IFN-γ对奶牛乳腺上皮细胞转分化的影响

[目的]用转化生长因子-β_1(TGF-β_1)诱导奶牛乳腺上皮细胞-肌纤维母细胞转分化(EMT),以不同浓度的IFN-γ为阻断剂,探讨干扰素-γ(IFN-γ)对奶牛乳腺上皮细胞(BMEC)表型重塑的作用。[方法]将原代培养的BMEC分为对照组、诱导组(TGF-β_110ng/m L)、药物组(IFN-γ20 ng/m L)及阻断组(TGF-β_110 ng/m L+IFN-γ10、20、50、100 ng/m L)。培养72 h后,观察α-平滑肌肌动蛋白(α-SMA)、结缔组织生长因子(CTGF)和胶原Ⅰ(COLⅠα1)的mRNA相对表达量。[结果]诱导组α-SMA、CTGF和胶原Ⅰ mRNA相对表达量显著增加(P0.05);药物组CTGF的mRNA相对表达量与对照组相比无显著差异(P0.05),α-SMA和COLⅠα1mRNA相对表达量显著下降(P0.05);阻断组IFN-γ明显抑制了TGF-βl诱导的转分化。[结论]IFN-γ能够负性调控TGF-β_1诱导的奶牛乳腺上皮细胞-肌纤维母细胞转分化(EMT),减少COLⅠα1分泌。     

Role of miR-433 in fibrosis of NIH-3T3 cells and silica-induced pulmonary fibrosis in mice

AIM To study the role and regulatory mechanism of microRNA-433 (miR-433) in fibrosis. METHODS TargetScan was used to predict the potential target genes of miR-433. The changes of miR-433 expression were detected after transforming growth factor β1 (TGF-β1) treatment of mouse embryonic fibroblasts (NIH-3T3 cells) for 24 h. The effects of miR-433 mimic on the expression of p-SMAD2, fibronectin (FN), α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in TGF-β1 treated cells were examined. The effects of miR-433 mimic transfection on the viability and S-phase fraction of NIH-3T3 cells induced by TGF-β1 were detected by CCK-8 assay and flow cytometry. A model of silica-induced pulmonary fibrosis in mice was established, and agomiR-433 was used for intervention. HE staining and Masson staining were used to observe the effect of miR-433 on pulmonary fibrosis in the mice. The expression of α-SMA in lung tissues was detected by immunohistochemistry. RESULTS miR-433 specifically bound to the 3'-UTR of SMAD2 and inhibited its expression at protein and mRNA levels. TGF-β1 down-regulated the expression of miR-433 in NIH-3T3 cells, up-regulated the protein level of p-SMAD2 and the expression of FN, α-SMA and CTGF at protein and mRNA levels, and increased the viability and the number of S-phase cells. miR-433 mimic reversed the effects of TGF-β1 on NIH-3T3 cell viability and S-phase arrest. In a model of silica-induced pulmonary fibrosis in mice, agomiR-433 inhibited the progress of pulmonary fibrosis and reduced the expression of α-SMA in mouse lung tissues. CONCLUSION miR-433 may interfere with TGF-β1/SMAD2 signaling pathway through targeting SMAD2, thus participating in the regulation of fibrosis process.     

复方中药水提物对腹水综合征肉鸡肝脏TGF-β1/Smad通路的影响

【目的】研究转化生长因子β1(transforming growth factor-β1,TGF-β1)/Smad通路在肉鸡腹水综合征肝纤维化中的作用及复方中药水提物的防治机理。【方法】选取健康Ross肉鸡258只,常规饲养至7日龄后,随机分为5组：空白组(C);模型组(M),低温、高脂和高蛋白饲料、高钠饮水的多因素法造模;复方中药水提物高、中、低剂量组(T1、T2、T3),在模型组基础上,每天饮水中分别添加2.0、1.0、0.5 g/kg复方中药水提物。观察临床症状和剖检变化,通过HE染色和Masson染色分别观察肉鸡肝脏组织结构变化与胶原纤维沉积,计算15、25、35、45日龄体重变化。利用实时荧光定量PCR检测各日龄肝脏中TGF-β1、转化生长因子β受体Ⅰ(transforming growth factorβreceptorⅠ,TβRⅠ)、Smad2、Smad3和Smad7基因相对表达量;利用ELISA法检测各日龄肝脏中TGF-β1、TβRⅠ蛋白含量。【结果】与空白组相比,模型组肉鸡腹部膨大、触压有波动感;腹腔内有大量淡黄色液体,肝脏充血肿胀、质脆,表面有瘀血点;肝脏组织结构紊...     

