[Effects of altitude on circulating endothelial progenitor cells and hypoxia-inducible factor-1α in patients with type 2 diabetes].

Objective: To compare the changes in the number of circulating endothelial progenitor cells and hypoxia-inducible factors in patients with type 2 diabetes at different altitudes, and to provide a basis for the research and treatment of type 2 diabetes vascular complications. Methods: Selected Type 2 diabetes patients who were diagnosed in a low altitude area of 386 m (Xianyang City) and a high altitude area of 1 520 m (Lanzhou) (25 persons/29 persons) and healthy persons (20 persons/20 persons) were selected. An automatic biochemical analyzer was used to detect the indexes of blood lipids, blood glucose, and glycosylated hemoglobin of the two groups of people, and the concentration of Hypoxia inducible factor-1α (HIF-1α) was detected by enzyme-linked immunosorbent assay (ELISA). The number of circulating endothelial progenitor cells (EPCs) in peripheral blood was determined by a cytometer. Results: No matter in low or high altitude areas, the number of circulating EPCs in the diabetes group was lower than that in the healthy group (P＜0.01). The levels of body mass index (BMI), waist to hip ratio (WHR), triglyceride (TG), fasting blood glucose (FBG) and glycosylated hemoglobin (HbAlc) were increased (P＜0.05). Compared with the low-altitude group, the expression levels of HIF-1α in diabetic patients at high-altitude and healthy people were increased significantly (P＜0.05), while the number of circulating EPCs was decreased significantly (P＜0.05), and the number of circulating EPCs in healthy people or the patients with type 2 diabetes without vascular complications was higher than that of patients with type 2 diabetes with vascular complications (P＜0.05). Conclusion: With the increase in altitude, the expression level of HIF-1α in type 2 diabetes mellitus(T2DM)patients is increased, and the number of circulating EPCs is decreased, which is closely related to the degree of vascular disease. Therefore, it is possible through transplantation of EPCs for high altitude T2DM patients to achieve the prevention and improvement of diabetic vascular complications.

Protective effect of rhodioloside and bone marrow mesenchymal stem cells infected with HIF-1-expressing adenovirus on acute spinal cord injury.

Rhodioloside has been shown to protect cells from hypoxia injury, and bone marrow mesenchymal stem cells have a good effect on tissue repair. To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury, a rat model of spinal cord injury was established using the Infinite Horizons method. After establishing the model, the rats were randomly divided into five groups. Rats in the control group were intragastrically injected with phosphate buffered saline (PBS) (5 µL). PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm. Rats in the rhodioloside group were intragastrically injected with rhodioloside (5 g/kg) and intramuscularly injected with PBS. Rats in the mesenchymal stem cell (MSC) group were intramuscularly injected with PBS and intramuscularly with MSCs (8 × 106/mL in a 50-µL cell suspension). Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs. Rats in the rhodioloside + Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside. One week after treatment, exercise recovery was evaluated with a modified combined behavioral score scale. Hematoxylin-eosin staining and Pischingert's methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue. Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord. Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord. The results showed that: (1) compared with the other groups, the rhodioloside + Ad-HIF-MSC group exhibited the highest combined behavioral score (P < 0.05), the most recovered tissue, and the greatest number of neurons, as indicated by Pischingert's methylene blue staining. (2) Compared with the PBS group, HIF-1 protein expression was greater in the rhodioloside group (P < 0.05). (3) Compared with the Ad-HIF-MSC group, Sry mRNA levels were higher in the rhodioloside + Ad-HIF-MSC group (P < 0.05). These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue. This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine, China (approval No. 2015KYLL029) in June 2015.

A meta-analysis of the effects of probiotics and synbiotics in children with acute diarrhea.

