膜联蛋白A8在小鼠早期妊娠子宫中表达研究

膜联蛋白A8(Annexin A8,ANXA8)是一种磷脂结合蛋白,与炎症反应、癌症的发生以及血管生成有密切联系。本实验旨在利用实时荧光定量PCR、原位杂交与免疫组织化学的方法研究ANXA8 mRNA与蛋白在小鼠早期妊娠和人工蜕膜子宫中的表达。原位杂交结果表明:ANXA8 mRNA在小鼠早期妊娠第1～4天子宫腔上皮和腺上皮有微弱表达,ANXA8 mRNA在妊娠第5、6天的初级蜕膜区与第7、8天的次级蜕膜区表达,并随妊娠进行逐渐增强;人工蜕膜化模型中ANXA8 mRNA表达在蜕膜区。实时荧光定量PCR证明:ANXA8 mRNA的表达量在早期妊娠模型中的第7、8天显著提高,人工蜕膜侧子宫与对照侧相比也显著提高。免疫组织化学结果表明:ANXA8蛋白与ANXA8 mRNA表达规律相似。体外分离培养小鼠子宫基质细胞,并诱导蜕膜化,实时荧光定量PCR结果表明ANXA8随着基质细胞的蜕膜化表达升高。以上体内和体外实验表明,ANXA8在小鼠子宫中的表达具有着床相关特异性,ANXA8参与小鼠子宫蜕膜化过程。     

Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells

We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells.     

N-乙酰半胱氨酸对山羊子宫内膜基质细胞增殖及相关基因表达的影响

旨在探究N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)对山羊子宫内膜基质细胞(goat endometrial stro-mal cells,gESCs)的影响.本研究以山羊子宫内膜基质细胞为对象,体外培养基中添加浓度梯度分别为100、200、400μmol·L-1的NAC,将0μmol·L1 NA...     

Histopathological and Immunohistochemical Characterization of Spontaneously Occurring Uterine Deciduomas in Young Adult Rats

Uterine deciduomas were found in two female virgin rats, a 15-week-old Lewis rat and a 7-week-old Sprague-Dawley rat. The firm white nodules were located at the base of unilateral uterine horns and were approximately 6 mm and 4 mm in diameter. Histopathologically, the nodules were composed of three areas, each with a distinct type of proliferating cells: large epithelioid decidual cells with round nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (antimesometrial region); compact spindle-shaped cells with oval nuclei and vacuolar cytoplasm (transitional region); and pleomorphic and spiny cells with round to oval nuclei and compact eosinophilic cytoplasm (mesometrial region). These cells proliferated in sheet-like arrangements and transformed into the other types of cells located in surrounding regions. Immunohistochemically, proliferating cells in all regions were strongly positive for proliferating cell nuclear antigen. The proliferating cells were positive for vimentin, and large decidual cells were positive for common acute lymphoblastic leukemia antigen 10, a marker of uterine interstitial cells. Large decidual cells were positive for α-smooth muscle actin and desmin, suggesting differentiation into muscular cells. Progesterone receptor was expressed in all cell types; however, estrogen receptor α was not expressed in the antimesometrial region. These extremely rare tumor-like nodules represent nonneoplastic lesions referred as decidual reactions of endometrial interstitial cells, and their biological behavior is that of a space-occupying benign tumor in young rats. Our cases might provide information as a historical control in toxicity and pharmacological studies in rats.     

分离培养兔子宫内膜细胞的鉴定及性激素对子宫内膜间质细胞形态的影响

为探讨子宫内膜细胞的形态学特点、分泌功能及性激素在子宫内膜细胞中的作用，采用不同的消化分离方法进行了孕早期家兔子宫内膜细胞的消化和分离培养试验。结果显示：子宫内膜主要包括两类细胞，基质细胞和上皮细胞，两类细胞分别在波形蛋白和细胞角蛋白免疫细胞化学染色中有阳性反应特点；在培养基中加入雌激素和／或孕酮刺激后，基质细胞形态发生变化，表现一定的蜕膜化现象。     

Quantification of insulin-like growth factor binding protein mRNA using real-time PCR in bovine granulosa and theca cells: effect of estradiol, insulin, and gonadotropins

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1-5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.     

