Human sperm function in co-culture with human, macaque or bovine oviduct epithelial cell monolayers

Human sperm function was compared in co-culture with monolayersof oviduct epithelial cells (OEC) from three species, human,macaque and bovine. For all species, freeze–thawed andpassaged OEC from females in the periovulatory phase were used.OEC cultured on an extracellular matrix (Matrigel) formed amonolayer which supported human sperm attachment to OEC fromall three species. Spermatozoa in co-culture with OEC from allthree species showed prolonged survival and improved motilitycharacteristics over those cultured in medium alone. This paperdescribes an efficient, repeatable co-culture system for humanspermatozoa which supports sperm attachment to OEC and subsequentlyimproves sperm function over that seen in control medium cultures.Because the improved sperm function in co-culture did not differsignificantly between human and bovine OEC for those attributesstudied, it is proposed that bovine OEC could be used as analternative to human OEC in certain human sperm co-culture studies.Follicular phase bovine OEC from reproductively normal donorsare far more accessible than their human counterparts, thusmaking this co-culture system more widely available for thestudy of human spermatozoa–female tract interactions.     

8株人黑色素瘤细胞系的建立及其生物学特性研究

目的：从人黑色素瘤组织建立一系列黑色素瘤细胞系，命名为黑色素瘤细胞系1-8号（HME1-8），并研究鉴定其体外细胞生物学与免疫学特性。方法：取黑色素瘤组织进行组织块培养法，待长满90%汇合面后消化传代培养，并进行形态学、生长动力学、免疫学及致瘤性等生物学特性研究。结果：该系列黑色素瘤细胞系已在体外培养生存1-2年，传60-100余代，其生物学特性显示：①这些细胞系体外生长的倍增时间为34.2-59.5h；②软琼脂集落形成率平均值在8.6%-26.2%之间；③接种至裸鼠后具有较高的致瘤性；④染色体核型分析为非整倍体，众数为64-117条；⑤ 透射电镜下可观察到细胞含丰富的核糖体及黑色素颗粒；⑥免疫组化分析显示细胞浆内含大量阳性棕色黑色素颗粒；⑦肿瘤抗原检测显示8株黑色素瘤细胞系的Melan-A抗原阳性率为75%, MAGE-1阳性率为50%, MAGE-2阳性率为37.5%，MAGE-3阳性率为62.5%。结论：8株黑色素瘤细胞系经体外长期培养后已永生化，形成了连续传代细胞系，具有明显的黑色素瘤细胞恶性表型特征。     

In vitro characteristics of two established hepatoma cell lines

The in vitro behaviours of a fibroblast-like (DENA-RH 13) and an epithelial-like (DENA-RH 13A) cell line, derived from a chemically induced hepatocarcinoma of rat are described. The DENA-RH 13A can be maintained and studied either in monolayer as a transplantable solid tumor or in fluid-suspension culture as ascites tumor in Wistar rats. The cytomorphology and growth pattern of the clones of the cell lines are described. While DENA-RH 13 cells produced undifferentiated tumors with mainly sarcoma-like structures in allogeneic hosts the epithelial-like cell line (DENA-RH 13A) grew in a carcinoma-like pattern in animals.     

A case of endometrial stromal tumor with epithelial elements

A rare endometrial stromal tumor with marked epithelial differentiation was found in a 56-year-old female. This polypoid tumor, occupying the uterine cavity, was composed of an admixture of endometrial stromal and glandular components. Ultrastructurally and immunohistochemically, it is suggested that these epithelial elements were derived from epithelial differentiation of the endometrial stromal cells. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

T淋巴细胞侵袭转移诱导蛋白1在小鼠子宫内膜基质细胞-胚泡共培养体系中对胚泡黏附的影响

目的:检测不同浓度T淋巴细胞侵袭转移诱导蛋白1 (Tiaml)抗体下,小鼠着床窗子宫内膜基质细胞内的Tiaml蛋白表达及其对基质细胞-胚泡共培养体系中胚泡黏附的影响,探讨Tiaml对小鼠胚胎着床的影响.方法:提取着床窗小鼠子宫内膜基质细胞并构建基质细胞-胚泡共培养体系;分别用免疫细胞化学和免疫印迹法检测不同浓度抗Tiaml抗体时着床窗基质细胞Tiaml蛋白的表达及其对胚泡黏附的影响.结果:抗体干预后着床窗基质细胞内的Tiaml蛋白表达水平降低;在不同抗体浓度下胚泡黏附率有差异.结论:随Tiaml抗体浓度增加,着床窗小鼠子宫内膜基质细胞Tiaml蛋白表达量及胚泡黏附率降低.表明Tiarnl可能在胚泡植入过程起重要的作用.     

Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model

The regulation of epithelial cell function and morphogenesis by the paracrine effectors from the mesenchyme or stroma has been well established using in-vivo studies. A more complete understanding of these relationships has been delayed due, in part, to a lack of appropriate co-culture models. In this study, we describe a co-culture model which demonstrates that normal paracrine relationships can be reconstituted in vitro and that human endometrial stromal cells regulate both growth and differentiation of primary human endometrial epithelial cells. Interesting differences in the proliferation of stromal and epithelial cells were noted in response to the basement membrane extract, Matrigel((R)). Exposure of stromal cells to Matrigel((R)) enhanced the paracrine capacity of these cells in vitro. When epithelial cells were co-cultured in contact with stromal cells embedded in Matrigel((R)), epithelial cell growth was inhibited by 65-80% compared to controls. Stromal cells in contact with Matrigel((R)) also regulated epithelial cell differentiation, as shown by induction of glycodelin expression. These co-culture studies show great promise as a method to investigate the cellular interactions between endometrial stromal and epithelial cells and their environment and to understand the molecular basis for the regulation of normal growth and differentiation of cells within complex tissues such as the endometrium.     

Expression of macrophage inflammatory protein-3alpha in an endometrial epithelial cell line,HHUA, and cultured human endometrial stromal cells

It has been demonstrated that human endometrial epithelial cells (EEC) and stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of macrophage inflammatory protein (MIP)-3alpha in endometrial cells, the production of MIP-3alpha by an EEC line, HHUA, and cultured ESC stimulated with various inflammatory mediators was evaluated by ELISA. Unstimulated HHUA and ESC constitutively secreted MIP-3alpha. Tumour necrosis factor-alpha and interleukin-1beta significantly stimulated the secretion of MIP-3alpha by HHUA and ESC. Lipopolysaccharide also stimulated the secretion of MIP-3alpha by ESC, but not by HHUA. These results show that the concentration of MIP-3alpha in the endometrium is modulated by these inflammatory mediators. MIP-3alpha may contribute to the normal and pathological processes of human reproduction by regulating the trafficking of immature dendritic cells and memory T lymphocytes into the endometrium.     

Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells

It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-alpha, and interleukin-1beta significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.     

八株小鼠胸腺基质细胞系产生白细胞介素1及白细胞介素6的水平

应用IL-1诱导LBRM33-IA5细胞产生IL-2、及支持IL-6依赖细胞系KD-83细胞的增殖,分别测定了本室所建8株小鼠胸腺基质细胞系自发分泌IL-1及IL-6的能力.8株MTSC 分泌IL-1水平不同,>100U/ml者,1株;30～40U/ml 者,2株.8株细胞均能产生IL-6,其中7株的分泌水平>80U/ml;1株为38U/ml.比较各株分泌IL-1及IL-6的水平,本文显示,在MTSC 各系中,尚存在IL-1非依赖性IL-6分泌途径.本文尚比较了两种测定IL-1活性的方法,支持胸腺细胞增殖法测得者,实为IL-1+IL-6的效应,不能反映IL-1的实际水平.     

Immunologic effects of human thymic stromal grafts and cell lines

These experiments report a method of preparing stromal remnants from human thymus. The remnants were composed of epithelial cells, fibroblasts, and macrophages. They were grafted under the renal capsule of nude mice. Some of the grafts were reconstituted with lymphocytes to obtain the microscopic morphology of the thymus. The mice with reconstituted grafts survived in a conventional environment, had increased numbers of T cells in their spleen, and showed improvement of T-cell mediated immunologic function. This was measured by a positive allogeneic effect, a mixed lymphocyte reaction, and cell-mediated lysis. In vitro cell lines were established from thymic remnants of two separate individuals. These lines grew as attached monolayers. One of them was composed of fibroblasts. The other had an epithelial morphology. This epithelial cell line (HT-7) was shown to produce factors which promoted thymocyte differentiation in vitro.     