OBJECTIVE: This meta-analysis assessed the effectiveness of probiotics and synbiotics for acute diarrhea (AD) in children and investigated probiotic formulations, types of interventions, and country factors. METHODS: Randomized, double-blind, placebo-controlled trials evaluating the effects of probiotics or synbiotics on AD were analyzed. We followed the recommendations of the Cochrane Handbook and the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement. The risks of systematic errors (bias) and random errors were assessed, and the overall quality of the evidence was evaluated using the Grades of Recommendations Assessment, Development, and Evaluation (GRADE) approach. RESULTS: The meta-analysis included 34 studies with 4911 patients. Five and 29 studies presented the results of synbiotic and probiotic interventions, respectively. After intervention, the durations of diarrhea (weighted mean difference (WMD) = -16.63 [-20.16; -12.51]) and hospitalization (risk ratio (RR) = 0.59 [0.48; 0.73]) were shorter, the stool frequency on day 3 (WMD = -0.98 [-1.55; -0.40]) was decreased, and the incidence of diarrhea lasting 3 days was lower in the probiotic and synbiotic groups than in the control groups. Furthermore, in the subgroup analyses, synbiotics were more effective than probiotics at reducing the durations of diarrhea and hospitalization, and Saccharomyces and Bifidobacterium were more effective than Lactobacillus at reducing the duration of diarrhea. CONCLUSION: This meta-analysis supports the potential beneficial roles of probiotics and synbiotics for AD in children. Further research is needed to determine problems associated with probiotic/synbiotic mixtures and appropriate dosages.

Exosomal miRNAs as Biomarkers of Cancer: a Meta-Analysis.

BACKGROUND: Tumor-derived exosomal miRNAs secreted by cancer cells play significant roles in the pathological processes of cancer, but no systematic meta-analysis has focused on the diagnostic efficiency of exosomal miRNAs. This meta-analysis assessed the diagnostic value of circulating exosomal miRNA in cancer. METHODS: Studies evaluating the diagnostic value of exosomal miRNA were identified in EMBASE, PubMed, Cochrane Library, and Web of Science up to August 1, 2018. The quality of each study was assessed according to the Quality Assessment of Diagnostic Accuracy Studies 2, and STATA 14.0 was used for the analyses. The true positive (TP), false positive (FP), true negative (TN), and false negative (FN) rates were extracted from each study to obtain the pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and their 95% confidence intervals (CIs). RESULTS: The meta-analysis included 16 studies with 1,591 patients. Five studies reported sensitivity values, and the pooled sensitivity was 0.86 (95% CI = 0.80 - 0.90, while 29 studies reported specificity values, and the pooled specificity was 0.89 (95% CI = 0.83 - 0.93). The pooled PLR was 7.8 (95% CI = 4.9 - 12.4), the pooled NLR was 0.16 (95% CI = 0.11 - 0.24), the pooled DOR was 48 (95% CI = 23 - 101), and the AUC was 0.94 (0.91 - 0.96). CONCLUSIONS: Our meta-analysis indicated that body fluid exosomal miRNAs are highly accurate for distinguishing patients from healthy individuals, and exosomal miRNAs have superior diagnostic value in plasma, prostate cancer patients, and non-Asian individuals.

Icariin protects cardiomyocytes against ischaemia/reperfusion injury by attenuating sirtuin 1-dependent mitochondrial oxidative damage.

BACKGROUND AND PURPOSE: Icariin, a major active ingredient in traditional Chinese medicines, is attracting increasing attention because of its unique pharmacological effects against ischaemic heart disease. The histone deacetylase, sirtuin-1, plays a protective role in ischaemia/reperfusion (I/R) injury, and this study was designed to investigate the protective role of icariin in models of cardiac I/R injury and to elucidate the potential involvement of sirtuin-1. EXPERIMENTAL APPROACH: I/R injury was simulated in vivo (mouse hearts), ex vivo (isolated rat hearts) and in vitro (neonatal rat cardiomyocytes and H9c2 cells). Prior to I/R injury, animals or cells were exposed to icariin, with or without inhibitors of sirtuin-1 (sirtinol and SIRT1 siRNA). KEY RESULTS: In vivo and in vitro, icariin given before I/R significantly improved post-I/R heart contraction and limited the infarct size and leakage of creatine kinase-MB and LDH from the damaged myocardium. Icariin also attenuated I/R-induced mitochondrial oxidative damage, decreasing malondialdehyde content and increasing superoxide dismutase activity and expression of Mn-superoxide dismutase. Icariin significantly improved mitochondrial membrane homeostasis by increasing mitochondrial membrane potential and cytochrome C stabilization, which further inhibited cell apoptosis. Sirtuin-1 was significantly up-regulated in hearts treated with icariin, whereas Ac-FOXO1 was simultaneously down-regulated. Importantly, sirtinol and SIRT1 siRNA either blocked icariin-induced cardioprotection or disrupted icariin-mediated mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Pretreatment with icariin protected cardiomyocytes from I/R-induced oxidative stress through activation of sirtuin-1 /FOXO1 signalling.