脂多糖对胚胎附植期Toll样受体4及相关免疫因子的影响

试验旨在探讨脂多糖（LPS）对绵羊胚胎附植期Toll样受体4（TLR4）及相关免疫因子表达的影响。以构建好的pcDNA3.1-TLR4过表达载体转染绵羊子宫内膜基质细胞,运用Western blotting技术鉴定细胞转染效果,然后用1 μg/L LPS刺激转染后的子宫内膜基质细胞,建立内膜基质细胞炎症模型。将经LPS处理的细胞分别培养12、24、48、72 h,采用实时荧光定量PCR技术和Western blotting法检测内膜基质细胞中TLR4 mRNA及蛋白表达量,以及免疫因子IL-1β和IL-6的mRNA表达水平,并以未经LPS处理的细胞作为对照。结果显示,与对照组相比,LPS促进了免疫因子IL-1β、IL-6的释放量,但随LPS作用时间的延长,细胞中IL-6的表达量逐渐下降,而IL-1β的表达量逐渐升高,使得Th1/Th2偏向不利于妊娠的Th1方向表达;TLR4 mRNA相对表达量在12、24、72 h均显著高于对照组（P<0.05）,48 h时极显著高于对照组（P<0.01）,且LPS处理后的细胞TLR4蛋白表达量也始终高于对照组。综上所述,pcDNA3.1-TLR4过表达载体成功转入绵羊子宫内膜基质细胞;LPS有效激活了子宫内膜细胞中TLR4信号通路,并促进了下游因子的表达;子宫蜕膜组织中TLR4受体蛋白对胚胎附植早期妊娠微环境的平衡维持也起到了重要作用。     

Low-density lipoprotein-related receptor protein 1 (LRP-1) is not required for insulin-like growth factor binding protein 3 (IGFBP-3) to suppress L6 myogenic cell proliferation

Insulin-like growth factor binding protein-3 (IGFBP-3) suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In nonmuscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein-1 (LRP-1)/activated α(2)M receptor is reportedly required for IGFBP-3 to inhibit proliferation. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. To investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, we have examined the effect of receptor-associated protein, an LRP-1 receptor antagonist, on recombinant porcine (rp)IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with receptor-associated protein results in a 37% decrease (P < 0.05) in the ability of rpIGFBP-3 to inhibit L6-cell proliferation. In L6 cells subjected to LRP-1 small interfering RNA treatment for 48 h (LRP-1 silenced), LRP-1 mRNA levels were reduced by greater than 80% compared with control cultures treated with nonsense small interfering RNA (mock silenced). In addition, the 85-kDa transmembrane subunit of LRP-1 was undetectable in Western immunoblots of total protein lysates from LRP-1-silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1-silenced L6 cells compared with mock-silenced controls, rpIGFPB-3 suppressed proliferation rate to the same extent in both LRP-1-silenced and mock-silenced cultures. Our results strongly suggest that, in contrast to data obtained for nonmuscle cell lines, the LRP-1 receptor is not required for IGFBP-3 to suppress proliferation of L6 myogenic cells.     

miR‐182 selectively targets HOXA10 in goat endometrial epithelium cells in vitro

Proper HOXA10 expression was essential for endometrial receptivity what was crucial for successful embryo implantation in mammalian. This study confirmed that miR‐182 regulated the expression levels of HOXA10 by binding to its 3′ UTR, selectively downregulated HOXA10 in goat endometrial epithelium cells (gEECs) but not stromal cell (gESCs) in vitro. However, HOXA10 and miR‐182 both up‐expressed in the goat endometrium at gestational day 15 (D15) compared with gestational day 5 (D5), suggesting that there were some other factors regulated the expression of HOXA10 during the development of goat endometrium in vivo. What's more, HOXA10 gene silencing (HOXA10‐siRNA) resulted in gEECs apoptosis in vitro, and it regulated the protein levels of oestrogen receptor a (ERa), progesterone receptor B (PRb), insulin‐like growth factor 1 receptor (IGF1R), BCL‐2, pleiotrophin (PTN), AKT and p‐JNK in gEECs. Furthermore, HOXA10 might regulate the protein levels of endometrial receptivity biomarker genes, including vascular endothelial growth factor (VEGF), osteopontin (OPN), cyclooxygenase‐2 (COX‐2) and prolactin receptor (PRLR) in gEECs. In conclusion, miR‐182 targeted HOXA10 selectively in EECs in vitro, and HOXA10 played an important role in maintaining the function of EECs in dairy goats.     

Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) have been shown to affect proliferation of several cell types via insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms. The goal of this study was to determine if levels of IGFBP-2, -3, -4 and -5 mRNA changed during differentiation of cultured porcine embryonic myogenic cells. Total RNA was isolated from muscle cultures at various stages of differentiation and Northern blots of this RNA were probed with 32P-labeled cDNA probes specific for individual IGFBPs. Fusion, myogenin mRNA, and creatine phosphokinase activity were used as markers of differentiation. The level of IGFBP-3 mRNA in differentiating cultures (120 h in culture) was only one-third of the level in myogenin negative, nonfused cultures (72 h in culture) (P < 0.05, n = 4). In contrast, the level of IGFBP-3 mRNA in extensively fused cultures (144 h in culture) was increased by three-fold as compared to the level in myogenin negative, nonfused cultures (P < 0.05, n = 4) and approximately seven-fold as compared to the 120-h cultures (P < 0.05, n = 4). No significant change in the level of IGFBP-5 mRNA was observed during differentiation of myogenic cultures. IGFBP-2 mRNA levels were not significantly different at 72, 96 and 120 h, but at 144 h IGFBP-2 mRNA level was increased three-fold as compared to nonfused cultures (72 h) (P < 0.05, n = 4). IGFBP-4 mRNA was not detectable on Northern blots of total RNA from porcine myogenic cultures at any stage of differentiation. Changes in IGFBP-3 and IGFBP-2 mRNA levels are associated with differentiation of embryonic porcine myogenic cells in culture and this may indicate that these IGFBPs play a role in differentiation of these cells.     

Characterization of a population of unique granular lymphocytes in a bitch deciduoma, using a panel of histo- and immunohistochemical markers

The ovaries and uterus were collected after ovariohysterectomy from a 16-month-old Labrador bitch in diestrus that never mated. Discrete swellings were found in the uterine horns, with the macroscopic appearance of normal early pregnancy. At histologic examination, the endometrium, devoid of any conceptus and chorion, showed a marked proliferation, on the basis of which a diagnosis of deciduoma was made. A remarkable population of stromal eosinophilic granular lymphocytes was present, especially in the axis of the endometrial folds. Periodic acid-Schiff and Dolichos biflorus-lectin histochemical reaction and a panel of 10 immunohistochemical markers were used to characterize eosinophilic granular cells. Our findings allowed us to compare these granular cells with the granulated decidual cells, whose presence was until now described only in primates, rodents, or a few other epitheliochorial species. On the basis of our results, the importance of eosinophilic granular cells in a decidualization process is hypothesized to occur also in the bitch.     

Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Expression of Glucocorticoid Receptor α and Its Regulation in the Bovine Endometrium: Possible Role in Cyclic Prostaglandin F2α Production

Cortisol (Cr), the most important glucocorticoid (GC), is well known to suppress uterine prostaglandin F2α (PGF) production. However, the details of the regulatory mechanisms controlling the cyclic changes in endometrial PGF production remain unclear. Here we investigated the expression of the GC receptor (GC-Rα), the actions of cortisol throughout the estrous cycle and the regulatory mechanism of GC-Rα in the bovine endometrium. The levels of GC-Rα protein were greater at the mid-luteal stage (Days 8–12) than at the other stages. Cr more strongly suppressed PGF production at the mid-luteal stage than at the follicular stage. GC-Rα expression was increased by progesterone (P4) but decreased by estradiol-17β (E2) in cultured endometrial stromal cells. The overall results suggest that ovarian steroid hormones control the cyclic changes in endometrial PGF production by regulating GC-Rα expression in bovine endometrial stromal cells.     

