To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.
Gene Library ; Humans ; RNA, Messenger ; analysis ; U937 Cells ; metabolism

OBJECTIVEMany studies have shown that tissue development is closely correlated with fluid transport. Aquaporins (AQPs) are a group of cell membrane proteins that actively and selectively transport water. This study aimed to investigate the changes of AQPs expression during lung development in rats in order to elucidate the role of AQPs in the rat lung development.

METHODSAQP1, AQP3, AQP4 and AQP5 proteins and mRNA in the lung cell membrane were measured by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) respectively in the 20-day-old embryo (E20), 7-day-old newborn rat, and one-month-old young and adult rats. The correlation between AQPs expression and lung development was studied.

RESULTSWith increasing age, the lung development showed a dynamic and successive course, with the most rapid from the fetus to the newborn rat, and then a slowed down afterwards. AQPs mRNA was weakly expressed in the lung of the E20 group. Lung AQPs mRNA and protein increased rapidly after birth until adulthood. The AQPs distribution patterns in the lung were unique with no duplication. There was a positive correlation between AQPs expression and lung development (P<0.05).

CONCLUSIONSIn addition to being involved in the transepithelial transport of water in the lung, AQPs is also related to its development.

Animals ; Aquaporins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Lung ; embryology ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY between layer-type and meat-type of chickens, which are two divergent kinds of the domestic chickens in feeding behavior and body weight, we detected mRNA levels of NPY in hypothalamic infundibular nucleus (IN), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of these two types of chickens using one-step real time RT-PCR. The meat-type chicken had more food daily (about 1.7 folds) and greater body weights (about 1.5 folds) and brain weights than the layer-type chicken at the age of 14 d. In the meat-type of chicken, NPY mRNA levels of the IN and PVN were significantly greater than those of the LHA, and were not significantly different between the IN and PVN. However, in the layer-type of chicken, NPY mRNA levels were significantly greater in the IN than those in the LHA and PVN, and were not significantly different between the PVN and LHA. In all these hypothalamic regions, the layer-type of chicken had significantly higher NPY mRNA levels than the meat-type chicken did. These results suggest the expression of NPY in the hypothalamus has a type-dependent pattern in domestic chickens.
Animals ; Body Weight ; Chickens ; classification ; metabolism ; Hypothalamus ; metabolism ; Male ; Meat ; Neuropeptide Y ; genetics ; RNA, Messenger ; analysis

OBJECTIVETo explore the changes in the PGE(2) and PGI(2), TXA(2) levels and PGT mRNA expression in the intestinal mucosa in scalded rats.

METHODSWistar rats inflicted with TBSA 30% III degree scald were employed as the model. The PGE(2) and PGI(2) and TXA(2) contents in the intestinal mucosa were measured by radioimmunoassay, and the expression of PGT mRNA was detected by in situ hybridization.

RESULTSThe PGE(2) and PGI(2) levels in intestinal mucosa were increased at 12 postburn hours (PBHs) and thereafter decreased dramatically (P < 0.05). The TXA(2) level in intestinal mucosa of scalded rats was obviously higher than that of normal level at 24 and 48 PBHs (P < 0.05), and the expression of PGT mRNA seemed to be increased after scalding.

CONCLUSIONThe decrease of PGE(2) level and the increase of TXA(2) level in the intestinal mucosa of scalded rats might be involved in rat mucosal injury, and PGT played an important role in the regulation of PGs levels.

Animals ; Burns ; metabolism ; pathology ; Intestinal Mucosa ; metabolism ; pathology ; Organic Anion Transporters ; metabolism ; Prostaglandins ; analysis ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar

To explore a new way to treat CML, inhibitory effect of small interfering RNA (SiRNA) on bcr-abl fusion gene expression of K562 cell line was studied. SiRNA for bcr-abl gene was designed and transfected into K562 cells, bcr-abl gene expression was tested by RT-PCR. The results showed that bcr-abl gene expression was inhibited by using siRNA in dose-dependent manner and reduced to 19.9% and 26.6% of the control at 24 and 48 hours after transfection with 0.2 micro g siRNA respectively. K562 cells proliferation was suppressed finally, but bcr-abl gene expression restored at 72 hours. In conclusion, anti-bcr-abl siRNA can effectively inhibit bcr-abl gene expression of K562 cell line.
Dose-Response Relationship, Drug ; Genes, abl ; Humans ; K562 Cells ; metabolism ; RNA, Messenger ; analysis ; RNA, Small Interfering ; pharmacology ; Transfection