Endothelial progenitor cells in peripheral blood may serve as a biological marker to predict severe acute pancreatitis.

AIM: To investigate the significance of endothelial progenitor cells (EPCs) in predicting severe acute pancreatitis (SAP). METHODS: We recruited 71 patients with acute pancreatitis (AP) and excluded 11 of them; finally, cases of mild acute pancreatitis (MAP) (n = 30) and SAP (n = 30), and healthy volunteers (n = 20) were internalized to investigate levels of EPCs, C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), fibrinogen (FIB) and white blood cells (WBC) in peripheral blood. RESULTS: The levels of TNF-α, WBC, FIB and CRP were higher both in SAP and MAP cases than in healthy volunteers (P < 0.05, all). Interestingly, the level of EPCs was higher in SAP than MAP (1.63% ± 1.47% vs 6.61% ± 4.28%, P < 0.01), but there was no significant difference between the MAP cases and healthy volunteers (1.63% ± 1.47% vs 0.55% ± 0.54%, P > 0.05). Receiver operating characteristics curve (ROC) showed that EPCs, TNF-α, CRP and FIB were significantly associated with SAP, especially EPCs and CRP were optimal predictive markers of SAP. When the cut-off point for EPCs and CRP were 2.26% and 5.94 mg/dL, the sensitivities were 90.0% and 73.3%, and the specificities were 83.3% and 96.7%. Although, CRP had the highest specificity, and EPCs had the highest sensitivity and highest area under the curve value (0.93). CONCLUSION: Data suggest that EPCs may be a new biological marker in predicting SAP.

SIRT1 mediates Sphk1/S1P-induced proliferation and migration of endothelial cells.

Angiogenesis is one of the most important components of embryonic organ formation and vessel growth after birth. Sphingosine kinase 1 (Sphk1) and S1P has been confirmed to participate in various cell signaling pathways and physiological processes including neovascularisation. However, the mechanisms that Sphk1/S1P regulates neovascularisation remain unclear. In this study, we elucidated that Sphk1/S1P upregulates sirtuin 1 (SIRT1), a NAD+ dependent deacetylases protease which exerts multiple cellular functions, to regulate the proliferation and migration of endothelial cells. By using CCK8 and Transwell assays, we demonstrated that Sphk1 and SIRT1 knockdown could significantly decrease proliferation and migration of HUVEC cells. Sphk1 inhibition results in SIRT1 downregulation which could be reversed by exogenous S1P in HUVEC cells. Treatment of HUVECs with S1P reverses the impaired proliferation and migration caused by SIRT1 knockdown. Furthermore, Sphk1 knockdown inhibits the phosphorylation of P38 MAPK, ERK and AKT. Treatment of HUVECs with PD98059, SB203580 and Wortmannin, which are the inhibitors of ERK, P38 MAPK and AKT respectively, resulted in decreased SIRT1 expression and reduced migration of HUVEC cells. Thus, we conclude that Sphk1/S1P induces SIRT1 upregulation through multiple pathways including P38 MAPK, ERK and AKT signals. This is the first report to disclose the existence and roles of Sphk1/S1P/SIRT1 axis in regulation of endothelial cell proliferation and migration, which may provide a theoretical basis for angiogenesis.

Salidroside induces neuronal differentiation of mouse mesenchymal stem cells through Notch and BMP signaling pathways.

