钙粘附蛋白在人类早期胚胎的表达

目的 检测钙粘附蛋白在人类早期胚胎的表达，探讨钙粘附蛋白在胚胎着床及发育中的作用。方法 应用特异性单克隆抗体，采用间接免疫荧光方法，检测人种植前胚胎(分别为2、4、6、7、8细胞胚及囊胚)上皮型钙粘附蛋白的表达，并应用激光共聚焦显微镜对荧光进行定量检测。结果 各期人种植前胚胎表面均见上皮型钙粘附蛋白表达，囊胚期表达明显增高。结论 人类早期胚胎在发育过程中能表达上皮型钙粘附蛋白，且表达量逐渐增加，可能对胚胎成功着床有重要意义。     

Expression of integrins and E-cadherin in squamous cell carcinomas of the head and neck

Integrins and cadherins are cell adhesion molecules suggested to play an important role in malignant progression and tumour differentiation. Our aim was to characterise the pattern of expression and the relations between integrin beta1, beta4, beta6 and E-cadherin and the different histopathological features important when judging tumour differentiation, using a well-defined scoring system. Formalin-fixed paraffin-embedded pre-irradiation biopsies from 85 patients with head and neck squamous cell carcinomas (HNSCC) were stained and evaluated for the expression of integrin beta1, beta4 and beta6 and E-cadherin. The integrins were upregulated in carcinomas compared to the adjacent mucosa and E-cadherin was downregulated. However, differences were found within the tumour: Expression of E-cadherin was lost and the three integrins were upregulated at the tumour borders, compared to central parts of the tumour biopsy. Expression of the integrins did not correlate with tumour or histopathological parameters, whereas expression of E-cadherin was correlated with high degree of keratinisation, high nuclear maturation and few mitoses - factors that characterise well-differentiated carcinomas -and E-cadherin can therefore be considered as a marker of differentiation. Furthermore, loss of adhesion expressed by low E-cadherin and integrin beta4 correlated with the presence of nodal metastases at the time of diagnosis.     

CD9 is expressed on human endometrial epithelial cells in association with integrins alpha(6), alpha(3) and beta(1)

Recently we reported that CD9 is involved in the invasion of a trophoblast-like choriocarcinoma cell line, BeWo, probably through the regulation of integrin functions. Integrins have also been reported to be expressed in the human endometrium and it has been suggested that they play important roles in blastocyst implantation. This study used immunohistochemistry to investigate the expression of CD9 in the endometrium during the menstrual cycle. CD9 was found to be intensely expressed on the cell surface of the glandular epithelium throughout the menstrual cycle without any apparent differences in staining intensity. In addition, Western blotting analysis of the affinity-purified proteins confirmed that CD9 was associated with integrins beta(1), alpha(3) and alpha(6) in the human endometrium. Therefore it can be concluded that CD9, in association with integrins alpha(6), alpha(3) and beta(1), is a constitutive molecule of the endometrial glandular epithelium. These results also suggest that CD9 may be an important regulator of these integrins in the human endometrium.     

Immunolocalization of integrins and fibronectin in tubal pregnancy

Integrins are a large family of cell adhesion molecules that serve as receptors involved in cell-to-cell and cell-to-matrix interactions during implantation. We studied immunohistochemical staining of integrins (alpha 3, alpha V, beta 1, and alpha 2 beta 1) and fibronectin in ectopic tubal pregnancy. Thirty fallopian tube samples with ectopic pregnancies and five normal tubal segments were obtained during ligation operations; the latter specimens served as controls in the study. Formalin-fixed paraffin-embedded tissue sections were stained with hematoxylin-eosin or primary antibodies against alpha 3, beta 1, alpha V, and alpha 2 beta 1 integrins and fibronectin, using the avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare staining intensities. In the control samples, immunostaining of all integrins was found in a single layer of tall columnar epithelial cells, the lamina propria (Lp) and the muscular layer. Fibronectin staining was detected in the Lp and the muscular layer. Staining intensities of alpha 3 and beta 1 integrins and fibronectin were increased in the normal part of fallopian tubes with ectopic pregnancies. Staining of beta 1 integrin was more intense than staining of alpha 3 and fibronectin, whereas there was no difference in alpha V and alpha 2 beta 1 integrin expression between normal tubal tissue in the ectopic pregnancy group and control tubal tissue. In the tubal pregnancy group at the site of implantation, staining intensity of alpha 3 and beta 1 integrins and fibronectin was strong in decidual cells, supporting tissue and placental villi, whereas alpha V and alpha 2 beta 1 staining was mild. We concluded that integrins, especially beta 1 and alpha 3, and fibronectin may play a role in progression of tubal implantation. Although the role of integrins has not yet been clearly defined, these molecules may function as markers of normal and abnormal states of receptivity. We like to suggest that integrins and fibronectin, which are needed in utero implantation, are expressed in tubal tissues during ectopic pregnancy and are involved in ectopic implantation.     

