Mesenchymal Stem Cell Transplantation: Neuroprotection and Nerve Regeneration After Spinal Cord Injury.

Spinal Cord Injury (SCI), with its morbidity characteristics of high disability rate and high mortality rate, is a disease that is highly destructive to both the physiology and psychology of the patient, and for which there is still a lack of effective treatment. Following spinal cord injury, a cascade of secondary injury reactions known as ischemia, peripheral inflammatory cell infiltration, oxidative stress, etc. create a microenvironment that is unfavorable to neural recovery and ultimately results in apoptosis and necrosis of neurons and glial cells. Mesenchymal stem cell (MSC) transplantation has emerged as a more promising therapeutic options in recent years. MSC can promote spinal cord injury repair through a variety of mechanisms, including immunomodulation, neuroprotection, and nerve regeneration, giving patients with spinal cord injury hope. In this paper, it is discussed the neuroprotection and nerve regeneration components of MSCs' therapeutic method for treating spinal cord injuries.

Mechanism of M2 macrophages modulating astrocyte polarization through the TGF-ß/PI3K/Akt pathway.

Recent studies have revealed that activated astrocytes (AS) are divided into two distinct types, termed A1 and A2. A2 astrocytes are neuroprotective and promote tissue repair and regeneration following spinal cord injury. Whereas, the specific mechanism for the formation of the A2 phenotype remains unclear. This study focused on the PI3K/Akt pathway and examined whether TGF-ß secreted by M2 macrophages could mediate A2 polarization by activating this pathway. In this study, we revealed that both M2 macrophages and their conditioned medium (M2-CM) could facilitate the secretion of IL-10, IL-13 and TGF-ß from AS, and this effect was significantly reversed after the administration of SB431542 (a TGF-ß receptor inhibitor) or LY294002 (a PI3K inhibitor). Moreover, immunofluorescence results demonstrated that TGF-ß secreted by M2 macrophages could facilitate the expression of A2 biomarker S100A10 in AS; combined with the results of western blot, it was found that this effect was closely related to the activation of PI3K/Akt pathway in AS. In conclusion, TGF-ß secreted by M2 macrophages may induce the conversion of AS to the A2 phenotype through the activation of the PI3K/Akt pathway.

Pancytopenia after Low-Dose Methotrexate Therapy in Two Hemodialysis Patients with Rheumatoid Arthritis.

Methotrexate (MTX) is an effective medication in the treatment of rheumatoid arthritis (RA), other rheumatic diseases and various solid tumors. However, its side effects, including gastrointestinal discomfort, oral ulcers, and especially bone marrow suppression, could be fatal and require special attention, particularly in patients with renal failure. We present two hemodialysis patients with RA who presented with a complication of severe pancytopenia after treatment with MTX. After receiving various supportive and blood purification treatments, both patients recovered. We reviewed twenty-four pancytopenia patients on dialysis associated with methotrexate. Among these patients, high morbidity and mortality were observed, indicating that MTX should be used cautiously in the absence of alternatives in such a population. Compared with the patients who recovered, the deceased patients showed a lower level of leukocytes. Which dialysis method might be the best choice is unclear. The mode of renal replacement therapy can be chosen according to the actual situation.

Altered Plasma Metabolic Profiles in Chinese Patients With Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune disease that leads to the demyelination of nerve axons. An increasing number of studies suggest that patients with MS exhibit altered metabolic profiles, which might contribute to the course of MS. However, the alteration of metabolic profiles in Chinese patients with MS and their potential roles in regulating the immune system remain elusive. In this study, we performed a global untargeted metabolomics approach in plasma samples from 22 MS-affected Chinese patients and 21 healthy subjects. A total of 42 differentially abundant metabolites (DAMs) belonging to amino acids, lipids, and carbohydrates were identified in the plasma of MS patients and compared with those in healthy controls. We observed an evident reduction in the levels of amino acids, such as L-tyrosine, L-isoleucine, and L-tryptophan, whereas there was a great increase in the levels of L-glutamic acid and L-valine in MS-affected patients. The levels of lipid and carbohydrate metabolites, such as sphingosine 1-phosphate and myo-inositol, were also reduced in patients with MS. In addition, the concentrations of proinflammatory cytokines, such as IL-17 and TNF-α, were significantly increased, whereas those of several anti-inflammatory cytokines and chemokines, such as IL-1ra, IL-7, and MIP-1α, were distinctly reduced in the plasma of MS patients compared with those in healthy subjects. Interestingly, some DAMs, such as L-tryptophan and sphingosine 1-phosphate, showed an evident negative correlation with changes in the level of TNF-α and IL-17, while tightly positively correlating with altered concentrations of anti-inflammatory cytokines and chemokines, such as MIP-1α and RANTES. Our results revealed that altered metabolomic profiles might contribute to the pathogenesis and course of MS disease by modulating immuno-inflammatory responses in the peripheral system, which is essential for eliciting autoimmune responses in the central nervous system, thus resulting in the progression of MS. This study provides potential clues for developing therapeutic strategies for MS in the near future.