Wound healing in cutaneous leishmaniasis: A double edged sword of IL-10 and TGF-β

Immune responses have a crucial role during the wound healing process in cutaneous leishmaniasis (CL). However, there are several paradoxes in immunity against CL. On the one hand, regulatory cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-β) increase susceptibility to CL through suppression of several proinflammatory cytokines that require for defense against CL. On the other hand, these cytokines play a pivotal role in the acceleration of wound healing process. This review discusses about the dual role of IL-10 and TGF-β during the wound healing process and immunity against CL to offer a new insight about wound healing in CL.     

The Analysis for the Single Nucleotide Polymorphism of TGF-β1 and Its Association with Reproduction in Different Sheep Breeds

A single nucleotide polymorphism(SNP)of 805 bp region in the intron 6 of transforming growth factor β1(TGF-β1)gene was identified by polymerase chain reaction-single-strand composition polymorphism(PCR-SSCP)in 196 sheep among Small-tailed Han sheep,Tong sheep,Tan sheep and Oula sheep.Comparative sequence analysis of cloned products revealed an AGAC deletion at 294 bases upstream of exon 7 of the TGF-β1 gene(site 14201 in gi76871756).Statistical results of the genotype and allele frequencies in different breeds showed that genotype AB was dominant in the Small-tailed Han sheep.Genotype BB,however,was in majority in low-fecundity sheep.The results of a Chi-square test indicated that all the populations were in Hardy-Weinberg equilibrium.     

鸡TGF-β1对大肠杆菌和鸡白痢沙门菌黏附DF1细胞的影响

旨在研究鸡转化生长因子-β1（transfer growth factor-β1,TGF-β1）对大肠杆菌和鸡白痢沙门菌黏附DF1细胞的影响。通过ELISA方法检测鸡新城疫病毒（Newcastle disease virus,NDV）感染DF1细胞后鸡TGF-β1表达量的变化,参考GenBank中鸡TGF-β1序列构建鸡TGF-β1的过表达和干扰表达载体,将构建成功的TGF-β1重组表达载体转染DF1细胞后48 h在荧光显微镜下观察其转染效率,荧光定量PCR检测鸡TGF-β1 mRNA水平表达量的变化,ELISA方法检测鸡TGF-β1胞外蛋白表达量的变化,通过黏附试验检测鸡TGF-β1对致病性大肠杆菌和鸡白痢沙门菌黏附DF1细胞的影响。结果显示,NDV感染DF1细胞后,鸡TGF-β1胞外表达量显著高于未感染细胞的表达量（P<0.05）,经酶切测序鉴定TGF-β1干扰表达和过表达重组载体构建成功。转染鸡TGF-β1重组过表达载体细胞的TGF-β1 mRNA和胞外蛋白表达量均显著高于未转染细胞（P<0.01）,致病性大肠杆菌和鸡白痢沙门菌对细胞的黏附率均显著高于未转染细胞（P<0.01）,转染鸡TGF-β1重组干扰表达载体细胞的mRNA和胞外蛋白表达量均显著低于未转染细胞（P<0.01）,致病性大肠杆菌和鸡白痢沙门菌对细胞的黏附率均显著低于未转染细胞（P<0.01）。综上,NDV感染DF1细胞后,鸡TGF-β1的表达量增加,鸡TGF-β1可促进致病性大肠杆菌和鸡白痢沙门菌黏附DF1细胞,这为进一步研究鸡TGF-β1在家禽病毒感染继发细菌性疾病中的作用提供试验基础。     