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.     

Thirteen-weeks subcutaneous treatment with oestradiol or an oestradiol/progesterone combination in beagle bitches

High doses of oestradiol (150 micrograms/kg) or an oestradiol/progesterone combination (150 micrograms/kg oestradiol plus 6.25 mg/kg progesterone) were administered subcutaneously on alternate days to immature ovariectomised and mature intact female Beagle dogs for 13 weeks. The effects of hormonal treatment on different parameters were studied. The results revealed severe anaemia (mainly at week 3) and the blood films showed morphological indication of blood loss and abnormal erythrogenesis. Neutrophil leucocytosis and non-progressive thrombocytopenia were also observed. Treatment with oestradiol alone induced changes in the endometrial stroma and myometrium, whereas treatment with the oestradiol/progesterone combination induced cystic endometrial hyperplasia. The pituitary glands of the dogs treated with oestradiol alone had a slight increase in the number of ACTH cells. These cells and STH cells showed cytological changes indicative of accelerated secretory activity; the PRL and gonadotropin-producing cells were not affected significantly. The oestradiol/progesterone combination increased the number of ACTH and PRL cells. These cells as well as STH cells exhibited several cytological criteria typical of a pituitary cell with accelerated activity; the gondadotropin-producing cells showed involutionary changes. The mammary glands of oestradiol-treated dogs showed stromal and ductal cell proliferation; whereas the oestradiol/progesterone combination induced secretory epithelial cell proliferation in addition to stromal and ductal cell proliferation.     

A local increase in the mammary IGF-1: IGFBP-3 ratio mediates the mammogenic effects of estrogen and growth hormone

A single epithelium-free mammary fat pad was surgically prepared in each of twenty-five one-month-old, Friesian heifers. At 18 mo of age, heifers were randomly assigned to one of four treatment groups. Treatments were: control (C), growth hormone (GH), estrogen (E) or growth hormone + estrogen (GE). Hormones were administered for 40 hr before the animals were sacrificed to provide mammary samples of parenchyma (PAR), intact fat pad (MFP), and epithelium-free or "cleared" fat pad (CFP). IGF-1 and IGF binding protein-3 (IGFBP-3) mRNA was highest in CFP and MFP whereas the protein products were highest in PAR. IGFBP-2, a 28-kDa IGFBP and a 24-kDa IGFBP were more abundant in CFP and MFP. E and GH increased incorporation of [(3)H]thymidine into DNA of PAR. Incorporation of [(3)H]thymidine into the DNA of MFP or CFP was minimal. Coincident with the changes observed in mammary epithelial proliferation, E increased IGF-1 protein in MFP and PAR, and to a lesser extent in CFP. E tended to increase IGF-1 mRNA levels in MFP, but not CFP implying that the regulation of IGF-1 expression is modulated by adjacent epithelium. GH and E reduced IGFBP-3 protein in PAR and increased the 24-kDa IGFBP in CFP and MFP. Increased proliferation of mammary parenchymal cells was associated with increased IGF-1 and reduced IGFBP-3 protein in mammary tissue. An increase in the ratio of mammary IGF-1: IGFBP-3 likely increases the proportion of the mammary IGF-1 available to stimulate proliferation. These data also indicate that stromal: epithelial interactions regulate the IGF-1 axis in mammary tissue.     