Fresh human amniotic membrane effectively promotes the repair of injured common peroneal nerve.

Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with α-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin-eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve; the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.

Combination of G-CSF and AMD3100 Improves the Anti-inflammatory Effect of Mesenchymal Stem Cells on Inducing M2 Polarization of Macrophages Through NF-κB-IL1RA Signaling Pathway.

Mobilized peripheral blood-derived mesenchymal stem cells (PB-MSCs) mainly derived from bone marrow-derived MSCs (BM-MSCs) exert a similar anti-inflammatory effect. However, the mechanism of anti-inflammatory effect of mobilized PB-MSCs by a combination of G-CSF and AMD3100 remains unclear. Cultured rat PB-MSCs mobilized by G-CSF/AMD3100 have shown typical surface markers and potential for multiple differentiations, similar to non-mobilized BM-MSCs. In a co-culture system, rat M0-type macrophages co-cultured with PB-MSCs have shown higher expression of M2 markers including CD206, Arg-1, IL-10, and CCL-22 than BM-MSCs, indicating that PB-MSCs induced greater M0 polarization to M2. Furthermore, compared with BM-MSCs, PB-MSCs in a co-culture system with lipopolysaccharide-induced M1-type macrophages more efficiently promoted M1 polarization to M2, accompanied by increasing expression of CD206, Arg-1, IL-10, and CCL-22 while decreasing expression of M1 markers including iNOS, TNF-α, IL-1ß and IL-6, indicating that PB-MSCs triggered greater M1 polarization to M2. Subsequently, polymerase chain reaction arrays showed higher expressions of both IL1rn and Tnfrsf11b in PB-MSCs versus BM-MSCs. In response to an inflammatory niche, such as TNF-α, PB-MSCs have shown higher expression and release of IL1RA, causing greater M2 polarization of macrophages, and the special effects may be almost entirely abolished through the neutralization antibody of IL1RA. Mechanistic studies determined that PB-MSCs showed higher levels NF-κBp65 and NF-κBp-p65 than BM-MSCs, which could be obviously enhanced by TNF-α. And the increased IL1RA expression by TNF-α in PB-MSCs could be markedly canceled by an NF-κB inhibitor PDTC. Interestingly, mimicking the mobilized PB-MSCs by a combination of G-CSF and AMD3100 in vivo, BM-MSCs were treated with G-CSF and/or AMD3100 in vitro, showing the increased expressions of NF-κBp65 and IL1RA, which could be prominently abolished by PDTC. Therefore, targeting IL1rn, gene modification or drug intervention for MSCs may provide a novel therapeutic strategy for human diseases, especially inflammatory diseases.

PSMD4 regulates the malignancy of esophageal cancer cells by suppressing endoplasmic reticulum stress.

Proteasome 26S subunit non-ATPase 4 (PSMD4) is an important proteasome ubiquitin receptor and plays a key role in endoplasmic reticulum stress (ERS). However, the study of PSMD4 in esophageal cancer (EC) is relatively rare. Here, we found that the expression of PSMD4 was markedly enhanced in EC tissues and cell lines. The cell counting kit-8 (CCK-8) assay showed that overexpression of PSMD4 significantly enhanced Eca109 cell viability, while inhibition of PSMD4 reduced Eca109 cell viability. Knockdown of PSMD4 induced Eca109 cell apoptosis and cell cycle arrest. More importantly, knockdown of PSMD4 significantly enhanced the expression of glucose regulated protein 78, activating transcription factor 6, and p-protein kinase R-like ER kinase, indicating an enhanced ERS response in esophageal cancer cells. Compared with the control cells, brefeldin A significantly inhibited the expression of PSMD4 and increased the expression of p53-upregulated modulator of apoptosis. However, such effects were largely reversed after overexpressing PSMD4 in Eca109 cells, suggesting that silencing PSMD4 could enhance ERS-induced cell apoptosis. In summary, upregulation of PSMD4 promoted the progression of esophageal cancer mainly by reducing ERS-induced cell apoptosis.