上皮间质转化促乳腺癌机制及药物干预治疗研究进展

乳腺癌是犬、猫等伴侣动物与人类常发疾病，作为人类及动物常患恶性肿瘤和主要致死肿瘤之一，其疾病负担仍呈逐步加重趋势，乳腺癌的预防及治疗形势愈加严峻。上皮间质转化（EMT）是乳腺癌发生发展中重要的生物学过程。EMT还可促进恶性肿瘤的侵袭、扩散及耐药，因此它在肿瘤的研究中日益受到关注，靶向于EMT是治疗乳腺癌的重要研究方向与热点。文章就EMT发生过程中细胞形态功能及标志物的变化、EMT分类及EMT与乳腺癌的关系分别展开论述，详细解析了EMT相关TGF-β/Smad、NF-κB及Wnt信号通路转导途径；随后对乳腺癌治疗药物研究进展，包括TGF-β/Smad通路抑制剂开发，相关药物、基因及细胞因子治疗前景、NF-κB通路与Wnt通路抑制剂的动物试验研究结果进行了详细论述；最后对乳腺癌的治疗发展与趋势进行了展望。深入认识信号通路调控乳腺癌EMT的生物学过程，明确其发生发展机制，寻找关键靶点及开发靶向药物，将为乳腺癌的精准治疗带来曙光。     

Relationship Between the mRNA Expression Level of TGF-β Receptor Genes in Tissues and Ovulation Rate in Hu Sheep

Eight ewes of Hu sheep which bred multi-lamb were used as the high-fecundity group and the other eight ewes of Hu sheep which bred single lamb were used as the control group to investigate the relationship between the mRNA expression level of TGF-β receptor genes in tissues and ovulation rate in Hu sheep. Cloprostenol sodium was injected to make the synchronization of estrus treatment, then all ewes were slaughtered within 24-36 h after empathema and the ovaries were collected. Furthermore, the number of ovulation points was counted to determine ovulation rate for each sheep. Tissue expression analysis was conducted by RT-PCR for one ewe form the high-fecundity group and the relationship between the mRNA expression of genes encoding TGF-β receptors and ovulation rate was detected by real-time fluorescent quantitative PCR. The results showed that the relative expression level of TGF-flR I gene in the reproductive organ was significantly higher than in the lung and muscle （P 〈 0.01）, while relative expression level of TGF-βR H in reproductive organ was significantly higher than that of other tissues （P 〈 0.01）, indicating that these are highly expressed genes in the ovary. In addition, mRNA expression level of TGF-βR I and TGF-flRH in the ovaries of the high-fecundity group were significantly higher （P〈 0.01） and obviously higher （P= 0.011） than the control group, respectively. The mRNA expression level of TGF-βR I and TGF-βR H had a positive correlation with ovulation rate and the correlation coefficients were 0.562 （P〉 0.05） and 0.711 （P〈 0.05）, respectively. It is suggested that TGF-β receptors have close relationship with highfecundity in Hu sheep.     

Isolation and Identification of Goat Dermal Papilla Cells and Expression of TGF/β-smad Pathway Related Genes

The purpose of this study was to isolate goat hair follicle cells,and to explore the expression of TGF-β/smad pathway-related genes,providing a good model for the research on the mechanism of goat hair follicle development in vitro.In this study,two steps of neutral protease and collagenase digestion were used to treat the skin on the back of goats,the hair follicle cells were isolated and purified under stereomicroscope.Cellular immunofluorescence and Western blotting method were used to verify the expression of α-SMA (α-smooth muscle actin,α-SMA) and VIM (vimentin,VIM),and the expression of related genes of the TGF-β/smad pathway in goat hair papilla cells were examined.The results showed that the isolated goat hair follicle cells in adherent culture after separation grew slowly and had mature morphology after 15 days of separation and could be subcultured.The results of immunofluorescence and Western blotting showed that both α-SMA and VIM were positive in vitro cultured hair papilla cells.The expression of smad4 and smad5 genes in the TGF-β/smad pathway were significantly higher than those in control group (P<0.05),and the expression of smad2,smad6,and smad7 were extremely significantly higher than those in control group (P<0.01).These key genes related to hair follicle development were highly expressed.This study successfully isolated goat dermal papilla cells,which would give a good theoretical basis and cell model for studying the hair follicle development mechanism in vitro.     

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4⁺CD25⁺Foxp3⁺ T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3⁺CD4⁺ cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4⁺ cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4⁺ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4⁺ and CD25⁺ T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4⁺ cells in the CD4⁺ T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4⁺CTLA-4⁺ T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.