Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Face ; microbiology ; Female ; Humans ; Middle Aged ; Mycobacterium avium Complex ; isolation & purification ; Mycobacterium avium-intracellulare Infection ; metabolism ; microbiology ; pathology ; Nasal Cavity ; microbiology ; Nose Diseases ; metabolism ; microbiology ; pathology ; Vimentin ; metabolism

Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; rho GTP-Binding Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the role of WT1 gene in breast carcinogenesis by analyses of the promoter methylation status and mRNA expression of WT1 gene in MCF10 model system of breast cancer progression.

METHODSMethylation specific PCR and sodium bisufite genomic sequencing were employed to detect methylation status of WT1 promoter in normal breast tissue, traditional breast cancer cell line MCF7 and MCF10 model series, including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell line, forming slowly progressing hyper and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in situ cell line, forming ductal carcinoma in situ), and three invasive cell lines with metastatic potential (MCF10CA1a, MCF10CA1d, and MCF10CA1h). Real time reverse transcription PCR assay was used to determine the mRNA expression levels of WT1 in various cell lines.

RESULTSHypermethylation of WT1 promoter was identified in MCF7 and all MCF10 model cell lines (MCF10A, MCF10AT, MCF10DCIS.com, MCF10CA1a, MCF10CA1d, and MCF10CA1h). Unexpectedly, an increased expression of WT1 mRNA was found in all MCF10 cell lines and MCF7 comparing with normal breast tissue [folds of overexpression: 3.23 (MCF10A), 1.94 (MCF10AT), 4.20 (MCF10CA1a), 1.53 (MCF10CA1d), 4.20 (MCF10CA1h), 4.35 (MCF10DCIS) and 28.69 (MCF7)].

CONCLUSIONSPromoter methylation does not silence the mRNA expression of WT1 during the development of breast cancer. Overexpression of WT1 occurs in the early stages of breast cancer development, suggesting its role as an oncogene rather than a tumor suppressor gene.

Base Sequence ; Breast ; pathology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Methylation ; DNA, Neoplasm ; genetics ; Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hyperplasia ; genetics ; metabolism ; pathology ; Molecular Sequence Data ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; WT1 Proteins ; genetics ; metabolism

OBJECTIVETo investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression.

METHODSMethylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ), MCF10CA1a, MCF10CA1d, MCF10CA1h cell lines (invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential). In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays.

RESULTSHypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS.com, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS.com showed reduced mRNA expressions of APC at 0.27, 0.96, 1.78, 2.70, and 2.03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0.02 and 0.33 times, respectively).

CONCLUSIONThe aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.

Adenomatous Polyposis Coli Protein ; biosynthesis ; genetics ; Breast ; pathology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Genes, APC ; Humans ; Hyperplasia ; genetics ; metabolism ; pathology ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics

Objective To analyze the clinical and genetic characteristics of 10 Chinese patients with 17? hydroxylase/17,20 lyase deficiency (17OHD). Methods Clinical features and laboratory data were collected from 7 kindreds with 17OHD. PCR products and subclone sequencing were performed to screen the mutation of CYP17A1 gene. Results All patients had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. The laboratory examinations indicated decreased plasma cortisol, 17-hydroxy progesterone, estradiol and testosterone, and elevated blood adrenocorticotrophic hormone(ACTH), follcie-stimulating hormone(FSH) and luteinizing hormone(LH). CT scan showed bilateral adrenal hyperplasia. 5 CYP17A1 mutations were identified, 4 of which are novel types D487_F489del, the most frequent mutation, was identified in 4 families and 45% alleles. Conclusion Our study indicates that 17OHD should be considered in the diagnosis of patients with sexual infantilism. D487_F489del is the most frequent mutation in Chinese 17OHD patients.