A New Method Aimed to Quickly Identify Pathogen and Drug Susceptibility Test Based on Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Combined with Flow Cytometry.

AIM: To discuss the significance and applied value in the rapid identification and drug susceptibility test for blood stream infection (BSI) using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) combined with flow cytometry (FCM). METHODS: The bacteria were separated from the positive blood culture bottle using the separation gel-adsorption method system, and then applying MALDI-TOF MS combined with FCM to identify pathogen and drug susceptibility test quickly. RESULTS: The efficiency of the separation gel-adsorption method for gram-negative bacterium, gram-positive bacteria, and fungi is 71%, 74%, and 88%, respectively. The results of identifying pathogens using MALDI-TOFMS are in agreement with results obtained using VITEK®2 (bioMérieux, Marcy l'Etoile, France); both methods can identify 90% of bacteria to species. For fungi, MALDI-TOF MS can identify 75% fungi to species, which is superior to VITEK2, which identifies 60% fungi to species. The results of drug susceptibility test using FCM are almost identical to VITEK2; additionally, the addition of fluorescein diacetate can identify the heterogenic drug-resistant strains. CONCLUSIONS: We can quickly identify pathogen and drug-susceptibility test based on MALDI-TOF MS combined with FCM, which is consistent with traditional methods and can shorten the report time from 36-72 hour to 3 hours. More importantly, these methods are of great significance and clinical importance for the rapid identification of BSI.

MicroRNA-224 inhibition prevents progression of cervical carcinoma by targeting PTX3.

Cervical carcinoma is known as one of the most lethal and common conditions in women worldwide. Increasing evidence shows that microRNAs (miRs) may be involved in the pathogenesis of cervical carcinoma. This study investigates the correlation between expression of miR-224 in peripheral blood mononuclear cells and both diagnosis and prognosis of cervical carcinoma to clarify the effect miR-224 has on the biological behaviors of the subjected cervical carcinoma cells. Initially, 132 patients diagnosed with cervical carcinoma and 120 healthy subjects were recruited. Peripheral blood expression of miR-224 and PTX3 was detected. A telephone follow-up was performed every 3 months after treatment. The diagnostic value of miR-224 in cervical carcinoma was analyzed using the Receiver Operating Characteristic curve. The effects of both miR-224 and PTX3 on cell proliferation, migration, and invasion were evaluated with an intervention of miR-224 ectopic expression or depletion and PTX3 silencing. The bioinformatics prediction website and dual-luciferase reporter assay revealed PTX3 to be a target gene for miR-224. Moreover, miR-224 was detected as over-expressed, but PTX3 was under-expressed in cervical carcinoma. Additionally, as a diagnostic biomarker, a high miR-224 expression was closely linked with the progression of cervical carcinoma. Both miR-224 overexpression and PTX3 silencing promoted cell proliferation, migration, and invasion, whereas, the aforementioned properties were depressed when miR-224 was inhibited. Altogether, the miR-224 overexpression may be a biological indicator in predicting the progression of cervical carcinoma. Thus, miR-224 inhibition may significantly prevent cervical carcinoma progression by targeting the PTX3 gene.

Preclinical Study of Cell Therapy for Osteonecrosis of the Femoral Head with Allogenic Peripheral Blood-Derived Mesenchymal Stem Cells.

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.

Aldehyde dehydrogenase 2 polymorphism as a protective factor for intracranial vascular stenosis in ischemic stroke in Han Chinese.

AIM: Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme that metabolizes acetaldehyde to acetic acid. ALDH2 gene polymorphism modifies its activity and the mutation of ALDH2 gene has been reported to be associated with the protection against ischemic stroke. However, the potential association of allelic variation of ALDH2 with intracranial vascular stenosis and the clinical characteristics of ischemic stroke without coronary artery disease remains unclear. METHODS: In this study, ischemic stroke patients were recruited, National Institutes of Health Stroke Scale scores were analyzed, intracranial arterial stenosis were evaluated by magnetic resonance angiography and gene typing of ALDH2 was determined by polymerase chain reaction and sequencing. RESULTS: We found that the rate of heavy drinking was significantly lower in the ALDH2 mutation group ((*)1/(*)2 and (*)2/(*)2) than in wild-type group ((*)1/(*)1) (18.6% vs. 38.0%, p = 0.01). Plasma homocysteine (Hcy) levels were significantly different in the two groups (15.45 ± 6.39 vs. 13.14 ± 4.45, p = 0.015). The ALDH2 mutation genotype was negatively correlated with severe intracranial vascular stenosis (OR, 0.34; p = 0.002), even after adjustment for high-density lipoprotein cholesterol, Hcy, and heavy drinking (adjusted OR, 0.44; p = 0.03). CONCLUSION: ALDH2(*)2 could be a protective factor and negative predictor for severe intracranial vascular stenosis in ischemic stroke in Han Chinese.