The effect of gonadotrophic stimulation on integrin expression in the endometrium.

BACKGROUND: Despite many recent advances in IVF treatment implantation rates per embryo transfer rarely exceed 30%. Three integrins (alpha(1)beta(1),alpha(4)beta(1) and alpha(v)beta(3)) have been shown to be expressed in the endometrium in a cyclically dependent manner and are thought therefore to play a vital role in the process of implantation. METHODS: The effect of gonadotrophin stimulation on the expression of these three integrins within the endometrium was investigated by examining biopsies from oocyte donation patients and comparing them with fertile controls. RESULTS: A delay in the maturation of the glandular epithelium was found in the oocyte donation patients. There was also a reduction in the expression of all three integrins in the glandular epithelium and also a reduced expression of the alpha(v)beta(3) integrin in the luminal epithelium. CONCLUSIONS: As these integrins have been shown to be important in implantation their reduced expression after IVF treatment may have an adverse effect on pregnancy rates.     

Expression pattern of integrin adhesion molecules in endometriosis and human endometrium

Integrins are cell adhesion molecules that undergo cell-specific dynamic changes during the normal menstrual cycle in the human endometrium. Here, using immunohistochemistry, we have investigated the expression pattern of the integrins alphav, alpha2beta1, alpha3beta1, alpha3, alpha6, beta1, beta2 and beta3 in the human ectopic endometrium of 30 patients and in nine cases in the corresponding eutopic endometrium. The biopsies were obtained during the early or late follicular phase (25 cases), during the corpus luteum phase (four cases) and in one case after 6 months' treatment with a gonadotrophin releasing hormone (GnRH) agonist. The integrin expression was independent of the ovarian steroid situation at the time of biopsy. The integrin alpha6 was expressed in all endometriotic and endometrium samples. The integrin alpha3 was absent in all endometrium tissues of patients with endometriosis. However, the corresponding endometriotic lesions re-expressed this adhesion molecule in 15 cases. No change in integrin beta3 expression pattern could be demonstrated in either endometriotic lesions or endometrium samples, regardless of the menstrual cycle phase. A correlation between serum oestradiol and progesterone concentrations and the expression of the investigated integrins was not observed, thus indicating that these two hormones play a minor role in the regulation of the cell adhesion molecules examined. Our investigation suggests that endometriosis is a dedifferentiated disease as it expressed different integrins in comparison with the eutopic endometrium, and independently of the hormonal situation. The ability of endometriotic tissues to express integrins may explain the high recurrence rates in patients with endometriosis, as these samples retain their adhesion potency after retrograde menstruation and are thus able to establish cell-cell and cell-matrix interactions with the surrounding peritoneum.     

Expression of the VLA beta 1 integrin family in bladder cancer.

Integrins are a family of transmembrane heterodimers, many of which function as receptors for extracellular matrix molecules and play a role in adherence to and motility on matrix components. Because of these functions, integrins are suspected of participating in metastatic processes. We investigated the expression of beta 1 integrins in human bladder cancer cell lines and tissues. Expression of beta 1 integrins on cultured bladder cancer cell lines was evaluated by flow cytometry, of 8 cell lines tested, alpha 1 was found in 4, alpha 2 and alpha 3 in all 8, alpha 4 in 1, and alpha 5 in 3. These results were in sharp contrast to the expression detected by immunostaining tissues containing normal urothelium and low stage (noninvasive) and high stage (invasive) bladder cancers. All normal urothelial tissues tested expressed alpha 2 and alpha 3 and none expressed alpha 1, alpha 4, or alpha 5. Similarly, a majority (77%) of low stage (noninvasive) bladder cancers stained positively for alpha 3, whereas only 6 of 13 expressed alpha 2 and none expressed alpha 1, alpha 4, or alpha 5. Among invasive bladder cancers, alpha 1 was detected in 7%, alpha 2 in 24%, alpha 3 in 68%, alpha 5 in 10%, and alpha 4 was not found in any samples. These results indicate that integrin expression in cultured human bladder cancer cell lines does not represent expression observed in tissue samples and may reflect adaption to or selection during tissue culture conditions. A progressive loss of alpha 2 expression is seen from normal urothelial cells through invasive bladder cancers. This loss may contribute to an invasive phenotype by a loss of the cell-cell adherence function mediated by the alpha 2 beta 1 and alpha 3 beta 1 integrins.     

