scholarly journals Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199767
Author(s):  
Ying Hu ◽  
Qing-Wei Zhao ◽  
Zheng-Cai Wang ◽  
Qing-Qing Fang ◽  
He Zhu ◽  
...  
Keyword(s):  
Real Time ◽  
Chitosan Nanoparticles ◽  
Human Adipose Tissue ◽  
Bone Morphogenetic Protein 2 ◽  
Expression Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Mrna Levels ◽  
Gene And Protein Expression

Objective To investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro. Materials and Methods Recombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis. Results The prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis. Conclusions Co-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

scholarly journals Effect of EPA on Neonatal Pig Sertoli Cells “In Vitro”: A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies

2021 ◽  
Vol 12 ◽  
Author(s):  
Iva Arato ◽  
Veronica Ceccarelli ◽  
Francesca Mancuso ◽  
Catia Bellucci ◽  
Cinzia Lilli ◽  
...  
Keyword(s):  
Protein Expression ◽  
Sertoli Cells ◽  
Fold Change ◽  
Control Group ◽  
Mrna Levels ◽  
Expression Levels ◽  
Chemotherapy Drugs ◽  
Gene And Protein Expression ◽  
Pubertal Boys

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 μM vs SCs and ***p < 0.001 at 3.33μM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 μM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 μM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

YAP1 Overexpression Is Associated with Kidney Dysfunction in Lupus Nephritis

Pathobiology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Ying Xie ◽  
Yuanyuan Ruan ◽  
Huimei Zou ◽  
Yixin Wang ◽  
Xin Wu ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Epithelial Mesenchymal Transition ◽  
Kidney Tissue ◽  
Mouse Kidney ◽  
Experimental Comparison ◽  
Control Group ◽  
Mrna Levels ◽  
Mesenchymal Transition ◽  
Protein Levels

<b><i>Objective:</i></b> The goal of the present study was to determine the expression of yes-associated protein 1 (YAP1) in renal tissues of mice with lupus nephritis (LN) and elucidate its role in the progression of renal fibrosis. <b><i>Methods:</i></b> C57BL/6 mice and MRL/lpr mice were selected for experimental comparison. Mouse kidney tissues were removed and sectioned for hematoxylin and eosin staining, Masson’s trichome staining, Sirius staining, and immunohistochemistry. The mRNA and protein levels of YAP1 in mouse kidney tissues were detected, and the correlation between YAP1 and fibronectin (FN) mRNA levels was analyzed. Mouse renal epithelial cells were used for in vitro experiments. After transfection and stimulation, the cells were divided into 4 groups, namely the C57BL/6 serum group (group 1), the MRL/lpr serum group (group 2), the MRL/lpr serum + siRNA-negative control group (group 3), and the MRL/lpr serum + siRNA-YAP1 group (group 4). Epithelial-mesenchymal transition (EMT) markers in each group were detected by Western blotting and immunofluorescence staining. Serum creatinine, blood urea nitrogen, and urinary protein levels were detected and assessed for their correlation with YAP1 mRNA levels by Spearman’s analysis. <b><i>Results:</i></b> Compared to C57BL/6 mice, MRL/lpr mice exhibited obvious changes in fibrosis in renal tissues. In addition, YAP1 expression was significantly higher in the renal tissues of MRL/lpr mice than in those of C57BL/6 mice, and YAP1 mRNA levels were positively correlated with those of FN. YAP1 silencing in lupus serum-stimulated cells could effectively relieve serum-induced EMT. Finally, we observed that YAP1 mRNA levels in mouse kidney tissue were significantly and positively correlated with the degree of renal function injury. <b><i>Conclusion:</i></b> YAP1 expression in the kidney tissues of LN mice was higher than that observed in normal mice, indicating that YAP1 may play an important role in the occurrence and development of LN.

scholarly journals Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yan Gong ◽  
Jesse Li-Ling ◽  
Dongsheng Xiong ◽  
Jiajing Wei ◽  
Taiqing Zhong ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Clinical Trial Registry ◽  
Altered Expression ◽  
Vitro Fertilization ◽  
Age Related ◽  
Tertiary Teaching ◽  
Poor Ovarian Responders

Abstract Background Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. Methods A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P <  0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P <  0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P <  0.05). Conclusions For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. Trial registration Chinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

MO332THE IRRADIATION-INDUCED RENAL ISCHEMIC PRECONDITIONING IS BLUNTED BY THE ORAL ADMINISTRATION OF THE ANTI-ANGIOGENIC AGENT, SUNITINIB

