸主动脉夹层(TAD)的关系。方法:采集人TAD的动脉壁组织和正常人胸主动脉壁组织,采用Western Blotting和免疫组织化学方法检测α-SMA在组织中的表达程度。结果:在DA组中,α-SMA的表达明显减少,血管平滑肌细胞(VSMC)以增殖表型为主。结论α-SMA的减少主要发生在血管中膜层的VSMC中,细胞发生表型变化,导致中膜弹性变差,发生夹层病变。因此,α-SMA可能在TAD的发病中具有重要作用,值得进一步深入探讨。     

缺氧时肺动脉内皮细胞与肺动脉平滑肌细胞增殖的相互影响

血管内皮细胞和血管平滑细胞在结构和功能上关系密切,两者的相互在与血管舒缩笔血和壁结构。本文观察了培养的小牛肺动脉内皮细胞（ＰＡＥＣｓ）和肺动平滑肌细胞（ＰＡＳＭＣＳ）缺氧时在细胞增殖方面的相互影响。ＰＡＳＭＣＳ常氧条件培养基（ＣＭ）可使ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入降低约５８％,缺氧ＣＭ对ＰＡＥＣＳ的＾３Ｈ－ＴｄＲ掺入无明显的抑制作用;ＰＡＥＣＳ的常氧ＣＭ使ＰＡＳＭＳ的＾３Ｈ－ＴｄＲ掺入升高约６０     

Mir-26a 调控子宫肌瘤中孕激素受体a（PRa）、雌激素受体α（ER-alpha） 的表达

目的：雌激素和孕激素在子宫肌瘤发病中起重要作用。但miRNA 在子宫肌瘤发病中的作用还知之甚少,我们前期已证实 mir-26a 在子宫肌瘤中低表达,本实验进一步探讨mir-26a 在体外对子宫肌瘤中孕激素受体a（PRa）、雌激素受体琢（ER琢）表达 的调控。方法：利用TargetScan 软件预测mir-26a 的潜在靶基因,找出靶基因3''UTR 区片段,插入PmirGLO 绿色荧光蛋白编码 区下游,构建报告基因载体,同时原代培养子宫肌瘤平滑肌细胞。将报告基因载体与mir-26a 共转染入原代培养的子宫肌瘤平 滑肌细胞,引入双荧光素酶报告基因系统对mir-26a 的靶基因进行验证。转染mir-26a mimics 于子宫肌瘤平滑肌细胞,western blotting检测子宫肌瘤平滑肌细胞中mir-26a 靶蛋白表达水平。结果：用TargetScan 软件和双荧光素酶报告基因系统证实ER琢、 PRa 为mir-26a 的靶基因。蛋白水平进一步验证,mir-26a mimics 的转染量不同,ER琢、PRa 的蛋白表达水平下调不同。结论： Mir-26a 通过结合靶基因的3''-UTR 区调控靶基因的mRNA水平。Mir-26a 抑制雌激素受体琢（ER琢）、孕激素受体a（PRa）在子宫 肌瘤中的表达。Mir-26a 可能通过调控雌激素受体琢（ER琢）、孕激素受体a（PRa）影响子宫肌瘤的发展。本实验通过确定mir-26a 对子宫肌瘤的作用机制,有望进一步提高子宫肌瘤的治疗技术,减少手术治疗的创伤。     

普罗布考对动脉粥样硬化大鼠平滑肌细胞凋亡及p53和Fas蛋白表达的影响

目的探讨普罗布考防治动脉粥样硬化（AS）的机制。方法选用雄性大鼠,复制大鼠AS模型,随机分为动脉粥样硬化模型组、普罗布考组和正常对照组。大鼠造模成功后给予普罗布考治疗,6周后处死大鼠,采用流式细胞术检测平滑肌细胞凋亡率及凋亡相关基因p53和Fas蛋白的表达。结果模型组大鼠血管平滑肌细胞凋亡率明显高于对照组（P〈0．05）,p53和Fas蛋白的表达增强（P％0．05）,主动脉壁可肉眼观测典型斑块。普罗布考组大鼠平滑肌细胞的凋亡率明显低于模型组（P〈0．05）,p53和Fas蛋白表达下调（P〈0．05）,主动脉斑块面积较模型组减小明显。结论普罗布考通过调节p53和Fas蛋白表达来调节AS大鼠平滑肌细胞的凋亡。     

雌激素对成年和老年雌性大鼠血管平滑肌细胞雌激素受体α表达的影响及其机制

目的探讨雌激素对成年和老年雌性大鼠血管平滑肌细胞(VSMC)雌激素受体α(ERα)表达的影响及其机制。方法采用RT-qPCR、Western blot及重硫酸盐修饰后测序(BSP)的方法,检测体外培养的3-4代成年(2-3月龄)和老年(≥20月龄)雌性SD大鼠VSMC在无或有生理剂量(10-10 mol/L、10-8 mol/L)17β-雌二醇(E2)存在下,ERα的表达及其启动子区CpG岛甲基化水平的变化。结果成年雌性大鼠VSMC无论在有或无雌激素存在时均表达ERα,且表达水平随雌激素浓度增加而上升。而老年雌性大鼠VSMC无论是在mRNA水平还是蛋白质水平几乎检测不到ERα表达,即使补充雌激素达最高生理剂量也无法诱导ERα的重新表达,其ERα基因启动区CpG岛呈现高水平甲基化。结论成年大鼠VSMC表达ERα,且生理剂量雌激素对其表达具有诱导作用。而老年大鼠VSMC ERα基因由于CpG岛已发生高度甲基化而抑制,生理剂量雌激素对ERα表达的诱导作用丧失。     

