Granulosa cells (GCs) are essential for proper oocyte, follicular development, and steroidogenesis in the ovary. Transforming growth factor beta (TGF-beta) superfamily members are critical in regulating GCs growth and differentiation. Smad3 is known to serve as a signaling intermediate for the TGF-beta; however, the functions of Smad3 in the human GCs remain unidentified. In this study, the luteinized GCs collected from follicular aspirates from patients undergoing in vitro fertilization were cultured and engineered to overexpress and knockdown Smad3, which were validated by RT-PCR and Western blotting. Immunocytochemistry showed that Smad3 protein was strongly expressed in human ovarian luteinized GCs. EdU incorporation demonstrated that Smad3 promoted the proliferation of GCs, and the expression of PCNA was also enhanced by Smad3. ELISA analysis indicated that the secretion of both estradiol and progesterone was stimulated by Smad3. In addition, Smad3 upregulated the level of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and protein kinase A (PKA) proteins. We subsequently added special PKA inhibitor H89 into the GCs and found that the stimulating effect on the growth of GCs by Smad3 was blocked partly. The morphology of cultured GCs was changed by Smad3, and the expression level of integrin beta 1 was enhanced by Smad3. Kindlin-2, an important cellular mediating molecule of integrin beta signaling, was expressed in human ovarian luteinized GCs and was upregulated by Smad3. Our results indicated that Smad3 promoted the proliferation and steroidogenesis of human ovarian luteinized GCs, and these effects may be mediated by the FSHR/LHR-PKA signaling pathway. (C) 2014 IUBMB Life, 66(6): 424-437, 2014

Objective:To observe distribution and number of T cells in human normal cervical tissue and provide experimental data for further studies like expressions of T cells in cervical lesions and cervical cancer tissues. Methods:The number, distribution and expression intensity of the surface differentiation antigen CD3, CD4, CD8 positive T cells in the normal cervical tissue were detec-ted by immunohistochemical SP technique and computerized imaging analysis system. Results:The CD3 positive cells mainly distribu-ted in the cervical mucosal epithelium and connective tissue above the adjacent lamina propria. The CD8 positive cells widely distribu-ted in the human cervical mucosal lamina propria connective tissue. The CD4 positive cells mainly distributed in the lamina propria connective tissue under the human cervical epithelium and the connective tissue around the cervical mucosa adenoid fossae. The num-ber of CD4 positive cells was higher than that of CD8 positive cells. Conclusions: In the human normal cervical tissue, The CD3, CD4, CD8 positive cells maintain a certain proportions.

目的探讨Smad3基因对大鼠卵巢颗粒细胞自噬的影响。方法 21d SD雌性大鼠腹腔注射孕马血清20 IU/只,48h后收集卵巢颗粒细胞进行原代培养。培养细胞分为3组:空白对照组:培养液中不加任何处理因素;敲低实验组:培养液中加入Smad3基因特异性的SiRNA及转染试剂RNAiMAX;过表达实验组:培养液中加入Smad3真核表达质粒及转染试剂Lipo 2000。免疫细胞化学方法鉴定培养细胞纯度;Western blotting方法检测Smad3蛋白表达变化,检测转染效率。Smad3基因敲低及过表达后,Western blotting方法检测自噬体膜形成标志蛋白LC3B及自噬调控相关蛋白Bcl-2的蛋白表达变化。结果 Smad3敲低时,LC3B蛋白Ⅱ型与Ⅰ型的比值（LC3BⅡ/Ⅰ）变化不明显,Bcl-2蛋白表达明显降低;Smad3过表达时,LC3BⅡ/Ⅰ明显增加,Bcl-2蛋白表达明显增加。结论Smad3基因特异的SiRNA及真核表达质粒可以有效地转入大鼠卵巢颗粒细胞中,Smad3基因促进大鼠卵巢颗粒细胞自噬,其机制可能与Bcl-2蛋白相关。

The function of Smad3, a downstream signaling protein of the transforming growth factor β (TGFβ) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague-Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48 h later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFβ receptor II (TGFβRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.