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Badr Khbouz ◽  
François Lallemand ◽  
Pascal Rowart ◽  
Laurence Poma ◽  
Agnès Noel ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Real Time ◽  
Ischemic Preconditioning ◽  
Functional Enrichment ◽  
Whole Body ◽  
Control Group ◽  
Mrna Levels ◽  
Wild Type ◽  
Post Irradiation ◽  
Real Time Qpcr

Abstract Background and Aims Whole-body irradiation has been suggested to induce renal ischemic preconditioning (RIP) in rodent models, possibly via neo-angiogenesis. First, we comprehensively investigate the pathways involved in kidney-centered irradiation. Next, we assess the functional and structural impact of kidney-centered irradiation applied before ischemia/reperfusion (I/R) injury. Finally, we test whether Sunitinib-mediated inhibition of the neo-angiogenesis prevents irradiation-associated RIP. Method Experiment 1: Unilateral irradiation of the left kidney (8.56 Gy) was performed in male 10-week-old wild-type C57bl/6 mice (n=10). One month later, total kidney RNA was extracted from irradiated and control (n=5) mice for comparative high-throughput RNA-Seq (using BaseSpace Sequence Hub Illumina). Functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID). Experiment 2: Two x-ray beams (225Kv, 13mA) specifically targeted both kidneys for a total dose of 8.56Gy. The right kidneys were removed and harvested, and the left kidneys undergo 30-minute ischemia followed by 48-hour reperfusion (n=8) at Days 7-14-21-28 post irradiation. Experiment 3: Following the same protocol of renal I/R at Day14, 3 groups of male 10-week-old wild-type C57bl/6 mice were compared (n=8 per group): 1/ bilateral pre-irradiation; 2/ bilateral pre-irradiation and gavage with Sunitinib from Day2 to Day13; 3/ control group without irradiation or gavage. Results Experiment 1: Comparative transcriptomics showed a significant up-regulation of various signaling pathways, including angiogenesis (HMOX1) and stress response (HSPA1A, HSPA1B). Expressions of angiogenesis markers (CD31, TGFb1, HMOX1) showed an increase at both mRNA (real-time qPCR) and protein (immuno-staining) levels in irradiated kidneys compared to controls (p&lt;0.01). Experiment 2: Following I/R, the blood urea nitrogen (BUN) and serum creatinine (SCr) levels were significantly lower in the irradiated animals compared to controls: (BUN: 86.2±6.8 vs. 454.5±27.2mg/dl; SCr: 0.1±0.01 vs. 1.7±0.2mg/dl, p&lt;0.01). The renal infiltration by CD11b-positive cells (187±32 vs. 477±20/mm²) and F4-80 macrophages (110±22 vs. 212±25/mm²) was significantly reduced in the irradiated group. The real-time qPCR mRNA levels of the angiogenic markers, TGFb1 and CD31, were significantly increased in the irradiated group compared to controls (p&lt;0,01). The CD31-immunostating (quantified by FiJi) was increased in irradiated mice compared to controls (p&lt;0.01). Experiment 3: One-way analysis of variance followed by Tukey’s test showed that, following I/R, the serum levels of BUN and SCr were lower in irradiated group compared to controls (BUN: 106.1±33.6 vs. 352.2±54.3mg/dl; SCr: 0.3±0.13 vs. 1±0.2mg/dl), and in irradiated group compared to the irradiated-exposed group to Sunitinib (BUN: 106.1±33.6 vs. 408.4±54.9mg/dl; SCr: 0.3±0.12 vs. 1.5±0.3mg/dl; p&lt;0.01). No difference was observed between the irradiated-exposed mice to Sunitinib and the controls. Conclusion Renal irradiation induces the activation of signaling pathways involved in angiogenesis in mice. Renal pre-irradiation leads to RIP, with preserved renal function and attenuated inflammation post I/R. Exposure to the anti-angiogenic drug Sunitinib post-irradiation prevents the irradiation-induced RIP.

scholarly journals TheBDKRB2 +9/-9 Polymorphisms Influence Pro-Inflammatory Cytokine Levels in Knee Osteoarthritis by Altering TLR-2 Expression: Clinical and in Vitro Studies

2016 ◽  
Vol 38 (3) ◽  
pp. 1245-1256 ◽  
Author(s):  
Shuo Chen ◽  
Lei Zhang ◽  
Ruonan Xu ◽  
Yunfan Ti ◽  
Yunlong Zhao ◽  
...  
Keyword(s):  
Knee Osteoarthritis ◽  
Inflammatory Cytokine ◽  
Molecular Mechanisms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Knee Oa ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Underlying Mechanisms