Human immunodeficiency virus type 1 (HIV-1) virus causes severe defect in the immune system and affects the host cell gene expression profoundly. The gene expression pattern will be characterized by changes in cellular mRNA levels that are dependent on both the stage of infection and the biological state of the infected cells. The expression levels of 7,404 cellular RNA transcripts were assessed in H9 cells at different time points after HIV-1 IIIB infection. In total 7 time-points, 959/7,404 (13%) genes were a 2-fold or greater expressed. 387 of 959 genes (40.4%) were up-regulated, and other 572 genes (59.6%) were down-regulated. Three hundred seventeen genes were up-regulated a 2-fold or greater at 72 hr postinfection and 2 to 139 genes were up-regulated at the other time-points. In contrast, 126 to 349 genes were down-regulated a 2-fold or greater in all time-points, excepting 6 hr postinfection. Twenty-three genes were up-regulated a 2-fold or greater over at least four of seven time-points, which were mostly ribosomal proteins and MHCs. Especially, MHCs including HLA-DRA were steadily up-regulated from 24 hr postinfection. Thirty genes were down-regulated a 2-fold or greater in all the time-points, which were mainly related with synthesis and metabolism. These results show that host cell gene expression was altered by HIV-1 infection according to time-points and will provide a framework for studies on interactions between host and HIV-1 infection.
Gene Expression ; HIV-1 ; HLA-DR alpha-Chains ; Immune System ; Metabolism ; Oligonucleotide Array Sequence Analysis ; Ribosomal Proteins ; RNA ; RNA, Messenger

Animals ; Brain ; growth & development ; metabolism ; Emotions ; Glucocorticoids ; therapeutic use ; Humans ; Memory ; RNA, Messenger ; analysis ; Receptors, Glucocorticoid ; analysis ; genetics ; physiology

OBJECTIVETo study the expression of human beta-defensin-3 (hBD-3) induced by lipopolysaccharide (LPS) in human bronchial epithelial (HBE) cells, and explore the role of hBD-3 in respiratory infection.

METHODSHBE cells were stimulated with different concentrations of LPS (0.01, 0.1, 1 and 10 microg/mL). hBD-3 mRNA expression was detected by RT-PCR 2 hrs later. hBD-3 protein expression was detected by Western blot 4 hrs later.

RESULTShBD-3 mRNA and protein was weakly expressed in normal HBE cells. LPS stimulation resulted in a significant increase of hBD-3 mRNA and protein expression (p<0.01). hBD-3 mRNA and protein expression increased with increasing LPS concentrations. There were significant differences in the hBD-3 mRNA and protein expression in cells stimulated by different concentrations of LPS (p<0.05).

CONCLUSIONSLPS can induce hBD-3 expression in a dose-dependent manner. hBD-3 might play a role in initial defensive reaction against bacterial invasion.

Bronchi ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Lipopolysaccharides ; toxicity ; RNA, Messenger ; analysis ; beta-Defensins ; analysis ; genetics

The objective of this study was to investigate the expressions of TPO receptor (c-mpl) proteins on CD34 positive bone marrow cells (CD34+ BMCs), platelets and the expression of c-mpl mRNA in bone marrow cells of the patients with polycythemia vera (PV). The expressions of c-mpl proteins on CD34+ bone marrow hematopoietic cells of 13 PV patients and 15 normal controls were assessed by bicolor flow cytometry and the expressions of c-mpl proteins on platelets of 15 PV patients and 15 normal controls were assessed by monocolor flow cytometry, and the expressions of c-mpl mRNA in bone marrow hematopoietic cells (BMHCs) were assessed by RT-PCR. The results showed that no difference was found between the expression of c-mpl proteins on CD34+ BMHCs of PV patients (0.99% +/- 0.14%) and that of normal controls (0.92% +/- 0.12%) (p > 0.05). There was no difference too between the expression of c-mpl protein on platelets in PV patients (20.33% +/- 4.84%) and that in normal controls (23.50% +/- 3.64%) (p > 0.05). No difference between the expression of c-mpl mRNA in BMHCs of PV patients and that in normal controls was seen. In conclusion, the expressions of c-mpl proteins on CD34+ BMHCs, platelets and c-mpl mRNA in BMHCs of PV patients were not obviously abnormal.
Antigens, CD34 ; analysis ; Blood Platelets ; metabolism ; Bone Marrow Cells ; metabolism ; pathology ; Hematopoietic Stem Cells ; metabolism ; pathology ; Humans ; Polycythemia Vera ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Thrombopoietin ; metabolism

High concentrations of oxygen, indispensable for the treatment of severe hypoxemia from neonatal as well as adult respiratory distress syndrome, increase the risk of oxygen toxicity. Biochemical mechanisms are lipid peroxidation, protein sulfhydryl oxidation, enzyme inactivation, and DNA damage. Recent reports suggest that cytokines might be involved in free radical injury as well as in adaptive response to hyperoxic injury. However, actual signal transduction pathways involving cytokines have not yet been clarified. In this study we exposed cultured human umbilical vein endothelial cells (HUVECs) to either ambient air or 100% oxygen, and compared for the rate of DNA synthesis ([3H]thymidine uptake) at different time points up to 72 h. After exposing the cells to each treatment condition, we extracted RNA, constructed complementary DNA using reverse transcriptase, amplified the specific DNA segments of cytokines by polymerase chain reaction (PCR), and used the PCR products for gel electrophoresis to examine the bands which signified mRNA levels of corresponding cytokines. There was a significant decrease in the rate of DNA synthesis as early as 24 h. The mRNA expression of IL-1 beta and TNFa seemed less influenced by hyperoxia, while IL-8 and TGF beta showed marked increase in mRNA levels at 6 h of 100% oxygen exposure.
Cells, Cultured ; Cytokines/genetics* ; DNA/biosynthesis ; Endothelium, Vascular/metabolism* ; Human ; Hyperoxia/metabolism* ; RNA, Messenger/analysis* ; Umbilical Veins