Survivin是1997年由耶鲁大学的Ambrosini等[1]利用效应细胞蛋白酶受体-1(effector cell protease receptor-1,EPR-1)的cDNA在人类基因组库中筛选克隆的一种凋亡抑制基因.Survivin蛋白属于凋亡抑制蛋白(IAP)家族成员,在大多数肿瘤中高表达,在成体已分化的正常组织细胞中几乎检测不到.然而,Survivin不仅仅是一个肿瘤特异性蛋白,有实验证明在鼠胚胎处于分化状态的组织中可以检测到Survivin的表达[2],而且在成体造血系统中也可表达Survivin[3].本研究采用免疫组织化学显色方法检测了不同年龄段人的正常睾丸组织中Survivin蛋白的表达定位,以探讨其在正常生精过程中的作用.

Objective To establish effective methods of purification and culture in vitro of human ovarian granulosa cells. Methods Human ovarian granulosa cells were obtained in conjunction with oocyte aspiration from women undergoing hormone treatment for in vitro fertilization.Cells were treated with trypsin and density gradient centrifugation,and then cultured with different media. Results Centrifuged by 50%(v/v) Percoll PLUS,and cultured with DMEM/F12 or McCoy'5a liquid medium,cells were of great purity,high survival rate,and good subsequent growth. Conclusion We have established a stable model of culturing human ovarian granulosa cells in vitro,and laid a good foundation for research of granulosa cells in vitro.

Objective: To study the effects of Smad3 on the proliferation of the ovarian granulosa cells (GCS) in the rat. Methods: Ovarian granulosa cells were cultured and treated in the Hollowing conditions: 1. GCs were cultured without any treatment; 2. GCs were cultured with transfection reagent lipofectamine RNAiMAX; 3. GCs were transfected with transfection reagent lipofectamine RNAiMAX and Non-silencing SiRNA; 4. GCs were transfected with transfection reagent lipofectamine RNAiMAX and the SiRNA of Smad3. The expression and localization of PCNA and Cyclin D2 proteins in the four groups were detected by immuocytochemistry. Results: The proteins of PCNA and Cyclin D2 were mainly located in the nuclei of GCs. The percentage of positive cells for PCNA was (25.80 ± 1.70) % in the siRNA of Smad3 treated group, which decreased significantly compared to the control group (P < 0.05), and the percentage of the stained cells for Cyclin D2 was (25.00 ± 2.90)% in the siRNA of Smad3 treated group, which decreased significantly compared to the control group (P <0.05). Conclusion: Smad3 gene could promote the proliferation of ovarian granulosa cells in rats.

Objective: To study the changes of Smad2/p-Smad2 expression in the kidney of Smad3 knock-out mice. Methods: The expressions and localization of Smad2 and p-Smad2 proteins in the kidney of wide-type and Smad3 knock-out mice were examined by immunohistochemistry. Results: Smad2 protein was widely and weakly expressed in cytoplasm in distal renal tubules and collecting ducts of the wide-type mice, and p-Smad2 protein was mainly expressed in the nuclei of collecting ducts and distal tubules. The expression of Smad2 and p-Smad2 protein was significantly increased in the Smad3 knock-out mice (P<0.01). Conclusion: Smad3 knock-out can cause a compensatory increase in the expression of Smad2 and p-Smad2 protein in the kidney of mice.

目的:观察人树突状细胞体外摄取量子点后,其形态、表面标志、超微结构的变化.方法:两种量子点与人未成熟树突状细胞体外共孵育,荧光显微镜下观察量子点在树突状细胞内的分布;免疫细胞化学方法检测树突状细胞表面标志;扫描电镜及透射电镜观察树突状细胞超微结构的改变.结果:与量子点共培养的树突状细胞的细胞质内充满了较强荧光颗粒,共培养前后的树突状细胞表面标志均呈阳性反应.扫描电镜下可见树突状细胞表面有丰富的典型树枝样的突起.透射电镜下可见树突状细胞以胞饮的方式摄入量子点,形成有膜包被的囊泡分布在细胞质内.结论:树突状细胞是通过胞饮作用摄取量子点,且量子点对树突状细胞的形态、表面标志及超微结构没有影响,生物相容性好.