过表达MyBPC1对牛骨骼肌卫星细胞增殖分化的影响

旨在探究肌球蛋白结合蛋白C1（myosin binding protein C1,MyBPC1）对牛骨骼肌卫星细胞增殖与成肌分化的影响,为进一步研究MyBPC1在细胞分化和肌肉发育过程中的调控作用提供依据。本研究利用西门塔尔胎牛原代牛骨骼肌卫星细胞体外诱导成肌分化模型模拟牛骨骼肌的生长发育过程。采用qRT-PCR和Western blot检测MyBPC1的细胞时序表达谱。试验分为两组。在RNA水平每组4个重复,每个重复20 μL;在蛋白水平每组3个重复,每个重复15 μg。采用qRT-PCR和Western blot检测牛骨骼肌卫星细胞转染MyBPC1的过表达效果,并进一步检测细胞增殖期标志因子Pax7、Ki67以及细胞分化期标志因子MyHC、MyOG的表达变化情况,观察牛骨骼肌卫星细胞肌管形成状态。结果,MyBPC1在牛骨骼肌卫星细胞分化前后表达水平存在极显著差异,牛骨骼肌卫星细胞诱导分化后MyBPC1的mRNA和蛋白表达量均极显著高于增殖期（P<0.01）。过表达MyBPC1后,细胞分化形成的肌管数量明显多于对照组,增殖标志因子Pax7的mRNA水平和蛋白表达水平无显著差异,分化标志因子MyHC的mRNA水平和蛋白表达水平极显著高于对照组（P<0.01）。过表达MyBPC1可以促进牛骨骼肌卫星细胞体外成肌分化,为进一步开展MyBPC1对牛骨骼肌卫星细胞的调控机制奠定基础。     

Insulin-like growth factor-I stimulated DNA replication in mouse endometrial stromal cells

Much evidence has suggested that sex steroid hormone-induced growth of uterine cells is mediated by polypeptide growth factors synthesized in uterine tissues. The present study aimed to clarify the effect of insulin-like growth factor-I (IGF-I) on the proliferation of mouse endometrial stromal cells obtained from immature mice. IGF-I and IGF-I receptor (type I) mRNAs were detected in the endometrial stromal cells. IGF-I increased bromodeoxyuridine (BrdU) uptake in the endometrial stromal cells, indicating an increase in DNA replication. E2 increased IGF-I mRNA levels in the endometrial stromal cells. IGF-I receptor is a tyrosine kinase receptor, and treatment with genistein, a tyrosine kinase inhibitor, reduced IGF-I-induced BrdU-uptake in the endometrial stromal cells. IGF-I signaling pathways involve mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI-3 kinase). Treatment with 10(-7) M of the MAP kinase inhibitor PD098059 and 10(-5) M of the PI-3 kinase inhibitor LY294002 decreased IGF-I-induced BrdU-uptake in the endometrial stromal cells. However, LY294002 (10(-5) M) also decreased the BrdU-uptake in the absence of IGF-I treatment. These results suggest that endometrial IGF-I is involved in the proliferation of endometrial stromal cells in a paracrine or autocrine manner, and that the MAP kinase pathway is involved in DNA replication of endometrial stromal cells.     

干扰HNRNPAB对牛骨骼肌卫星细胞增殖与分化的作用研究

为了研究核不均一核糖核蛋白AB(heterogeneous nuclear ribonucleoprotein AB, HNRNPAB)对牛骨骼肌卫星细胞增殖与分化的影响。本研究以体外分离培养的鲁西黄牛胎牛原代骨骼肌卫星细胞为试验材料,体外诱导成肌分化,分别收取分化前和分化后第1、2、3天的细胞,提取RNA(每组设置4个重复)或蛋白质(每组设置3个重复),通过qRT-PCR、Western blot检测HNRNPAB在成肌分化前后的表达情况。随后合成HNRNPAB的干扰RNA,并转染牛骨骼肌卫星细胞干扰HNRNPAB的表达,设置阴性对照组;在试验组和对照组中,分别利用EdU试验检测细胞增殖情况,qRT-PCR技术、Western blot技术检测HNRNPAB、增殖标志因子Pax7、Cyclin D1及分化标志因子MyoG、MyHC的mRNA表达水平及蛋白表达水平。结果显示,HNRNPAB在牛骨骼肌卫星细胞成肌分化过程中的mRNA表达呈现先升后降的趋势,在分化第1天表达量最高,分化前后蛋白表达水平也具有显著差异。干扰HNRNPAB后,EdU细胞阳性率极显著上升(P<0.01),增...