大鼠心脏的雌激素受体免疫组织化学研究

观察雌激素受体在雌性与雄性大鼠心脏中的表达.取大鼠心房与心室组织制作冰冻切片,应用抗雌激素受体单抗进行免疫组织化学(SP法)染色并进行图像分析.结果显示,雌性与雄性大鼠心脏都存在雌激素受体,且受体的表达无性别差异(P＞0.05);心房与心室都存在雌激素受体阳性表达,其表达也无明显差异(P＞0.05);阳性反应见于心肌细胞和成纤维细胞.结果表明,大鼠心脏存在雌激素受体,心房与心室都可能是雌激素的靶组织;心血管疾病的性别差异与雌性、雄性的受体含量无关,可能与生理条件下受体的活性及功能状态有关.     

Estrogen receptor mRNA in uteri of normal estrous cycling and ovariectomized rats by in situ hybridization.

Biological effects of estrogen are mediated via its binding to the estrogen receptor (ER), the contents of its protein and mRNA varying during the estrous cycle. In the present study, the ERalpha mRNA expression in different cell components of the uterus was investigated in normal estrous cycling rats using nonisotopic in situ hybridization. Additionally, ovariectomized (OVX) rats treated with 17beta-estradiol (E2: 5 microg/kg, sc injection daily) were also investigated to clarify the effects of exogenous E2. At proestrus and diestrus, and especially the former, the luminal and glandular epithelial (LE and GE) cells were strongly positive, along with stromal cells beneath the luminal epithelium. At estrus, the expression was slightly diminished in LE cells, but almost completely lacking in GE cells. At metestrus, positive signals appeared again in GE cells. In the myometrium, ER mRNA was demonstrated to be constantly positive in all estrous cycle stages. OVX rat uteri underwent marked atrophy, but ER mRNA still remained in all cell types. After 2 consecutive days of E2 treatment, markedly increased intensity was observed, especially in LE and GE cells. The uteri of OVX rats treated with E2 for 14 days, however, showed slightly diminished expression, whereas the serum concentration of E2 was comparable to that in rats after 2 days. These results provide evidence that cell-type specific patterns of ER mRNA expression characterize the uteri of both normal estrous cycling rats and OVX rats after estrogen treatment.     

Immune-deficient animals to study "hormone-dependent" breast and endometrial cancer

Athymic (nu/nu) mice are T cell deficient and can accept xenografts of human tumor material. Hormone-dependent tumor growth can be demonstrated in ovariectomized athymic mice by estrogen administration. Estrogen receptor (ER) positive MCF-7 breast cancer cells implanted into the axillary mammary fat do not grow into palpable tumors unless sustained release preparations of estrogen are administered. The non-steroidal antiestrogen tamoxifen, though it exhibits estrogenic properties in the mouse, does not facilitate MCF-7 tumor growth (during short term, i.e. 8 weeks of therapy) and can prevent estradiol-stimulated growth. In contrast, ER negative MDA-MB-231 cells grow with or without estrogen administration and tamoxifen does not control tumor growth. These statements reflect current dogma concerning the value of athymic mice to confirm the hormone dependent growth of cancer cells in vivo. Our aim has been to define the limits of this dogma and to investigate the growth relationship of hormone-dependent and independent cells with their host environment. The potential endocrine or paracine effect of ER negative tumors on the growth of ER positive tumors was evaluated by transplantation on opposite sides of athymic mice or by the inoculation of different ratios of ER positive/negative cells (MCF-7:MDA-MB-231 9:1, 99:1, 999:1). MCF-7 cells could not be encouraged to grow by a rapidly growing MDA-MB-231 tumor on the opposite side of the animal. Similarly ER negative tumors grew out of the mixed tumor inoculates suggesting that ER positive tumors could not be encouraged to grow preferentially by the paracrine influences of ER negative cells. However, estrogen facilitates the growth of an ER positive tumor following inoculation of mixed cell populations. Antiestrogen treatment can blunt estrogen-stimulated growth but cannot control the growth of ER positive/negative containing tumors. ER positive endometrial tumors grow in response to estrogen treatment and some (EnCa101) have been shown to grow in response to tamoxifen or a combination of tamoxifen and estrogen. More unusual though is our recent observation that an ER negative primary endometrial tumor (BR) and its metastasis (BR-MET) grow more rapidly in estrogen-treated athymic mice. This finding seems to have far-ranging consequences for our view of hormone-dependent growth. Either our view of estrogen-stimulated growth needs to be modified or the host is specifically altered during estrogen treatment. We have taken the position that since natural killer cells (present in athymic mice) can be lowered by estrogen this may result in an increased tumor cell survival in the heterotransplant model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative image analysis of estrogen and progesterone receptors as a prognostic tool for selecting breast cancer patients for therapy

OBJECTIVE: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis. STUDY DESIGN: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system. RESULTS: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse. CONCLUSION: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.     

Factors influencing the expression of stress fibers in vascular endothelial cells in situ

The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age- matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.     

Estrogen-modulated estrogen receptor x Pit-1 protein complex formation and prolactin gene activation require novel protein synthesis

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER.Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER.Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER.Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER.Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER.Pit-1 protein complex formation in a dose-dependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.