[Enrichment and Biological Characteristics of Peripheral Blood-derived Mesenchymal Stem Cells in Rats].

OBJECTIVE: To explore the effective method for enrichment of rat peripheral blood-derived mesenchymal stem cells(PBMSC) and study the cell biological characteristics. METHODS: Peripheral mononuclear cells were isolated by density gradient centrifugation from blood of 4 week old rats after G-CSF mobilization. Thereafter, the fibroblast-like cells were acquired by plastic-adherent culture, and the proliferation curve was assayed. For analyzing surface markers of the second generation cultured isolated PBMSC, both flow cytometry(CD90, CD44, CD29, CD45, CD11b and CD79a) and immunocytochemical staining(CD73, CD105, CD34 and HLA-DR) methods were used. Furthermore, the differentiation capacities of PBMSC into osteocytes, chondrocytes and adipocytes were identified. RESULTS: (1) The adherent cells displayed typical colony-forming unit fibroblast(CFU-F) growth pattern after 6-7 day of primary culture and reached 80% confluence after 21 days of culture. The passaged PBMSC possessed high proliferative capacity and spindle growth pattern and was able to grown into exponential phase next day with a doubling time of 39.2 h. (2) PBMSC expressed mesenchymal markers such as CD90, CD44, CD29, CD73 and CD105, but failed to expressed markers of CD45, CD11b, CD79a, CD34 and HLA-DR. (3) After 21 days of culture in osteogenic, chondrogenic and adipogenic differentiation media, calcifying nodules, intracellular glycosaminoglycans and lipid droplets could be found by alizarin red, alcian blue and oil red-O staining, respectively. CONCLUSION: PBMSC can be enriched from rat peripheral blood with high purity and abundance by our methods. The growth and phenotypic characteristics of the isolated PBMSC are consistent with that of well-known MSC, and these cells possess the capability to multi-lineage mesoderm differentiation.

Isolation and Characterization of Rat Mesenchymal Stem Cells Derived from Granulocyte Colony-Stimulating Factor-Mobilized Peripheral Blood.

Mesenchymal stem cells (MSCs) have been isolated from many tissues and organs. However, there is much dispute as to whether MSCs exist in peripheral blood. This may be due to the limited identification methods of MSCs, especially the lack of detection markers for phenotypic characteristics. In this study, as many as 10 surface markers of MSCs derived from rat peripheral blood (rPBMSCs) were analyzed after granulocyte colony-stimulating factor mobilization. Our results suggest that mobilized rPBMSCs overexpress mesenchymal markers, including CD90, CD44, CD29, CD73 and CD105, but do not express CD45, CD11b, CD79a, CD34 or HLA-DR. This is in conformity with the standard definition of MSCs by the International Society for Cellular Therapy. In addition, the colony-forming efficiency of the mobilized rat peripheral blood was 15.83 ± 1.61/106, significantly outnumbering that of the nonmobilized group, which was 0.28 ± 0.1/106 (p < 0.01). Combining the growth characteristics with the differential capacities of mobilized rPBMSCs towards forming osteocytes, chondrocytes and adipocytes, we further confirmed the existence of rPBMSCs. Additionally, this treatment could improve locomotive function after spinal cord injury (SCI) in rats. Due to their convenient collection, fewer complications, cost effectiveness and suitability for autograft, PBMSCs might be a substitute for MSCs derived from bone marrow and provide promising prospects for the cell-based therapy of SCI.

Rutaecarpine inhibits angiotensin II-induced proliferation in rat vascular smooth muscle cells.