Salidroside (p-hydroxyphenethyl-ß-D-glucoside, SAL), a phenylpropanoid glycoside isolated from a popular traditional Chinese medicinal plant Rhodiola rosea L., possesses multiple pharmacological actions. Previous study showed that SAL could induce rat mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons and induce mouse MSCs D1 to differentiate into neuronal cells. However, the mechanisms of SAL-induced neuronal differentiation of MSCs still need investigation. In this study, we observed the effects of SAL on neuronal differentiation of D1 cells and the possible involvement of Notch and BMP signaling pathways. SAL inhibited the proliferation, induced neuronal phenotypes, and upregulated the expressions of neuronal-specific marker molecules, such as neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (MAP2), and beta 3 class III tubulin (Tubb3/ß-tubulin III) in D1 cells. SAL not only downregulated the expressions of Notch1 and hairy enhancer of split 1 (Drosophila) (Hes1) but also upregulated the expression of Smad1/5/8 and its phosphorylation (p-Smad 1/5/8). The neuronal differentiation effects of SAL on D1 cells were promoted by a Notch signaling antagonist, DAPT, but attenuated by a BMP signaling pathway antagonist, Noggin. Our findings suggest that SAL might be promising in inducing neuronal differentiation of mouse MSCs mediated by both Notch signaling pathway and BMP signaling pathway.

Distribution of endothelial progenitor cells in tissues from patients with gastric cancer.

It is accepted that endothelial progenitor cells (EPCs) are recruited into tumor sites and take part in the neovascularization of tumors. However, few articles have discussed the specific distribution of EPCs in vivo in tissues of gastric cancer patients. For this reason, the present study sought to elucidate EPC distribution in vivo in tissues of patients with gastric cancer. Fresh tumor tissues were collected from 26 newly diagnosed patients with histologically confirmed gastric cancer (mean age, 51 years; range, 21-81 years; 7 females, 19 males). All patients were treated surgically with curative intent. One portion of the fresh tissues was prepared for flow cytometric analysis and another was immediately snap frozen in liquid nitrogen and stored at -80°C for later use in quantitative polymerase chain reaction. The analysis was based on two groups of tissues, namely the cancer group and cancer-adjacent group. The presence of CD34/CD133 double-positive cells was determined in cancer-adjacent and cancer tissues by flow cytometry. The analysis revealed that the total number of EPCs in cancer tissue was slightly greater than the number in the cancer-adjacent tissue, but not to the point of statistical significance. The number of EPCs in cancer-adjacent and cancer tissues of patients with early-stage gastric cancer was higher than the EPC count in late-stage gastric cancer patients, and significant differences were identified in the number of EPCs in cancer tissue between patients of different tumor stages. Levels of cluster of differentiation (CD)34, CD133 and vascular endothelial growth factor receptor 2 were not significantly different in cancer-adjacent tissue compared with cancer tissue. These results suggest that cancer-adjacent and cancer tissue of gastric cancer patients may be used as a reference index in the clinical and pathological staging of tumors.

Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron-like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/ß-tubulin III). It also increased expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine-beta-hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1-12 days. DA steadily increased after treatment with salidroside for 1-6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons.

Attenuated Salmonella typhimurium carrying the hepatocyte growth factor and keratinocyte growth factor genes repairs gastrointestinal mucosal damage caused by chemotherapy.

Radiotherapy and chemotherapy are the main therapeutic approaches for patients with malignant tumours, especially advanced tumours. However, they can cause adverse effects, one of which is gastrointestinal mucosal damage, which can greatly affect patients' quality of life. Until now, there have been no effective therapies to avoid or treat these adverse effects. In this study, we used attenuated Salmonella typhimurium (S. typhimurium) to deliver the hepatocyte growth factor (HGF) or keratinocyte growth factor (KGF) to murine gastrointestinal mucosa. We found that attenuated S. typhimurium carrying the HGF or KGF genes can effectively reduce the ratio of tumour to non-tumour carcass weight, repair damage to the gastrointestinal mucosal from chemotherapy, improve the immune response, and reduce the mortality rate of mice. Oral administration of attenuated S. typhimurium with HGF and KGF may be promising as a way of improving the quality of life of patients undergoing radiotherapy and chemotherapy.