Objective: To explore gemcitabine's effect on tumor suppressor candidate 2(Tusc2), large tumor suppressor2(Lats2) gene expression in breast cancer of mice, both in mRNA and protein level. Methods: Mice models of breast cancer were established and the samples were selected at 0 day, 5 days and 10 days after gemcitabine hydrochloride treatments as well as control. RT-PCR and Western blotting were used to detect the variation of the Tusc2 and Lats2 gene expression both for blank control, negative control and test groups. Results: The results were similar both in RT-PCR and Western blotting. Compared with blank control, relative amounts of Tusc2 and Lats2 RNA in test group increased around 4.5 and 8 times 10 days after treated with gemcitabine in breast cancer mice respectively (P < 0.05). However, no differences were showed with Tusc2 and Lats2 RNA level 5 days after treated with gemcitabine in breast cancer mice. As to protein expression level, TUSC2 and LATS2 protein relative amounts were raised around 5 and 10 times (P < 0.05) compared with blank control. Significant statistical difference was found between test group and control (P > 0.05). No significant differenc was found between blank control and negative control (P > 0.05). Conclusion: Gemcitabine hydrochloride can upgrade Tusc2 and Lats2 gene expression effectively in breast cancer mice.

Objective: To study the effects of follicle stimulating hormone (FSH) on the expression and phosphorylation of Smad2/Smad3 proteins and the interaction between FSH and TGF-β/Smads signaling pathway in rat ovarian granulosa cells (GCs). Methods:The GCs from female SD rats of 21 d were cultured with TβRⅡ antibody and FSH at different concentration, and then cells were obtained at defined points to determine the expression of Smad2/Smad3 and P-Smad2/P-Smad3 by Western Blot assay. Results: FSH enhanced the expression of TβRⅡ, Smad2/Smad3 and P-Smad2/P-Smad3 proteins in manner of time dependent; Neutralization of TβRⅡ antibody to TβRⅡ on the surface of GCs inhibited the stimulation of FSH on Smad2/Smad 3 in GCs; The effects of FSH on Smad2/Smad3 proteins recovered when TβRⅡ was expressed again. Conclusion: In the culture condition, FSH stimulated the expression and phosphorylation of Smad2/Smad3; The activation of FSH on Smad2/Smad3 depended on TβRⅡ.

Objective To investigate the expression and localization of Smad2/3 and phosphorylated-Smad2/3 in human kidney.Methods Immunohistochemistry was used to analyze 20 specimens of human renal tissue.Results Smad2 and Smad3 proteins were widely expressed in renal tubules and corpuscles,and mainly localized within the cytoplasm of the epithelia lining the renal tubules and collecting ducts,especially remarkable in the epithelia of distal convoluted tubule.P-Smad2 and p-Smad3 were mainly distributed in the nuclei in renal cortex and modulla,with more frequent localization in distal convoluted tubules.Conclusion Smad2 and Smad3 play active roles in normal human renal function.

本实验以选取转化生长因子βⅡ受体（transforming growth factor-βreceptor typeⅡ,TGF-βRⅡ)抗体的不同剂量和不同孵育时间,对原代培养的大鼠卵巢颗粒细胞表面TGF-βRⅡ进行中和,从而达到建立体外阻断TGF-β信号转导通路模型的目的.为研究与TGF-β信号转导通路相关的各种调节因素提供了体外研究的新模型.

Objective To observe the distribution and the effect of the quantum dots(QDs) on mouse abdominal cavity macrophages.Methods The QDs were co-cultured with mouse abdominal cavity macrophages in vitro.The differentiation and effect of the QDs on macrophage ultrastructures were observed under electronic microscope. Results The QDs were enveloped with unit membrane and internalized in the cytoplasm of the macrophage under transmission electron microscope.And it formed vacuolelike structures in the macrophage.There were many lamellar processes on the surface of the macrophage under scanning electron microscope.Conclusion The QDs can promote macrophage activation,and make its surface projection increased.The QDs were internalized by the macrophage,distributed in the cytoplasm,and formed vacuolelike structures enveloped with unit membrane.