OBJECTIVE: To evaluate the effects and possible mechanisms of rutaecarpine on angiotensin II (Ang II)-induced proliferation in cultured rat vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from Male Sprague-Dawley rat aorta, and cultured by enzymic dispersion method. Experiments were performed with cells from passages 3-8. The cultured VSMCs were randomly divided into control, model (Ang II 0.1 µmol/L), and rutaecarpine (0.3-3.0 µmol/L) groups. VMSC proliferation was induced by Ang II, and was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and cell counting. To examine the mechanisms involved in anti-proliferative effects of rutaecarpine, nitric oxide (NO) levels and NO synthetase (NOS) activity were determined. Expressions of VSMC proliferation-related genes including endothelial nitric oxide synthase (eNOS), and c-myc hypertension related gene-1 (HRG-1) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Rutaecarpine (0.3-3.0 µmol/L) inhibited Ang II-induced VSMC proliferation and the best effects were achieved at 3.0 µmol/L. The Ang II-induced decreases in cellular NO contents and NOS activities were antagonized by rutaecarpine (P <0.05). Ang II administration suppressed the expressions of eNOS and HRG-1, while increased c-myc expression (P <0.05). All these effects were attenuated by 3.0 µmol/L rutaecarpine (P <0.05). CONCLUSION: Rutaecarpine is effective against Ang II-induced rat VSMC proliferation, and this effect is due, at least in part, to NO production and the modulation of VMSC proliferation-related gene expressions.

[The research of fibroblasts from human hypertrophic scar showing a mesenchymal stem cell phenotype and multilineage differentiation potentialities].

OBJECTIVE: To explore the CD phenotypic, protein expression and pluripotent differentiation of human hypertrophic scar fibroblasts cultured in vitro, so as to study the mechanisms of scar formation. METHODS: Fibroblasts were isolated and cultured from human hypertrophic scar of 3 cases. The cells morphology was observed by inverted microscope, and the growing state of the third passage was detected by the cell counting meter of Vi-CELL. The cell surface markers CD105, CD14, CD73, CD34, CD44, CD45 and CD90 were identified by flow cytometry. The expression of CK19, Oct-4, Nanog and vimentin was detected by immunocytochemistry, and the expression of alpha-smooth muscle actin(alpha-SMA) was tested by immunofluorescence. The differentiated potential of fibroblasts of the third passage into adipogenic, osteogenic and chondrogenic lineages was assayed. RESULTS: The primary passage fibroblasts showed the shape of spindle shaped or irregular polygon with a radiated or circinate of growing arrangement. The growth curve showed the cells growth was slow on the first and second day, and quick during the third to fifth day, which reached platform stage on the sixth or seventh day. The fibroblasts highly expressed mesenchymal stem cell surface markers-CD73, CD105, CD44, CD90, but not expressed hematopoietic stem cell surface markers-CD14, CD34, CD45 by flow cytometry. And positive expression of vimentin, Oct-4 and negative expression of CK19 were detected by Immunocytochemistry. Positive expression of alpha-SMA was also detected by immunofluorescence. Multidirectional differentiation induction indicated that the third passage cells could differentiate into adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Human hypertrophic scar-derived fibroblasts show the biologic characteristics of mesenchymal stem cells, which may play an important role in wound healing and hypertrophic scar formation.

[Effects of weile powder on bicarbonate transporters CFTR SLC26A3 and SLC26A6 in gastric ulcers of rats].

OBJECTIVE: To investigate the effects of Weile Powder (WLP) on bicarbonate transporters in rats with gastric ulcers, and to probe its functional mechanisms. METHODS: The 48 SD rats were randomly divided into the normal control group, the model group, the low dose WLP group (at the daily dose of 0.075 g/mL), the middle dose WLP group (at the daily dose of 0.150 g/mL), the high dose WLP group (at the daily dose of 0.030 g/mL), and the ranitidine group (at the daily dose of 0.030 g/mL), 8 in each group. The gastric ulcer rat model was prepared by the glacial acetic acid cauterization method. Rats in each medication group were administered from the 2nd day of modeling. Rats were sacrificed after 14-day successive medication. The protein was extracted from the ulcer tissue. The protein expressions of solute carrier26A3 (SLC26A3)and solute carrier26A6 (SLC26A6) were detected using Western blot. The gastric ulcer and its peripheral tissue were sectioned. The changes of cystic fibrosis transmembrane conductance regulator (CFTR) were measured by immunofluorescence. RESULTS: Compared with the model control group, the expression levels of SLC26A3 increased in the high dose WLP group and the ranitidine group with statistical difference (P < 0.05). The expression levels of SLC26A6 increased in the high and middle dose WLP groups and the ranitidine group with statistical difference (P < 0.05). The expression level of CFTR also obviously increased in the high and middle dose WLP groups (P < 0.01). CONCLUSION: WLP could elevate the expression levels of SLC26A6, SLC26A3, and CFTR, increase the secretion of bicarbonate, thus protecting the gastric mucosa.