Beta1 integrin activation on human neutrophils promotes beta2 integrin-mediated adhesion to fibronectin

Although the importance of beta1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of beta1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the alpha4, alpha5, alpha9 and beta1 integrin subunits. To examine whether the integrins VLA-4 (alpha4/beta1) and VLA-5 (alpha5/beta1) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates beta1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2-induced adhesion did not depend on beta1 integrins, but on the beta2 integrin CD11b/CD18. Similarly, activation of beta1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca2+. These data suggest that engagement of beta1 integrins on neutrophils results in a cross-talk signal that leads to activation of the beta2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both beta1 and beta2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.     

Interleukin-6 levels in co-culture of human in vitro fertilization embryos with Vero cells are not predictive of future successful development

PROBLEM: In an attempt to predict successful embryo transfer and implantation, we measured interleukin (IL)-6 levels in culture supernatants of co-cultured preimplantation human embryos. We tested whether all in vitro fertilized human embryos in co-cultures do secrete IL-6, and whether there was any difference in such production between embryos that successfully reached the blastocyst stage and blocked embryos. We also addressed the question of IL-6 secretion by co-culture support cells, namely Vero cells themselves. METHOD OF STUDY: Each fertilized oocyte was cultured individually and transferred in culture wells supplemented with a feeder layer of Vero cells at day 2. In vitro IL-6 production was measured by bioassay of the culture media. RESULTS: Because Vero cells themselves secrete IL-6, it became impossible, in co-culture, to quantify production of IL-6 by the sole embryos. On the other hand, the co-culture technique has shown us that embryos are likely to consume IL-6. There was no difference between blastocysts and blocked embryos. CONCLUSIONS: IL-6 levels in human embryo co-cultures do not correlate with future successful embryo transfer.     

Normalization of keratinocyte-type integrins during the establishment of the oral mucosa phenotype in vitro.

In stratified epithelia, integrins play a fundamental role in mediating basal cell attachment to a variety of extracellular matrix molecules. To assess whether keratinocyte-specific integrins are expressed in a similar way as in the normal situation also under in vivo related conditions, we processed oral mucosa equivalents consisting of keratinocytes and fibroblasts from non-cornified gingiva. In this model histomorphology, the expression of differentiation-specific keratins and keratinocyte-type integrins exhibited similarity to the tissue of origin. The stages of tissue normalization were assessed on frozen sections by indirect immunofluorescence. The initial activated stage (1 week) was characterized by (i) incomplete epithelial organization and a weak presence of the suprabasal mucosa type keratin K4, (ii) diffuse expression of the integrin chains beta 1 and alpha 6 and (iii) abundance of the wound healing-associated integrin alpha v throughout the whole epithelium. After 2 weeks, the increase in epithelial organization was characterized by (i) the presence of a basal and suprabasal cell compartment, (ii) extension of K4 in the suprabasal compartment, (iii) extended expression of the keratinocyte integrins beta 1 and alpha 6 and (iv) concentration of alpha v integrin underneath basal cells. Further normalization of tissue architecture was indicated by (i) a slight increase in K4 extension, (ii) appearance of keratinocyte integrins beta 1 and alpha 6 in basal and parabasal cells and (iii) interruption of the band-like alpha v integrin immunolocalization at the subepithelial site. The findings in the in vitro model system indicate that these oral mucosa equivalents exhibit similarities to the in vivo situation of non-cornified gingiva, thus rendering them a suitable model for the assessment of stages during epithelial reconstruction or in vivo relevant studies on material effects.     

Possible involvement of crosstalk cell-adhesion mechanism by endometrial CD26/dipeptidyl peptidase IV and embryonal fibronectin in human blastocyst implantation

When human blastocysts hatch through the zona pellucida, gaining the ability to adhere to the endometrium, crosstalk between the embryo and the uterus may represent a successful outcome of their synchronized development and differentiation. CD26/dipeptidyl peptidase IV is known as a marker molecule of the implantation phase endometrium. To study the role of CD26 in implantation, 35 human hatched blastocysts were prepared by enzymatic treatment of expanded blastocysts that had been grown on schedule from frozen-thawed surplus embryos at the 2- or 4-cell stage. The blastocysts were placed on CD26-overexpressing or mock-transfected control monolayer cell cultures. The CD26-overexpression caused significantly higher blastocyst adhesion rate (53.3% versus 25.0%, P < 0.05) and significantly larger outgrowth area of trophectoderm (1.7-fold, P < 0.05). The second part of the present study was to show the expression of fibronectin, a CD26 ligand, in human preimplantation embryos, using the same donated resources. Fibronectin mRNA was detected by RT-PCR from the single hatched blastocyst (2/2) and from the single early blastocyst (3/6) but not from the single morula (0/5) samples. An indirect immunofluorescence technique verified the localization of fibronectin on the surface of the blastocyst. These results indicate that the adhesion mechanism by endometrial CD26 and embryonal fibronectin may be involved in human blastocyst implantation.     