OBJECTIVE: To investigate the effects of fenofibrate on the proliferation and apoptosis and endothelial nitric oxide synthase (eNOS) mRNA expression of cultured human umbilical vein endothelial cells (HUVECs) induced by lysophosphatidylcholine (LPC). METHODS: HUVECs were cultured in vitro. The study was designated to 5 groups according to fenofibrate concentration: control group, LPC group, LPC + low-concentration fenofibrate (10 micromol/L), LPC + middle-concentration fenofibrate (50 micromol/L), and LPC + high-concentration fenofibrate (100 micromol/L). The study was designated to 6 groups according to the intervention time: control group, LPC group, LPC + fenofibrate (50 micromol/L) 6 h, LPC + fenofibrate 12 h, LPC + fenofibrate 24 h, and LPC + fenofibrate 48 h. The proliferation and apoptosis of HUVECs were evaluated by MTT assay, flow cytometry and fluorescence microscopy, respectively. eNOS mRNA were assayed by real time-PCR. RESULTS: Compared with the control group, LPC could inhibit the proliferation and induce apoptosis, and downregulate eNOS mRNA expression and decrease NO production of HUVECs. Fenofibrate could increase the proliferation and decrease the apoptosis, and up-regulate eNOS mRNA expression and enhance NO production in HUVECs. CONCLUSION Fenofibrate could improve the proliferation and inhibit the apoptosis, and up-regulate eNOS mRNA expression of HUVECs induced by LPC, which may be responsible for fenofibrate to prevent and treat atherosclerosis.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fenofibrate ; pharmacology ; Humans ; Hypolipidemic Agents ; pharmacology ; Lysophosphatidylcholines ; pharmacology ; Nitric Oxide Synthase Type III ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo realize quadratic formula optimization of Renshen Jianxin Capsule (RJC) by screening Chinese herbs with major anti-myocardial ischemia effect in RJC and optimize their optimal dosages.

METHODSBy following "uniform design-pharmacodynamic experiment-mathematical modeling-formula optimization", authors employed U10(10(8)) uniform design in the experiment. Eight Chinese herbs contained in RJC were taken as observatory factors. Electrocardiograph (ECG) changes of myocardial ischemia induced by isoproterenol were taken as pharmacodynamic indices. The mathematical model between herbal factors and pharmacodynamic indices was established using stepwise regression analysis to screen Chinese herbs with major anti-myocardial ischemia effect. Their optimal dosages were optimized using the grid algorithm.

RESULTSThe regression equation was y =1. 7889 -0. 3247 Ginseng xSalvia Miltiorrhiza -0. 0663 Astragalus membranaceus xOriental Waterplantain tuber. Forecasting factors included were Ginseng, Salvia Miltiorrhiza, Astragalus membranaceus, and Oriental Waterplantain tuber. The optimal formula dosage calculated by the grid algorithm was Ginseng 1. 62 g, Astragalus membranaceus 4. 62 g, Salvia Miltiorrhiza 2. 43 g, and Oriental Waterplantain tuber 1. 66 g.

CONCLUSIONUniform design combined with stepwise regression analysis and grid algorithm were able to realize quadratic formula optimization of RJC.

Astragalus membranaceus ; Chemistry, Pharmaceutical ; standards ; Coronary Artery Disease ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Electrocardiography ; Humans ; Isoproterenol ; Myocardial Ischemia ; drug therapy ; Panax ; Salvia miltiorrhiza

OBJECTIVETo explore the inhibitory effect of combined administration of bear bile powder (BBP) and cyclophosphamide (Cytoxan, CTX) on colorectal cancer liver metastasis by regulating tumor promotion inflammation microenvironment.

METHODThe CRC liver metastasis mode in mice was established through in situ spleenic injection of SL4 tumor cells into spleens. The mice were randomly divided into 5 groups: the model group, the CTX (80 mg x kg(-1)) treatment group, the CTX + BBP high dose (300 mg x kg(-1)) group, the CTX + BBP middle dose (150 mg x kg(-1)) group and the CTX + BBP low dose (75 mg x kg(-1)) group. Mice were orally administered with drugs for 12 days, and sacrificed on the 13'h day for weighing their spleens and lives, HE staining, and immunofluorescence analysis. Their peripheral blood, and metastatic tumor in spleens and lives were analyzed with flow cytometry.

