STAT6 ASODN对哮喘小鼠脾淋巴细胞影响的实验研究

目的研究STAT6反义寡核苷酸对哮喘小鼠脾淋巴细胞的影响作用。方法实验细胞分组:正常鼠空白组(A组)、正常鼠OVA组(B组)、哮喘空白组(C组)、哮喘OVA组(D组)、哮喘治疗组(E组)。正常设计并人工合成一段互补于小鼠STAT6 mRNA翻译起始区271-290的反义寡核苷酸片段,全链硫代修饰。用卵白蛋白和氢氧化铝复制哮喘模型,用淋巴细胞分离液分离脾淋巴细胞,进行体外培养并导入由阳离子脂质体转染剂Geneshuttle携带的反义寡核苷酸,观察反义寡核苷酸的转染对脾淋巴细胞STAT6蛋白表达水平及细胞培养上清中IL-4分泌水平的影响。免疫细胞化学观察脾淋巴细胞中STAT6蛋白的表达水平,同时采用酶联免疫吸附(ELISA)法测定脾细胞培养上清液中IL-4的浓度。结果D组细胞STAT6蛋白表达明显高于其余各组,均具有显著性差异(P均<0.01),STAT6 ASODN转染后,E组细胞该蛋白的表达量明显下降(P<0.01);D组脾淋巴细胞培养上清中IL-4分泌水平明显高于其余各组,均具有显著性差异(P均<0.01);STAT6 ASODN转染后,E组培养上清中IL-4分泌水平显著低于D组(P<0.01)。结论STAT6 ASODN可特异性抑制哮喘鼠脾淋巴细胞中STAT6蛋白的表达,并可特异性抑制脾淋巴细胞中IL-4的分泌,为反义基因技术治疗哮喘提供了依据。     

Hydrophobic chromatography of the HL-60 cellular fraction co-binding with hexamethylene bisacetamide

Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

Odor Memory Stability after Reinnervation of the Olfactory Bulb

The olfactory system, particularly the olfactory epithelium, presents a unique opportunity to study the regenerative capabilities of the brain, because of its ability to recover after damage. In this study, we ablated olfactory sensory neurons with methimazole and followed the anatomical and functional recovery of circuits expressing genetic markers for I7 and M72 receptors (M72-IRES-tau-LacZ and I7-IRES-tau-GFP). Our results show that 45 days after methimazole-induced lesion, axonal projections to the bulb of M72 and I7 populations are largely reestablished. Furthermore, regenerated glomeruli are re-formed within the same areas as those of control, unexposed mice. This anatomical regeneration correlates with functional recovery of a previously learned odorant-discrimination task, dependent on the cognate ligands for M72 and I7. Following regeneration, mice also recover innate responsiveness to TMT and urine. Our findings show that regeneration of neuronal circuits in the olfactory system can be achieved with remarkable precision and underscore the importance of glomerular organization to evoke memory traces stored in the brain.     

Long non-coding RNA CCAT1 promotes cervical cancer cell proliferation and invasion by regulating the miR-181a-5p/MMP14 axis

Cervical cancer is a serious threat to women’s health and is the third most common malignancy in women worldwide. Recent studies indicate that the long non-coding RNA CCAT1 plays a role in the malignant behavior of many tumors. However, the role of CCAT1 in cervical cancer is still unknown. Our aim is to evaluate the expression and investigate the regulatory role and potential mechanism of CCAT1 in cervical cancer. CCAT1 expression was measured by qRT-PCR. In addition, CCK-8 assays, colony formation assays, qRT-PCR assays, Transwell assays and xenograft experiments were performed to determine the role of CCAT1 in the proliferation and invasion in cervical cancer cells. The expression of CCAT1 in the cervical cancer tissues was higher than in the adjacent normal tissues. Overexpressing CCAT1 promoted cervical cancer cell proliferation, colony formation, and invasion in vitro. Elevated CCAT1 suppressed miR-181a expression, which was accompanied by an increased expression of MMP14 and HB-EGF. In contrast, knocking down CCAT1 resulted in increased expression of miR-181a, along with decreased expression of MMP14 and HB-EGF. Thus, CCAT1 is a key oncogenic lncRNA associated with cervical cancer and plays a role in promoting cervical cancer cell proliferation and invasion by regulating the miR-181a-5p/MMP14 axis.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.     

Combination of diethyldithiocarbamate with 12-O-tetradecanoyl phorbol-13-acetate inhibits the growth of human myeloid leukemia HL-60 cells in vitro and in xenograft model

ABSTRACT

12-O-tetradecanoylphorbol-13-acetate (TPA), is a major active constituent of the seed oil of Croton tiglium L., has pharmacological activity for the treatment of acute myeloid leukemia patients. Diethyldithiocarbamate (DTC) is a potent inhibitor of NF-κB show activity of anticancer. In this study, we determined the effect of DTC and TPA in combination on HL-60 cells cultured in vitro and in vivo. In this study, we have shown that DTC and TPA synergistically inhibited the growth of HL-60 cells and strongly induced apoptosis in the cells. Mechanistic studies showed that the combined effects of DTC and TPA were associated with a decrease in Bcl-2. The animal experiment showed that the combination of DTC and TPA more potently inhibited the growth of HL-60 tumors than either agent alone. Our results indicate that the administration of TPA and DTC in combination may be an effective strategy for inhibiting the growth of acute myeloid leukemia cells.