Clomiphene citrate does not affect the secretion of alpha3, alphaV and beta1 integrin molecules during the implantation window in patients with unexplained infertility.

BACKGROUND: The expression of integrin molecules on the endometrium suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation. A prospective, controlled study was carried out to investigate the effect of clomiphene citrate (CC) on secretions of beta1, alpha3 and alphaV integrin molecules in the endometrium of patients with unexplained infertility during the implantation window. METHODS: A total of 40 endometrial samples was evaluated in both spontaneous (n = 13) and ensuing clomiphene-treated cycles (100 mg on days 5-9) and also from fertile women serving as controls (n = 14) during postovulatory 7th or 8th day of menstrual cycle. A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities. Endometrial thickness and serum oestradiol and progesterone concentrations were also measured on the day of sampling. RESULTS: Staining of alpha(v) but not beta1 and alpha3 integrins was significantly less intense in infertile cases than fertile control cases (1.42 +/- 0.12 versus 2.21 +/- 0.13 respectively, P = 0.012) and this was not restored to normal concentrations with treatment. CONCLUSIONS: Our study indicated that cc treatment significantly decreased the endometrial thickness and increased oestradiol and progesterone concentrations. However, secretion of alpha(v), beta1 and alpha3 integrin molecules, which might play a role in implantation, was not affected.     

Distribution of integrin cell adhesion molecules in endometrial cancer.

Integrins are ubiquitous cell adhesion molecules that are involved in maintaining normal tissue morphology and have been implicated in the behavior of certain malignancies. We examined the expression of nine integrin subunits in 38 endometrial adenocarcinomas using immunohistochemistry. The pattern of integrin expression in the cancers was compared with that seen in the endometrium of 20 normal cycling women and 7 postmenopausal women. Integrin expression was correlated with grade, stage, nodal status, depth of invasion, steroid receptor status, and histological pattern. In endometrial cancers there was an inverse relationship between the number of integrins expressed and histological grade (P = 0.011). Of the normally expressed, constitutive endometrial epithelial integrin subunits (alpha 2, alpha 3, alpha 6, and beta 4), the least frequently seen in the cancers was the alpha 3 subunit (44.7%) and the most frequently found was alpha 6 (81.6%). The alpha 5 beta 1 integrin, a fibronectin receptor normally found only on endometrial stromal cells, was seen in 17.8% of cases of these epithelial cancers. In addition, a significant association was found between the loss of the alpha 2 beta 1 integrin and the presence of lymph node metastases (P < 0.001). These data suggest that a decline in integrin expression occurs more frequently in poorly differentiated endometrial cancers and that the loss of specific integrins may be associated with metastatic nodal spread.     

Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle.

A heterogeneous group of cells interact with each other and with the surrounding matrix to form the complex structure of human endometrium. Since the integrin superfamily of molecules is involved in the cell-cell and cell-matrix interactions, this study was designed to screen, in situ, the cellular distribution of CDW49a-f molecules in human endometrium throughout the menstrual cycle. The integrin molecules were localized by immunohistochemistry using monoclonal antibodies. Glandular epithelium expressed all integrin molecules. With the exception of CDW49d (alpha 4 beta 1), surface epithelium also expressed all these molecules. Endothelial cells were positive for all integrin molecules except CDW49a (alpha 1 beta 1). Endometrial lymphoid cells were positively immunostained for CDW49a, d and e (alpha 5 beta 1) and were negative for CDW49b (alpha 2 beta 1), CDW49c (alpha 3 beta 1) and CDW49f (alpha 6 beta 1). Regional differences in the expression of integrin molecules were observed. As compared to the functionalis epithelium, basalis epithelium characteristically exhibited higher expression of CDW49a, d and e. Two integrins in endometrium, CDW49a and d exhibited changes related to the menstrual cycle. CDW49a, which was not expressed in glandular epithelial cells in the proliferative phase, was strongly expressed in these cells after ovulation and its expression was diminished in the late secretory phase. This molecule was not expressed in the stromal cells, however, predecidual cells characteristically expressed this molecule in the late secretory phase. CDW49d was only expressed in the glandular epithelial cells in the mid-proliferative to mid-secretory phases.(ABSTRACT TRUNCATED AT 250 WORDS)