Background/Aims: The bradykinin B2 receptor (BDKRB2) +9/-9 gene polymorphisms have been shown to be associated with the susceptibility and severity of osteoarthritis (OA); however, the underlying mechanisms are unclear. In this study, we investigated the correlation between the BDKRB2 +9/-9 polymorphisms and pro-inflammatory cytokine levels in OA and the molecular mechanisms involved. Methods: A total of 156 patients with primary knee OA and 121 healthy controls were enrolled. The BDKRB2 +9/-9 polymorphisms were genotyped. The tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 levels were determined using Enzyme-linked immunosorbent assay (ELISA). The toll-like receptor (TLR)-2 and TLR-4 mRNA levels were determined by quantitative real-time PCR. The basal and bradykinin-stimulated pro-inflammatory cytokine secretion in human OA synoviocytes and the involvement of TLR-2 and mitogen-activated protein kinases (MAPKs) were investigated. Results: The presence of -9 bp genotype is associated with higher TNF-α, IL-6, and IL-8 levels and higher TLR-2 expression in OA patients. The basal and bradykinin-induced TLR-2 expressions in human OA synoviocytes were significantly reduced by specific inhibitors of p38, JNK1/2, and ERK1/2. Both the B2 receptor antagonist MEN16132 and TLR-2 silencing inhibited IL-6 and IL-8 secretion in human OA synoviocytes. Conclusion: The data suggested that the BDKRB2 +9/-9 polymorphisms influence pro-inflammatory cytokine levels in knee osteoarthritis by altering TLR-2 expression.

scholarly journals A More Aggressive Breast Cancer Spheroid Model Coupled to an Electronic Capillary Sensor System for a High-Content Screening of Cytotoxic Agents in Cancer Therapy: 3-Dimensional In Vitro Tumor Spheroids as a Screening Model

2005 ◽  
Vol 10 (7) ◽  
pp. 705-714 ◽  
Author(s):  
P. Bartholomä ◽  
Impidjati ◽  
A. Reininger-Mack ◽  
Zhihong Zhang ◽  
H. Thielecke ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Therapy ◽  
Real Time ◽  
Cytotoxic Agents ◽  
High Content Screening ◽  
Expression Vectors ◽  
Tumor Spheroids ◽  
Capillary System ◽  
Cdna Expression

One major problem in cancer therapy is the immortality of tumor cells showing an active telomerase, which is responsible for the elongation of the telomeres after each cellular division and the knocking down of apoptotic suppressors. A further phenomenon occurring during cancer therapies is the problem of multicellular resistance. To develop therapeutic anticancer approaches inducing cellular apoptosis, gene-modified biological in vitro systems were established and evaluated for drug screening in a capillary system for a real-time, impedimertic monitoring. Multicellular spheroids of the human breast cancer cell line T-47D clone 11 were transfected with 1) antisense caspase-3 cDNA expression vectors for knocking down the main cell death molecule and 2) sense Bcl-xl cDNA expression vectors for overexpressing the apoptotic suppressor, resulting in more aggressive tumor models. These gene-modified tumor spheroids less sensitive for apoptosis were developed for screening drugs such as methotrexate in tumor spheroid–based biosensor systems via impedance spectroscopy. In this report, it is demonstrated that this could successfully exhibit that this real-time monitoring system with tumor spheroids positioned in a capillary system with a 4-electrode configuration is the most efficient high-content screening module for impedimetric measurements of physiological alterations during gene modification and drug application.

scholarly journals THE TOXICITY OF MAULI BANANA (MUSA ACUMINATA) STEM WATER EXTRACT ON BONE MARROW MESENCHYMAL STEM CELL IN VITRO

2019 ◽  
pp. 184-186
Author(s):  
AMY NINDIA ◽  
DIDIT ASPRIYANTO ◽  
MAHARANI LAILLYZA APRIASARI ◽  
SELVIANA RIZKY
Keyword(s):  
Growth Factor ◽  
Water Extract ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Cell Culture Process ◽  
Control Group Design ◽  
Water Solvent ◽  
Stem Water ◽  
Banana Stem