Molecular characteristics of eight gastric cancer cell lines established in Japan

Molecular characterization of eight gastric cancer cell lines established in Japan are summarized according to the genetic and epigenetic alterations and growth factor status. TMK-1 poorly differentiated adenocarcinoma cell line harbors mutant p53 tumor suppressor gene and rearrangement of p15MTS2. MKN-1 adenosquamous carcinoma line with mutant p53 reveals silencing of E-cadherin by promoter CpG hypermethylation. MKN-7 well-differentiated adenocarcinoma cell line has amplification of c-erbB2 oncogene and cyclin E gene. MKN-28 well-differentiated adenocarcinoma cell line reveals mutations in p53 and APC tumor suppressor genes and silencing of CD44. The MKN-45 poorly differentiated adenocarcinoma cell line with wild-type p53 is characterized by homozygous deletion of p16CDKN2/MTS1/INK4A and p15MTS2, amplification of c-met oncogene and promoter mutation of E-cadherin. MKN-74 derived from moderately differentiated tubular adenocarcinoma has wild-type p53. KATO-III signet ring cell carcinoma line has genomic deletion of p53, amplification of K-sam and c-met oncogene and mutation of E-cadherin. HSC-39 signet ring cell carcinoma cell line harboring p53 missense mutation has homozygous deletion of p16CDKN2/MTS1/INK4A and p15MTS2, amplifications of c-myc, c-met, K-sam and CD44 gene and mutation in beta-catenin gene.     

喉上皮细胞条件液诱导胚胎干细胞分化为上皮细胞的实验研究

目的:探讨体外用喉上皮细胞条件培养液诱导胚胎干细胞分化为上皮细胞的方法及诱导条件。方法:将E14小鼠胚胎干细胞体外培养形成拟胚体后，在人喉上皮细胞条件培养液及因子诱导条件下共培养 14 d，形态学观察并采用免疫细胞化学方法检测上皮细胞的角蛋白Ck 4、14、19蛋白质水平的表达情况。结果:在诱导14 d的培养系统中较对照细胞可见更多上皮样细胞出现，免疫细胞化学染色显示上皮样细胞胞浆中可见角蛋白Ck 4、14、19均有表达。结论:胚胎干细胞在喉上皮细胞条件培养液诱导条件下可形成上皮样细胞，为组织工程制作人工喉上皮材料奠定了实验基础。     

Absence of glucose decreases human fertilization and sperm movement characteristics in vitro

The effect of glucose in a modified Ham's F10 medium (MM) without hypoxanthine, phosphate and transition metals on human fertilization and sperm survival in vitro was determined. Mature human oocytes from in-vitro fertilization (IVF) patients or Percoll-washed human spermatozoa were randomly allocated to one of the treatment groups: normal Ham's F10, MM, MM with 5 mM glucose (HGMM) and MM with 0.5 mM glucose (LGMM). Oocytes were inseminated in one of the four media for 12-20 h and checked for fertilization. Sperm were incubated likewise for 4 and 24 h, and sperm motility and sperm movement characteristics including average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), straightness (STR), and linearity (LIN) were determined using computer-assisted semen analysis. Fertilization rates were significantly lower in oocytes cultured in MM (23.8%) compared to LGMM (75.5%), HGMM (73.6%) or Ham's F10 (71.1%). Sperm characteristics after 4 h incubation in all four media were similar, except VAP, VSL, VCL and ALH were significantly lower in MM (no glucose) in comparison with the other three media. After 24 h VAP, VSL, VCL, ALH, LIN and percentage rapid spermatozoa were significantly higher in spermatozoa incubated in HGMM or Ham's F10 compared with MM or LGMM. Also after 24 h, the percentage of spermatozoa which were highly motile was greater in HGMM than in Ham's F10. Absence of glucose significantly lowered fertilization rates and sperm movement characteristics in vitro.      

Immunohistochemical studies on the phenotype of murine and human thymic stromal cell lines

Four different murine and human thymic stromal cell (TSC) lines were analyzed by means of indirect immunofluorescence for the presence of tissue specific intermediate filament proteins, extracellular matrix products and thymic hormone. Among these cell lines, only one (IT-45R1) was shown to be epithelial in nature, as revealed by its immunoreactivity with antikeratin antibodies. In addition, IT-45R1 cells (but not the other cell lines) produced thymulin--a well defined thymic hormone--that was detected intracellularly and in the culture medium. Concerning extracellular matrix elements, we noted that all TSC lines were able to produce basement membrane proteins as type IV collagen and fibronectin. Type I collagen however was synthesized by TEPI, HT-5 and HT-7 cells but not by IT-45R1 cells. Our data demonstrate that only the IT-45R1 TSC line is epithelial and active in thymic hormone production. The other three TSC lines are not epithelial in nature, but their definitive phenotypes remain to be determined.     