Inhibitory effects of the attenuated Salmonella typhimurium containing the IL-2 gene on hepatic tumors in mice.

To observe the inhibitory effects of an attenuated S. typhimurium strain carrying IL-2 gene (TPI) on hepatoma cell line (HepG2) and transplanted tumors in mice. TPI, TPG (an attenuated S. typhimurium strain carrying green fluorescent protein gene), and TP (an attenuated S. typhimurium strain) strains were transfected into HepG2 cells. At 48 h after transfecting, the transfection rate was 82.58 ± 1.74%. The expression level of IL-2 was (99.5 ± 12.2) ng/1 × 10(6) cells. Compared with TPG, TP, and normal mouse groups, the proportion of CD4(+) T and CD8(+) T cells in the blood from the TPI group was higher, the levels of IgM and IgG(1) were significantly increased, and the proliferation activity of splenic lymphocyte was significantly stronger. The transplanted tumor weight in the TPI group was significantly smaller than that in the other two groups. The infiltration of lymphocytes increased in the tumor from TPI group mice. TPI was effectively transfected into cancer cells, which expressed the protein of interest. Oral administration of TPI prolonged survival of mice transplanted with hepatoma cell tumours.

The characteristics of the synonymous codon usage in hepatitis B virus and the effects of host on the virus in codon usage pattern.

BACKGROUND: Hepatitis B virus (HBV) infection is one of the main human health problem and causes a large-scale of patients chronic infection worldwide.. As the replication of HBV depends on its host cell system, codon usage pattern for the viral gene might be susceptible to two main selections, namely mutation pressure and translation selection. In this case, a deeper investigation between HBV evolution and host adaptive response might assist control this disease. RESULT: Relative synonymous codon usage (RSCU) values for the whole HBV coding sequence were studied by Principal component analysis (PCA). The characteristics of the synonymous codon usage patterns, nucleotide contents and the comparison between ENC values of the whole HBV coding sequence indicated that the interaction between virus mutation pressure and host translation selection exists in the processes of HBV evolution. The synonymous codon usage pattern of HBV is a mixture of coincidence and antagonism to that of host cell. But the difference of genetic characteristic of HBV failed to be observed to its different epidemic areas or subtypes, suggesting that geographic factor is limited to influence the evolution of this virus, while genetic characteristic based on HBV genotypes could be divided into three groups, namely (i) genotyps A and E, (ii) genotype B, (iii) genotypes C, D and G. CONCLUSION: Codon usage patterns from PCA for identification of evolutionary trends in HBV provide an alternative approach to understand the evolution of HBV. Further more, a combined selection of mutation pressure with translation selection on codon usage might shed a light on understanding the evolutionary trends of HBV genotypes.

Keratinocyte growth factor gene therapy ameliorates ulcerative colitis in rats.