Improvement of ginsenoside Rg1 on hematopoietic function in cyclophosphamide-induced myelosuppression mice.

This study was aimed to investigate the effects of ginsenoside Rg1 (Rg1) on hematopoietic function of bone marrow in cyclophosphamide-induced bone marrow depression mice. Mice were given cyclophosphamide (150mg/kg, i.p. for three days) to produce bone marrow depression. Rg1 was then administrated at 7.5 and 15mg/kg by i.p. for seven days. Bone marrow cells number was counted, and the percentage of hematopoietic stem cells (Lin(-)Sca-1(+)c-kit(+)) was quantified by flow cytometry. The histology of femoral bone was examined by H&E staining. The expression of calcium-sensing receptor mRNA was determined by the real time RT-PCR. Rg1 (7.5 and 15mg/kg) protected against cyclophosphamide-induced bone marrow depression, as evidenced by increased bone marrow cell numbers and improved femoral bone morphology. The percentage of Lin(-)Sca-1(+)c-kit(+) cells and lymphoid lineage CD3(+) cells were lower in cyclophosphamide group, but returned towards normal after Rg1 treatment in both bone marrow and peripheral blood cells. Expression of calcium-sensing receptor mRNA was increased in bone marrow cells on the 10th day after cyclophosphamide, but it was returned to normal level after Rg1 treatment. Rg1 alone did not produce these changes in normal mice. These results demonstrated that Rg1 improved hematopoietic function of bone marrow in cyclophosphamide-induced myelosuppression.

[Study on inducing differentiation of human amniotic epithelial cells into insulin secreting cells in vitro].

OBJECTIVE: To investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro. METHODS: The hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: (1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups. CONCLUSION: hAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.

Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma (PPARγ).

OBJECTIVES: Peroxisome proliferator-activated receptor gamma (PPARγ) plays a critical role in regulation of diverse biological processes, including lipid metabolism and adipogenesis, cell division and apoptosis, and is involved in variety of disease conditions, such as obesity, atherosclerosis, inflammation and tumour. Developing a cell-based reporter gene model targeting PPARγ would be useful to screen human PPARγ agonists that could be beneficial to patients with these diseases. METHODS: We stably co-transfected human embryonic kidney (HEK) cell line 293T cells with phPPARγ-IRES2-EGFP vector to express human PPARγ (hPPARγ), a reporter vector pPPRE×3-TK-LUC, and control vector pRL-CMV. The efficiency of the co-transfection was evaluated with flow cytometry of hPPARγ expressing cells. Specificity of hPPARγ activity was determined by dual luciferase reporter assay of co-transfected cells exposed to PPARγ agonist rosiglitazone, PPARα agonist WY14643 and retinoic acid receptor alpha (RARα) agonist all-trans-retinoic acid (ATRA). KEY FINDINGS: The phPPARγ-IRES2-EGFP co-transfected HEK293T cells showed concentration- and time-dependent luciferase induction upon exposure to the rosiglitazone, while WY14643 and ATRA were unable to activate the co-transfected HEK293T cells. CONCLUSIONS: These data indicated that the HEK293T cells could be stably transfected with hPPARγ. This cell-based drug screening platform could be used targeting specific nuclear receptor of hPPARγ with effectiveness and specificity for hPPARγ agonists discovery.

[Application of an improved tracheal intubation method during the preparation of myocardial infarction model in the mouse].

OBJECTIVE: An improved tracheal intubation method was used in preparation of myocardial infarction model in the mouse for increasing the success rate. METHODS: The mice were intubated by an improved trachea cannula through oral cavity. The left anterior thoracotomy performed. The myocardial infarction model was made by legating the left anterior descending coronary artery in mouse. The color of heart was observed, and electrocardiogram was recorded. The survival rate and pathologic change were observed after two weeks of operation. RESULTS: 40 myocardial infarction model mice were made by improved trachea cannula. The color of ventricles anterior wall had got madder red, and ST stages were raised on II leads of electrocardiogram in all of model mice. After two weeks, 27 mice were survival. The survival rate was 87.1% except for accidental death during operation. The heart chamber expanded and ventricular wall became thin in myocardial infarction mice by eyes. After pathological sections were stained, by HE cardiac muscle fibers ruptured or lysed. There were some of necrosis of myocardiac cells and many of infiltration of inflammatory cells. CONCLUSION: Application of an improved tracheal intubation method simplified operation of tracheal intubation during preparation of myocardial infarction model in the mouse. The trauma was tinier than the other one, and achievement ratio of the model preparation was improved.