Objective To investigate the underlying mechanism of bone marrow mesenchymal stem cells (BMSC) modulating the inflammatory response during the multiple organ dysfunction syndrome (MODS), especially the expression of inflammatory cytokines, which will provide new theoretical and experimental basis of MODS in clinic. Methods BMSC of Sprague-Dawley (SD) rat (female, 4 weeks) was extracted and cultivated, and the 4th passage were used in experimental study. According to the random number table, 60 female SD rats were divided into three groups (n = 20 per group): sham group, MODS group, BMSC group. MODS model in rats was induced by lipopolysaccaride (LPS, 1 mg/kg) via femoral vein injection. Sham group was injected with the sterile phosphate buffer saline (PBS) in the same volume. BMSC group, in which BMSC infusion was started at 2 hours after 0.5 mL LPS stimulation (1×106/cells) through the tail vein. The survival rate was observed after 72 hours in each group. Abdominal aortic blood was collected for routine blood and biochemical examination at 72 hours after operation. Protein microarray was used to detect the related 34 inflammatory cytokines. Signal ratio was defined as the differentially expressed factors when it was more than 2.0 or less than 0.5. And enzyme linked immunosorbent assay (ELISA) was be applied to validate the significant inflammation factor. Meanwhile, the heart, kidney, intestine tissue was harvested, then their pathological changes were observed by hematoxylin eosin (HE) staining.Results 20, 12, 16 rats lived in sham group, MODS group and BMSC group respectively at 72 hours after operation. Compared with the sham group, the indicators (routine blood, liver and kidney function, myocardial enzyme) were apparently unusual, and the heart, kidney, intestine tissue were injured obviously in the MODS group. After BMSC administration, the organ function was improved and tissue damaged was alleviated significantly. Protein microarray showed that interleukin-4 (IL-4) and receptor for advanced glycation end products (RAGE) were significantly different in 34 goal cytokines. The signal ratio change of IL-4 was 0.397, 1.124, 2.826 respectively, and the signal ratio of RAGE was 6.197, 1.552, 0.250, respectively in MODS/sham group, BMSC/sham group, BMSC/MODS group. ELISA validated the result that the expression level of IL-4 decreased significantly (ng/L:3.59±1.21 vs. 29.10±5.78) and the expression level of RAGE increased significantly (ng/L: 1.09±0.04 vs. 0.11±0.03) in MODS group as compared with sham group (bothP < 0.05). Compared with the MODS group, the level of IL-4 was obviously higher than that in BMSC group (ng/L: 9.59±2.21 vs. 3.59±1.21,P < 0.01), and RAGE decreased significantly (ng/L: 0.29±0.07 vs. 1.09±0.04,P < 0.05).Conclusions BMSC administration can regulate the expression of IL-4 and RAGE in the rats subjected to MODS. Moreover, BMSC can promote the restoration of tissue and organ function, thus improve the survival rate. BMSC may be the target in cell therapy for the inflammatory disease.

Candida shehatae xyl1 gene and Pichia stipitis xyl2 gene were amplified by PCR and the xyl1 and xyl2 were both placed under the promoter GAL of vector pYES2 to produce the recombinant expression vector pYES2-P12. Subsequently the pYES2-P12 vector was transformed into S. cerevisiae YS58 by LiAc to produce the recombinant yeast YSS8-12. It was indicate that the recombinant yeast YSS8-12 could converse xylose to ethanol with the xylose consumption rate of 81. 3%.

Objective To investigate the expression of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) in astrecytomas, as well as the correlation between them. Methods The expression of COX-2, EGFR and PCNA were respectively detected by immunohistochmical (S-P) method in 68 astrocytomas and 5 cases normal brain tissue. Proliferation index (PI) was calculated and the correlation of COX-2, EGFR and PI was analyzed. Results COX-2 and EGFR were negative expression in normal brain tissue. The positive expression rate of COX-2 and EGFR in high grade astrocytomas was significantly higher than that in low grade astrocytomas(73.53% vs 44. 18% ,67.65% vs 38.24%, P <0. 01 ), and the PI was significantly higher than that in low grade astrocytumas as well as normal brain tissue(46.11 ± 10. 68vs 23. 04±6. 25,4. 52±0. 95, P <0. 01 ). The PI in COX-2 positive group was higher than that in negative group( P <0. 01 ). The positive expression rate of COX-2 in the group with EGFR positive expression was higher than that in the negative group. Conclusions The expression of COX-2 and EGFR was related to pathological feature of astrocytomas. COX-2 may promote the proliferation of tumor cells. There was a static correlation between the expression of EGFR and COX-2 in astrocytomas. EGFR signal transduction probably modulated the expression of COX-2 in astrocytomas cells.