RESULTSpleen and liver weights of the: CTX treatment group and other doses groups were significantly lower than that of the model group. HE staining and immunofluorescence analysis showed that lymphocyte infiltration was detected in normal tissues, and macrophages infiltration was observed around the tumor tissues. Flow cytometry analysis showed that the number of T-lymphocytes in peripheral blood of different doses groups were much higher than that of the CTX treatment group (P < 0.05), with the rise in the ratio of CD4/CD8; the total number of lymphocytes in spleen cell suspension increased in different doses groups, compared to the CTX treatment group, with notable increase in B cells (P < 0.05) and significant decrease in CD11b, F4/80 cells (P < 0.05). The combined treatment showed less monocyte macrophages in liver metastasis than that of the CTX treatment group.

CONCLUSIONThe combined treatment of bear bile powder and cyclophosphamide has the effect in not only protecting liver and increase immunity, but also in anti-inflammation and antitumor by regulating tumor microenvironment and reducing the collection of mononuclear macrophages. Particularly, the combined administration of low dose of bear bile powder and CTX shows the most significant effect in reducing inflammatory cell infiltration.

Animals ; Bile ; chemistry ; Colorectal Neoplasms ; drug therapy ; mortality ; pathology ; Combined Modality Therapy ; Cyclophosphamide ; administration & dosage ; Humans ; Liver Neoplasms ; drug therapy ; mortality ; physiopathology ; secondary ; Male ; Mice ; Mice, Inbred C57BL ; Tumor Microenvironment ; drug effects ; Ursidae

Traditional Chinese herbs are readily contaminated by mold that produced mycotoxins which are closly related to the herbs' external factors and external environments during the storage process. In this study, Citri Reticulatae Pericarpium was used as example, and the characteristics of traits, water content, active components (naringin, hesperidin, sinensetin, naringin, tangeretin) and the accumulation of aflatoxins (AFs) were selected as the evaluation indexes. Citri Reticulatae Pericarpium was stored under different environments and packaging materials for 12 months, and then the quality changes and mildew of Citri Reticulatae Pericarpium were examined. The results showed that the color of Citri Reticulatae Pericarpium was deepened after storage, but without mildew phenomenon. Besides, the sample storage in kraft paper and woven bags had varying degrees of moth phenomenon after 12 months storage, and the water content exceeded the limit of Chinese Pharmacopoeia. In addition, the contents of the five active constituents obviously decreased, especially for hesperidin, which did not meet the pharmacopoeia standard after storage. AFs were not detected in any of the tested samples. According to the results, we conclude that low temperature and humidity environment is more suitable for the storage of Citri Reticulatae Pericarpium, and that packaging materials should be further investigated. This study is of great significance for preventing the mold to contaminate the traditional Chinese medicine and ensuring the quality, effectiveness and safety of TCMs.

The present study designed and prepared near-infrared responsive sinomenine hydrochloride(SIN) reservoir microneedles and evaluated the feasibility of this type of microneedles in increasing the drug loading and transdermal absorption by characterizing their mechanical properties and in vitro release characteristics.SIN was selected as the model drug, and methoxy poly(ethylene glycol) poly(caprolactone)(mPEG-PCL) copolymers and indocyanine green(ICG) were employed as amphiphilic block copolymers and light inductor to prepare near-infrared responsive nanoparticles.Based on the preparation principle of bubble microneedles, near-infrared responsive SIN reservoir microneedles were designed and prepared.The features of the near-infrared responsive SIN reservoir microneedles were characterized by measuring the morphology, length, mechanical properties, and skin penetration of microneedles.Meanwhile, the drug release performance of reservoir microneedles was evaluated by in vitro release assay.The results showed that the prepared SIN microneedles were conical, with an exposed tip height of about 650 μm.Each needle could load about 0.5 mg of drugs per square centi-meter, and this type of microneedle showed good mechanical properties and performance in skin penetration.The results of the in vitro release assay showed that the 24 h cumulative release per unit area and release rate of the microneedle were 825.61 μg·cm~(-2) and 74.3%, respectively, which indicated that its release kinetics was in line with the first-order kinetic model.This study preliminarily proved that the reservoir microneedle could effectively increase the drug loading with good mechanical properties and release perfor-mance.
Drug Delivery Systems/methods* ; Drug Liberation ; Indocyanine Green ; Morphinans ; Needles ; Polyethylene Glycols