AIM: To investigate the effect of keratinocyte growth factor (KGF) gene therapy in acetic acid-induced ulcerative colitis in rat model. METHODS: The colitis of Sprague-Dawley rats was induced by intrarectal infusion of 1 mL 5% (v/v) acetic acid. Twenty-four hours after exposed to acetic acid, rats were divided into three experimental groups: control group, attenuated Salmonella typhimurium Ty21a strain (SP) group and SP strain carrying human KGF gene (SPK) group, and they were separately administered orally with 10% NaHCO(3), SP or SPK. Animals were sacrificed and colonic tissues were harvested respectively on day 3, 5, 7 and 10 after administration. Weights of rats, colonic weight/length ratio and stool score were evaluated. Histological changes of colonic tissues were examined by hematoxylin and eosin (HE) staining method. The expression of KGF, KGF receptor (KGFR) and TNF-α were measured either by enzyme-linked immunosorbent assay or Western blotting. Immunohistochemistry was used to detect the cellular localization of KGFR and Ki67. In addition, superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents in the homogenate were measured. RESULTS: Body weight and colonic weight/length ratio were declined in SPK group compared with SP and control groups (body weight: 272.78 ± 17.92 g vs 243.72 ± 14.02 g and 240.68 ± 12.63 g, P < 0.01; colonic weight/length ratio: 115.76 ± 7.47 vs 150.32 ± 5.99 and 153.67 ± 5.50 mg/cm, P < 0.01). Moreover, pathological changes of damaged colon were improved in SPK group as well. After administration of SPK strain, KGF expression increased markedly from the 3rd d, and remained at a high level till the 10th d. Furthermore, KGFR expression and Ki67 expression elevated, whereas TNF-α expression was inhibited in SPK group. In the group administered with SPK, SOD activity increased significantly (d 5: 26.18 ± 5.84 vs 18.12 ± 3.30 and 18.79 ± 4.74 U/mg, P < 0.01; d 7: 35.48 ± 3.35 vs 22.57 ± 3.44 and 21.69 ± 3.94 U/mg, P < 0.01; d 10: 46.10 ± 6.23 vs 25.35 ± 4.76 and 27.82 ± 6.42 U/mg, P < 0.01) and MDA contents decreased accordingly (d 7: 7.40 ± 0.88 vs 9.81 ± 1.21 and 10.45 ± 1.40 nmol/mg, P < 0.01; d 10: 4.36 ± 0.62 vs 8.41 ± 0.92 and 8.71 ± 1.27 nmol/mg, P < 0.01), compared with SP and control groups. CONCLUSION: KGF gene therapy mediated by attenuated Salmonella ameliorates ulcerative colitis induced by acetic acids, and it may be a safe and effective treatment for ulcerative colitis.

Effects of hepatocyte growth factor-mediated activation of Dll4-Notch-Hey2 signaling pathway.

BACKGROUND: Hepatocyte growth factor (HGF) treats ischemic disease by promoting arteriogenesis, however, its mechanism of action is not known. The notch signaling pathway plays an important role in neovascularization. The relationship between the proliferation and migration ability of artery endothelial cells and the Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis was investigated as a mechanism of action of HGF. METHODS: Based on the prophase study cells and supernatant were harvested at the indicated time after human femoral artery endothelial cells (HFAECs) were infected with adenovirus-HGF (Ad-HGF) at 200 pfu/cell. Cells were analyzed for HGF expression and Notch1, Dll4 and Hey2 expression by ELISA and reverse transcription-PCR (RT-PCR). The changes in the proliferation and migration ability of HFAECs were observed by MTT and Transwell migration experiments. Ad-GFP-infected HFAECs were used as control. RESULTS: Compared with the control group the Ad-HGF group's HGF expression was not increased with time, and the induction by HGF of Notch1, Dll4 and Hey2 gene transcription was not enhanced with an increase of HGF. The proliferation ability of Ad-HGF-transduced HFAECs was enhanced and their migration ability was also enhanced in the presence of HGF. CONCLUSIONS: Through activating the Dll4-Notch-Hey2 signaling pathway, HGF indirectly promotes the proliferation and migration ability of cells, so that offspring artery branches are formed.

Keratinocyte growth factor (KGF) gene therapy mediated by an attenuated form of Salmonella typhimurium ameliorates radiation induced pulmonary injury in rats.

The aim of this study is to investigate the effect of KGF (Keratinocyte growth factor) gene therapy mediated by the attenuated Salmonella typhimurium Ty21a on radiation-induced pulmonary injury in rats model. Sprague-Dawley rats were divided into three groups: TPK group (treated with TPK strain, attenuated Salmonella typhimurium Ty21a-recombined human KGF gene); TP group (treated with TP strain, attenuated Salmonella typhimurium Ty21a-recombined blank plasmid); and Saline group (treated with saline). After intraperitoneal administration for 48 h, the thoraxes of the rats were exposed to X-ray (20 Gy), and the rats were administered again two weeks after radiation. On the 3rd, 5th, 7th, 14th and 28th day after radiation, the rats were sacrificed and lung tissues were harvested. Histological analysis was performed, MDA contents and SOD activity were detected, mRNA levels of KGF, TGF-ß, SP-A and SP-C were measured by Real-time RT-PCR, and their concentrations in the BALF were quantified with ELISA. Administration of TPK strain improved the pathological changes of the lung on the 28th day. In the TPK group, KGF effectively expressed since the 3rd day, MDA contents decreased and SOD activity increased significantly, on the 7th day and 14th day respectively. SP-A and SP-C expression elevated, whereas TGF-ß expression was inhibited in the TPK group. These results suggest that this novel gene therapy of KGF could ameliorate radiation-induced pulmonary injury in rats, and may be a promising therapy for the treatment of radiative pulmonary injury.

[The toxic effects of lead acetate on the apoptosis and the ultrastructure in human renal tubular epithelial cells (HK-2)].

OBJECTIVE: To explore the toxic effects of lead acetate on the apoptosis and ultrastructure of human renal tubular epithelial cells (HK-2). METHODS: After HK-2 cells were exposed to 5, 10 and 20 µmol/L lead acetate for 24 h, the morphological changes of HK-2 cells were observed by Hochest 33342-PI staining, and the ultrastructure changes of HK-2 cells were examined under a electron microscope, LDH activity and MDA content in supernatant of HK-2 cellular culture were detected by spectrophotometer, DNA damage of HK-2 was determined by DNA ladder and the apoptotic rates of HK-2 cells were measured by flow cytometry. RESULTS: The morphological changes of apoptotic HK-2 cells in exposure group were observed by Hochest 33342-PI staining. The cytoplasm vacuoles, karyopycnosis, nuclear membrane vague and apoptotic bodies in HK-2 cells of exposure group were found under electron microscopy. LDH activity and MDA contents in exposure group increased significantly, as compared to control group (P < 0.01). The results of DNA Ladder showed that DNA damage of HK-2 cells in exposure group appeared. The apoptotic rates of HK-2 cells exposed to 5, 10, 20 µmol/L lead acetate were 14.16% ± 2.94%, 19.45% ± 2.73%, 25.01% ± 3.97%, respectively, which were significantly higher than that (5.81% ± 2.18%) in control group (P < 0.05). CONCLUSION: Lead acetate could remarkably induce the apoptosis of HK-2 cells and affect the kidney.

Effects of mesenchymal stem cells transfected with human hepatocyte growth factor gene on healing of burn wounds.

OBJECTIVE: To explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) transfected with adenoviral vector carrying hepatocyte growth factor (HGF, Ad-HGF) on burn wound healing. METHODS: BMSCs from male Wistar rats were separated and purified with Percoll separating medium by density gradient centrifugation and cultured with DMEM containing 20% fetal bovine serum (FBS). Then BMSCs were transfected with Ad-HGF at the optimal gene transduction efficiency of 100 multiplicity of infection (MOI). The efficiency of transfection and the expression of HGF in the suspension were detected by flow cytometry and enzyme linked immunosorbent assay (ELISA) respectively. Thirty-two female rats were subjected to 90 degree centigrade water for 12 seconds to induce a partial thickness skin burn. The animals were randomly divided into mesenchymal stem cells (MSCs) treatment group (Group A), Ad-HGF treatment group (Group B), Ad-HGF-modified MSCs treatment group (Group C) and saline control group (Group D). On days 3, 5, 7, 14 and 21 postburn, HE and Sirius red stain were performed to observe the burn wound healing and collagen content. The content of hydroxyproline in wounds was also detected. Transplanted cells and the expression of (sex-determining region Y) SRY gene were detected by in situ hybridization and polymerase chain reaction (PCR), while the expression of HGF in wound tissues was detected by ELISA. RESULTS: The result of flow cytometry showed that the transfection efficiency was 86.41% at 100 MOI. Compared with the control group, the content of HGF in the supernatant after transfection increased time-dependently and peaked at 48 h, showing significant differences at 24 h, 48 h, 72 h and 96 h (P less than 0.01). Results of HE stain revealed that the range of re-epidermidalization in Group C was significantly larger than that in other groups in the first week. Three weeks postburn, the epidermis was significantly thicker in Group C than in other groups and the nails of dermis inserted into the derma of burn wounds. Sirius red stain showed that the content of collagen I in Group C was much less compared with that in other groups 21 days postburn. In situ hybridization revealed an expression of SRY gene in burned female rats, consistent with the finding of PCR. Immunohistochemistry demonstrated the largest increase of HGF expression in Group C, whose contents of hydroxyproline, however, decreased on day 7 postburn. Compared with other groups, the content of HGF in the wounds of Group C increased obviously on day 14 after transfection (P less than 0.05) and there was no significant difference among Groups A, B and D. CONCLUSION: Our study suggests that transplantation of MSCs modified with Ad-HGF has positive effects on the healing of burn wounds probably through differentiation and release of relevant cytokines.