Karyotype consistency in human colorectal carcinoma cell lines established in vitro

Karyotypes of nine human colorectal cell lines deposited with the ATCC were studied by trypsin-Giemsa banding. CCL 229,230,231 and 235 (Modal chromosome number, Sm, was 49, 68, 47, and 40, respectively) belong in the stable type that is characterized by karyotypes consisting mostly of normal chromosomes and stable markers. CCL 228, 234, and 238 (Sm = 55,79, and 70 respectively) belong in the unstable type that has karyotypes consisting of numerous markers in addition to normal chromosomes and stable markers. The remaining intermediate type (CCL 233 and 237, Sm = 60 and 64, respectively) has karyotypic characteristics between the above two types usually with two or less unstable markers per cell. The stable markers (together with normal chromosomes) are constitutive components of a cell genome and are common to most cells within the same cultured population. Unstable markers, which generally constitute only a small portion of the total chromosome complement are the likely cause of karyotypic variations between cells and often are produced by balanced inter- or intrachromosome changes, or both. Consequently, total chromosome length per cell genome is remarkably consistent within a cell population, and karyotypes between cells, such as from four stable lines, are profoundly stable and mostly identical.Chromosome deletions and interhomologue exchanges (including isochromosomes) had the highest incidences among both stable and unstable markers. The complex markers occurred relatively infrequently. There were neither common markers nor unique chromosome breakages common to all of these established cell lines. However, chromosomes No. 7 and 1 had the highest incidence (15 and 12, respectively) of structural modifications resulting in the formation of stable markers (82 total exchanges in nine cell lines), and chromosomes No. 7 and 2 were involved at high incidence (21 and 15, respectively) in the formation of both stable and unstable markers (181 total exchanges). Moreover, No. 7 is overrepresented in eight of nine lines. The significance of chromosome changes involving No. 7 in this as well as other tumor pathotypes is briefly discussed.     

Telomerase activity is found in the epithelial cells but not in the stromal cells in human endometrial cell culture

Telomerase activity is associated with the proliferative activity of cells. In the endometrium, telomerase activity is higher in the proliferative phase than in the secretory phase of the menstrual cycle, suggesting that telomerase activity may occur primarily in the glandular epithelial cells. To test this, a dissociated cell culture of the endometrium was performed, and the telomerase activity in each cell fraction was analysed. Telomerase activity was found in all 10 endometrial tissues of the proliferative phase of the menstrual cycle. Both the fragments of epithelial glands and single cells, which were prepared by enzymatic dissociation, showed telomerase activity. In the 7 day cell culture, it was found in nine out of 10 epithelial cell enriched fractions, but in none of the stromal cell enriched fractions. Flow cytometric analysis showed that the epithelial enriched fraction was contaminated with a predominant number of stromal cells, while the stromal cell enriched fraction was comprised mostly of stromal cells with apparent proliferative activity. Our results suggest that telomerase activity of the endometrium occurs primarily in the epithelial cells in the endometrium and that the stromal cells do not express telomerase activity regardless of their potent proliferative activity.      

Type II collagen-specific human T cell lines established from healthy donors

Four type II collagen-specific T cell lines established from the peripheral blood of a healthy donor were studied in detail. These CD4+ T cell lines with an alpha/beta T cell receptor proliferated in response to native and denatured human and chicken type II collagen and human type I collagen, but not to human type IV collagen or other potentially arthritogenic antigens. The T cell response showed typical dose response characteristics, peaked between 30 and 48 h, was major histocompatibility complex class II restricted and not due to a mitogenic effect. Type II collagen-reactive T cells could hardly be detected in freshly isolated peripheral blood mononuclear cells from healthy donors, as revealed by limiting dilution analysis (frequency less than 1/100,000). By prestimulation in bulk cultures for 10 days, type II collagen-reactive T cells could be approximately 1000-fold enriched. However, in these limiting dilution cultures, collagen-reactive T cells could only be observed in a narrow window of cell concentrations, suggesting that type II collagen-reactive T cells may be controlled by suppressive mechanisms in healthy persons.