Objective: To study the effects of FSH on the phosphorylation of Smad2/ Smad3 protein in rat ovarian granulosa cells. Methods: The granulosa cells of 21-day-old SD female rats were cultured with TGFβR II antibody and FSH of different concentrations, and then the cells were obtained at defined points to determine the phosphorylation of Smad2/Smad3 by immunocytochemistry. Results: 1. Both Smad2 and Smad3 proteins were mainly expressed in the cytoplasm of ovarian granulosa cells, and a small amount of phosphorylated-Smad2/3 were expressed in the nucleus; 2. The treatment with FSH increased the nuclear transfer of Smad2/Smad3 and the expression of P-Smad2/P-Smad3 in granulosa cells, The effects of FSH were time and dose dependent; 3. The antibody-neutlised TGFβR II inhibited the stimulation of FSH on Smad2/3 in granulosa cells. Conclusion: 1 FSH stimulates the phosphorylation of Smad2/Smad3; 2. The activation of FSH on Smad2/Smad3 is depends on TGFβR II.

Objective: To explore the possible function and significance of pancreatic polypeptide during the healing process of experimental gastric ulcer in rat. Methods: The immunohistochemical PAP method, morphometry and image analysis were applied to study the changes of the morphology, numerical density on area(N A) and mean grey degree of islet PP cells during the healing process of experimental gastric ulcer in rats. Results: Compared with normal control group(NCG) and saline control group( SCG), the N A of PP cells markedly decreased, and the mean grey degree markedly increased (P < 0.05) on the 6th day after the experimental gastric ulcer group(EUG). During the 10th and the 28th day after the process of experimental gastric ulcer, the N A of PP-positive cell increased, and the mean grey degree decreased(P < 0.05) especially on the 10th day. The results of morphological observation and image analysis in pancreas islet PP cells were basically the same during the healing process of experimental gastric ulcer. Conclusion: During the healing process of experimental gastric ulcer, the N A and mean grey degree of PP-positive cell both changed. It indicated that PP cells played a role in the modulating process of experimental gastric ulcer.

Elevated serum homocysteine (Hcys) levels have been suggested to contribute to congenital cardiovascular malformations, neural tube defects, and cardiovascular diseases. To investigate the mechanisms resulting in cardiovascular diseases and birth defects, Kuang-Hueih Chen et al. identified and characterized a novel gene, named rHCY2, whose expression was markedly up-regulated when Hcys was elevated in rat. In vivo, rHCY2 gene could induce chicken embryonic cells apoptosis and embryonic malformations. Its N-terminal kinase domain is apparently similar to human receptor-interacting serine–threonine kinase 3 (hRIP3). In view of this, we hypothesize that a link between the teratogenic effects of Hcys and hRIP3 is theoretically plausible. However, given the lack of data on the topic, it remains to be seen whether an elevated serum Hcys level will increase the expression of hRIP3. Using normal and abnormal human fetal hearts and cultured normal human fetal cardiomyocytes, we show that congenital cardiovascular malformations are associated with the overexpression of hRIP3, and evidence is found for a certain association between overexpression of hRIP3 and homocysteine-induced congenital cardiovascular malformations. Folic acid and anti-hRIP3 antibodies seem to favor maintenance of the shape and ultrastructure of cultured human fetal cardiomyocytes.

目的研究同型半胱氨酸(HCY)、同型半胱氨酸诱导基因-2抗体(HCY-2Ab)及叶酸对人胚心肌细胞超微结构的影响.方法取培养的人胚心肌细胞,进行HE染色,常规电镜包埋,透射电镜观察.结果当HCY的浓度小于1.0mmol/L时,随着HCY浓度的升高,心肌细胞核内常染色质增多、细胞质内线粒体和粗面内质网均多于空白对照组,而糖原颗粒减少;当浓度超过1.0mmol/L时,心肌细胞逐渐退化,细胞核内常染色质减少,异染色质增多,核膜包裹染色质形成凋亡小体,细胞质内线粒体肿胀、数目减少,粗面内质网减少;加入外源性的HCY-2Ab或叶酸,心肌细胞形态较相同HCY浓度下的好转.结论HCY对培养的人胚心肌细胞超微结构的维持具有正调节和负调节双相作用;外源性的HCY-2Ab和叶酸在一定程度上可以逆转HCY的作用.