Objective: Since mesenchymal stem cells (MSC) can differentiate into bone, cementum, and periodontal ligament, they can be used to treat aggressiveperiodontitis. The limited number of MSCs requires replenishment of growth factor in the cell culture process. Since growth factor is quite expensive,an alternative material is needed. Mauli banana stem has antioxidant and immunomodulatory properties. Methanol extract of Mauli banana stem isknown to be toxic toward MSCs; therefore, another solvent with a non-toxic effect is needed, such as a water solvent. We analyzed the toxicity of Maulibanana stem water extract on MSC in vitro.Methods: In this laboratory experimental (true experimental) study with a Post-test Only Control Group Design, MSC cultures were treated withMauli banana stem water extract at 10, 20, 40, 60, 80, and 100 mg/mL dosages. One group without any treatment served as a control group and onewas a media control group. Each group was incubated for 24 h and then was given 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidereagent and analyzed by an enzyme-linked immunosorbent assay (ELISA) reader.Results: One-way analysis of variance showed a significant difference.Conclusion: Mauli banana stem water extracts at 10, 20, 40, and 60 mg/mL were not toxic toward MSC in vitro, while dosages of 80 and 100 mg/mLdosage were toxic.

4 MYOSTATIN GENE KNOCKDOWN THROUGH LENTIVIRAL VECTOR-MEDIATED DELIVERY OF shRNA FOR IN VITRO PRODUCTION OF TRANSGENIC BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 120 ◽  
Author(s):  
M. P. Milazzotto ◽  
W. B. Feitosa ◽  
B. E. Strauss ◽  
M. Bajgelman ◽  
C. M. Mendes ◽  
...  
Keyword(s):  
Real Time ◽  
Cell Morphology ◽  
Real Time Pcr ◽  
Lentiviral Vector ◽  
Gene Knockdown ◽  
Control Group ◽  
Bovine Embryos ◽  
Myostatin Gene ◽  
Transgenic Embryo

The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed. This work was supported financially by FAPESP 03/0156-9.

scholarly journals Human Uterine Decidual NK Cells in Women with a History of Early Pregnancy Enhance Angiogenesis and Trophoblast Invasion

2020 ◽  
Vol 2020 ◽  
pp. 1-7 ◽  
Author(s):  
Ningyi Jia ◽  
Jian Li
Keyword(s):  
Early Pregnancy ◽  
Mrna Level ◽  
Trophoblast Invasion ◽  
Migration And Invasion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Mrna Levels ◽  
History Of ◽  
And Migration ◽  
Primiparous Women

Objective. The present study aimed to identify changes in decidual natural killer (dNK) cells and related cytokines in women who have undergone induced abortions (IAs). The effects of dNK cells on subsequent pregnancies remain unknown. Accordingly, we sought to investigate whether a history of early pregnancy can change dNK cells and facilitate their role in the regulation of angiogenesis and trophoblast invasion. Materials and Methods. dNK cells were obtained from primiparous women who had undergone IA(s) prior to this study and primiparous women who had never been pregnant before this IA (control). Real-time polymerase chain reaction (PCR) was used to measure the mRNA levels of IFN-γ, IP-10, VEGF, and PLGF in dNK cells. The levels of these cytokines were quantified using the enzyme-linked immunosorbent assay. HUVEC and HTR-8/SVneo cells were used to evaluate the angiogenesis, migration, and invasion activities influenced by dNK cells. Results. In dNK cells, the mRNA level of IFN-γ was higher in the control group than that in the IA group. The secretion of IP-10 and VEGF was higher in the IA group compared to the control group. After coculturing with the dNK supernatant, the HTR-8/SVneo cells exhibited better invasiveness and migration in the IA group than those in the control group. Angiogenesis assay demonstrated that dNK cells from IA group might help HUVEC attain better tube formation ability. Conclusion. The findings of this study suggest that a history of early pregnancy has an impact on dNK cells. These trained dNK cells can regulate angiogenesis and trophoblast invasion and migration by promoting the production of certain cytokines.

scholarly journals A Promising Antiprion Trimethoxychalcone Binds to the Globular Domain of the Cellular Prion Protein and Changes Its Cellular Location

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
N. C. Ferreira ◽  
L. M. Ascari ◽  
A. G. Hughson ◽  
G. R. Cavalheiro ◽  
C. F. Góes ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Prion Protein ◽  
Molecular Mechanisms ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Globular Domain ◽  
Mrna Levels ◽  
Spongiform Encephalopathies

ABSTRACTThe search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identifiedin vitroto be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPCmRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23–231) as the substrate and PrPScseeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90–231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPCglobular domain. J8 also reduced the fibrillation of mouse rPrP23–231seeded within vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPCon the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPCcontent on the cell surface.

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