Objective To investigate the effect of microcireulation blood flow and altering immunity by Chuanxiongqin,ulinastain and thymosin α1 on sepsis patients.Methods 90 patients were randomly divided into 3 groups(n=30),namely ICU group,Chuanxiongqin group,ulinastain and thymosin α1 group.HLA-DR/CD14+and IL-6,TNF-α,Lac,DD were measured.Results (1)DD showed no significant difference at every time point between ICU group and ulinastain+thymosin α1 group(P＞0.05).DD decreased in Chuanxiongqin group,and was significantly different from the others on the third day.(2)Lac unchanged significantly at every time point in ICU group(P＞0.05).Lac in Chuanxiongqin group and ulinastain+thymeain α1 group tended to decrease,and was statistically different from ICU group on the second day.(3)IL-6 and TNF-α tended to increase at every time point in ICU group(P＜0.05).In ulinastain+thymosin α1 group,IL-6 and TNF-α returned to the level before treatment,HLA-DR/(D14+increased significantly,and was higher than Chuanxiongqin group and ICU group statistically.Conclusion Chuanxiongqin could ameliorate circulation;ulinastsin and thymosin α1 could depress IL-6,TNF-α.So ulinastain and thymosin α1 might protect the immunity of sepsis patients.

BACKGROUNDThrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury. In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species (ROS) can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.

METHODSROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin (20 U/ml) exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 (ERK1/2) signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.

RESULTSThrombin, at 20 U/ml, stimulated human lung fibroblasts (HLF) to generate ROS in a time dependent manner. The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.

CONCLUSIONThe activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.

Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; analysis ; physiology ; Fibroblasts ; drug effects ; physiology ; Flow Cytometry ; Glutathione ; metabolism ; Humans ; Lung ; cytology ; NADPH Oxidases ; analysis ; physiology ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology ; Thrombin ; pharmacology

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca2+, Mg2+, K+, and HCO3 - in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.
ATP-Binding Cassette Transporters/*chemistry/*genetics/metabolism ; Adenosine Triphosphate/metabolism ; Amino Acid Sequence ; Animals ; Calcium/metabolism ; *Cloning, Molecular ; Cryptosporidiosis/parasitology ; Cryptosporidium/chemistry/genetics/*metabolism ; Humans ; Iron/metabolism ; Mice ; Molecular Sequence Data ; Protein Structure, Tertiary ; Protozoan Proteins/*chemistry/*genetics/metabolism ; Sequence Alignment

The aim of this study was to investigate the effect of urotensin II (U II) on the nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. The endothelial nitric oxide synthase (eNOS) mRNA expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction. The activity of nitric oxide synthase (NOS) and NO content in cardiomyocytes were measured. The current results showed that U inhibited eNOS mRNA expression, the NOS activity and the NO production of cardiomyocytes. U II (0.1 micromol/L) inhibited the NOS activity and the NO production in cardiomyocytes in a time-dependent manner. These results suggest that the cardiovascular effect of U II might be partially associated with NO production in cultured neonatal rat cardiomyocytes.
Animals ; Animals, Newborn ; Cells, Cultured ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Urotensins ; pharmacology

The experience with the umbilical cord blood (UCB) stem cells for unrelated transplantation from our 3 000 UCB storage was described. UCB, collected from closed blood bags, were mixed with hydroxyethyl starch for nucleated cell (NC) enrichment. After finishing CD34 analysis, culture of hematopoietic progenitors (CFU-GM and CFU-GEMM) assays, microbial culture, HLA Class I (A, B) serology and class II (DR) low resolution SSP typing, cord blood units are stored in the liquid nitrogen for clinical applicatoin. Cord blood contained an average of nuclear cell (NC) (1.2 +/- 0.6) x 10(9), CD34(+) cells (3.0 +/- 3.7) x 10(6), CFU-GM (1.1 +/- 0.7) x 10(6) and CFU-GEMM (1.1 +/- 1.2) x 10(6) for storage and the recovery rates were 91%, 88%, 85% and 82%, respectively. The recovery rates for red blood cell and Hb were (39 +/- 9)% and (40 +/- 8)%, respectively. The storage volume was (35.1 +/- 7.1) ml in a 50 ml storage bags. The mean time from collection to processing of 15 hours (range 4 - 24 hours) had no influence on cell viability. The cell viability before processing is more than 95% and 92% after UCB thawing. The recovery rates of NC, CD34(+) cells and CFU-GM post-thawing were 96%, 90% and 91%, respectively. There were no HIV antibody (HIVAb) positive in all of UCB units. For an incidence of processed samples, infection with syphilis, HBsAg, HBcAb, HCVAb, CMV, bacterial contamination and abnormal hemoglobin were 0.1%, 0.8%, 3.2%, 0.2%, 87.1%, 1.2% and 0.1%, respectively. More than 3 HLA loci matched can be found for random patients in our cord blood bank and 6 HLA loci matched have 5%. For transplantation with nucleated cell counts of > 2.7 x 10(7) cells/kg, our cord blood bank will be able to provide all of the umbilical cord blood stem cell samples for children and 50% of units can be used for some of adult recipients transplantation in the country. It is concluded that: (1) The large cord blood banking for 20 000 UCB storage is feasible in China. (2) Our system of whole procedure and methods is functionable for supplying qualified cord blood units in transplantation. (3) The volume for collection is critical to the yield of CD34(+) cells or hematopoietic progenitor cells, however cord blood NC is also important and proportional with CD34(+) cells. Only the units containing more than 8 x 10(8) cells and more than 60 ml of cord blood can be in the procession for storage.