Effect of endothelial progenitor cells in neovascularization and their application in tumor therapy.

OBJECTIVE: To review the effect of endothelial progenitor cells in neovascularization as well as their application to the therapy of tumors. DATA SOURCES: The data used in this review were mainly from PubMed for relevant English language articles published from 1997 to 2009. The search term was "endothelial progenitor cells". STUDY SELECTION: Articles regarding the role of endothelial progenitor cells in neovascularization and their application to the therapy of tumors were selected. RESULTS: Endothelial progenitor cells isolated from bone marrow, umbilical cord blood and peripheral blood can proliferate, mobilize and differentiate into mature endothelial cells. Experiments suggest endothelial progenitor cells take part in forming the tumor vascular through a variety of mechanisms related to vascular endothelial growth factor, matrix metalloproteinases, chemokine stromal cell-derived factor 1 and its receptor C-X-C receptor-4, erythropoietin, Notch signal pathway and so on. Evidence demonstrates that the number and function change of endothelial progenitor cells in peripheral blood can be used as a biomarker of the response of cancer patients to anti-tumor therapy and predict the prognosis and recurrence. In addition, irradiation temporarily increased endothelial cells number and decreased the endothelial progenitor cell counts in animal models. Meanwhile, in preclinical experiments, therapeutic gene-modified endothelial progenitor cells have been approved to attenuate tumor growth and offer a novel strategy for cell therapy and gene therapy of cancer. CONCLUSIONS: Endothelial progenitor cells play a particular role in neovascularization and have attractively potential prognostic and therapeutic applications to malignant tumors. However, a series of problems, such as the definitive biomarkers of endothelial progenitor cells, their interrelationship with radiotherapy and their application in cell therapy and gene therapy of tumors, need further investigation.

[Genetic detection and analysis of the VHL gene in patients with sporadic pheochromocytoma].

OBJECTIVE: To carry out a genetic detection and analysis of Von Hippel-Lindau (VHL) gene in Chinese patients with sporadic pheochromocytoma. METHODS: DNA samples were extracted from peripheral blood cells and fresh pheochromocytoma specimens from 41 patients with sporadic pheochromocytoma were assayed by polymerase chain reaction and direct sequencing. The DNA samples of 50 healthy volunteers were extracted from peripheral blood as a control. The PCR products of exon 1, exon 2 and exon 3 were used for molecular analysis of the VHL gene. The genetic detection of family members of VHL gene mutations was also performed. RESULTS: One of mutations was located at nucleotide 572 (G-->C) in exon 2, presenting a codon 120 from arginine (R) to threonine (T). Tow small insertions were locatated at nucleotide 623T (TTTGTtG) in exon 2, leading to a frameshift mutation. There were also three carriers of G572C and three carriers of 623T (TTTGTtG) in family members of the three cases. CONCLUSION: There are some Chinese patients with sporadic pheochromocytoma with tumorigenic VHL gene mutations. It is recommended to use the genetic detection and analysis of VHL gene as a routine examination for patients with sporadic pheoehromoeytoma under the age of 50 years with questionable family history. The genetic detection and analysis of VHL gene may be useful as a marker for the diagnosis of hereditary pheochromocytoma.