OBJECTIVETo investigate the effects of saturated hydrogen saline on the prevention of abdominal aortic aneurysm (AAA) induced by calcium chloride in a rat model.

METHODSIn healthy male Sprague-Dawley rats, AAA was induced by infiltration of abdominal arota with 0.5 mol/L calcium chloride. Saturated hydrogen saline (5 ml·kg(-1)·d(-1)) or saline was administred intraperitoneally once daily. Twenty-eight days later, the diameter of the aorta was measured, and the aortic tissue was exercised for histological examination. Pro-inflammatory cytokines (tumor necrosis factor α (TNF-α), IL-1β) in AAA tissue were detected with ELISA. The protein expression and mRNA expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 in AAA tissue were observed by immunohistochemistry staining and real-time PCR.

RESULTThe aorta diameter of the experiment group and control group were (2.2 ± 0.3) mm and (3.4 ± 0.5) mm, the tissue IL-1β levels were (81 ± 29) ng/L and (165 ± 51) ng/L, the tissue TNF-α levels were (109 ± 46) ng/L and (360 ± 51) ng/L, the relative mRNA expressions were 2.4 ± 1.0 and 11.8 ± 2.9, the relative mRNA expressions were 2.9 ± 0.6 and 6.7 ± 1.0 (t = 4.055 to 10.406, P < 0.05). Compared with the control group, the infiltration of inflammation, the injury of elastic fibers in the vessel wall, and the positive expression of MMP-2 and 9 protein of the experiment group were all reduced.

CONCLUSIONSSaturated hydrogen saline prevents the degradation of elastin in vessel wall and ameliorates the formation and development of AAA, which may be associated with its anti-inflammatory effects, thereby reduces the MMP-2 and 9 mRNA and protein expression.

Animals ; Aorta, Abdominal ; pathology ; Aortic Aneurysm, Abdominal ; prevention & control ; Disease Models, Animal ; Hydrogen ; pharmacology ; Interleukin-1beta ; metabolism ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

In order to explore the transfection and expression of hRI gene on human umbilical blood stem cells, and observe it's effect on the tumor growth. After enriching human umbilical cord blood CD34+ cells with a high-gradient magnetic cell sorting system (MACS), transfected them with supernatant of retrovirus containing human Ribonuclease inhibitor (hRI) cDNA. Hematopoietic progenitor clonogenic assay and PCR were used to evaluate transfection efficiency, and Western-blot and immune fluorescence were used to evaluate the expression quantity of hRI gene after transfection. Observe the effect of RI on the growth of melonoma in B16C57BL mice. The results showed that human umbilical blood CD34+ cells were highly purified by MACS, which made the purity of human umbilical blood CD34+ cells average 96.15%. hRI can be transfected on umbilical blood CD34+ cells, and the transfection efficiency was 35%. The positive expression of hRI gene on transfected CD34+ cells is identified by Western-blot and immune fluorescence assay. Mice injected with transfected CD34+ cells show a significant restraint of the tumor growth, a lower efficiency of tumor formation, a lower weight of the tumor and a longer incubation period of tumor formation with respect to the control groups. The results demonstrated the capacity of RI to inhibited the tumor growth by blocking the vasculature in tumor.
Animals ; Antigens, CD34 ; metabolism ; Cell Proliferation ; drug effects ; Fetal Blood ; cytology ; Genetic Therapy ; Hematopoietic Stem Cells ; metabolism ; Humans ; Melanoma ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured