Angiopoietin-1 Modified Human Umbilical Cord Mesenchymal Stem Cell Therapy for Endotoxin-Induced Acute Lung Injury in Rats

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.

Autophagy inhibitor 3-methyladenine regulates the expression of LC3, Beclin-1 and ZnTs in rat cerebral cortex following recurrent neonatal seizures / 世界急诊医学杂志（英文）

BACKGROUND: Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and excitotoxicity, and in patients with Alzheimer's disease and critical illness. The role of autophagy in developmental epilepsy, however, is unknown. The present study was to investigate the effects of recurrent neonatal seizure, in the presence and absence of autophagy inhibitor 3-methyladenine (3-MA), on the acute phase gene expression of ZnTs, LC3 and Beclin-1 in rat cerebral cortex and the interaction among them. METHODS: Thirty-six Sprague-Dawley neonatal rats at postnatal day 6(P6) were randomly divided into three groups: a recurrent-seizures group (RS, n=12), a 3-MA treated-seizure group (3-MA group, each rat pretreated with 3-methyladenine before seizures, 100nmol/ l/day, i.p., n=12) and a control group (n=12). At 1.5 and 6 hours after the last seizures, the mRNA levels of ZnT1-ZnT3, microtubule-associated protein 1A/1B light chain 3 (LC3) and beclin-1 were detected using the real-time RT-PCR method. The LC3 protein level was examined by Western blotting. RESULTS: The levels of LC3, beclin-1 and ZnT-2 transcripts in the RS group elevated significantly at 1.5 and 6 hours after the last seizures compared with those in the control and 3-MA groups. At the interval of 1.5 hours, the mRNA level of ZnT-1 increased significantly after the last seizure compared with that in the control group. There was no significant difference in the transcript levels of ZnT-3 among the three groups. Linear correlation analysis showed that the expression of the five genes in the control group exhibited a significant inter-relationship. In the 3-MA group, however, the inter-relationship was only found between beclin-1 and ZnT-1. In the RS group, the inter-relationship was not observed. CONCLUSIONS: The autophagy/lysosomal pathway is immediately activated along with the elevated expression of ZnT1 and ZnT2 in the cerebral cortex after recurrent seizures. 3-MA is involved in the regulation of the autophagy/lysosomal pathway and ZnTs by down-regulating the expression of LC3 and beclin-1.

Cytokines Combined with Striatal Conditioned Medium Promote Directional Differentiation of Mesenchymal Stem Cells into Dopaminergic Neurons in Vitro / 实用儿科临床杂志

Objective To explore the possibility of basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF)combined with striatal conditioned medium promoting the directional differentiation of rat bone marrow mesenchymal stem cells(BMMSCs)into dopaminergie neurons.Methods 1.Separation and culture of BMMSCs:BMMSCs were harvested from healthy adult Wistar rats for serial subcultivation.2.Preparation of Striatal conditioned medium:newborn Wistar rats within 24 hours were selected,and their brain tissues were removed to prepare striatal conditioned medium.3.Induced differentiation of BMMSCs:the 5th passage BMMSCs were collected and pre-induced in low glucose-Dulbecco's modified eagle medium(L-DMEM)containing bFGF and EGF.Twenty-four hours later,pre-induction liquor was replaced with striatal conditioned medium for further induced differentiation.4.Result assessment:the morphological changes of stem cells were observed under inverted phase microscope.The expression of neuron specific enolage(NSE)and tyrosine hydmxylage(TH)were identified by immunocytochemical technique.Results The cell body of rat BMMSCs contracted into round and spindle shape after induction by bFGF and EGF combined with striatal conditioned medium.Partial neuron-like cells with prominence could be found.Immunocytochemieal detection showed that the percentages of NSE and TH positive cells were(72.70±14.81)% and(34.50±15.93)%,respectively.Conclusion BMMSCs can be induced directionally into dopaminergiC neurons by bFGF and EGF combined with striatal conditioned medium in vitro.

Study of single cell PCR for HLA typing / 中华血液学杂志

<p><b>OBJECTIVE</b>To apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.</p><p><b>METHODS</b>Single cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.</p><p><b>RESULTS</b>Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.</p><p><b>CONCLUSION</b>The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.</p>

Biological characteristics of rabbit bone marrow mesenchymal stem cells and their response to different growth factors / 中国实验血液学杂志

This study was aimed to analyze the biological characteristics of rabbit bone marrow mesenchymal stem cells (rBM-MSCs) and their response to different growth factors. Rabbit BM-MSCs were separated from bone marrow mononuclear cells by using adherent cultivation. Biological characteristics were investigated by optical and electron microscopy. Immunophenotype of rBM-MSCs was measured by flow cytometry. The expression of collagen was detected by RT-PCR. Differentiation potential was identified by specific staining and RT-PCR. The response of rBM-MSCs to IL-1, 3, 8 and HGF with different concentrations were tested by MTT. The results showed that the rBM-MSCs gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology and could be cultured for over 15 passages. CD44 was highly expressed on F5 rBM-MSCs (32%) and CD45 was lowly expressed (4.7%). Type I collagen was highly expressed, while type II collagen was lowly expressed and type X collagen was not detected on rBM-MSCs using RT-PCR method. In various conditions inducting differentiation, rBM-MSCs could differentiate into the osteoblast, chondrocyte, adipocyte and neuron-like cells. The rBM-MSCs were sensitive to IL-3, even low concentration (10 ng/ml) of IL-3 could promote the proliferation of rBM-MSCs effectively (>32%, P < 0.01), whereas high concentration IL-3 inhibited it significantly. It is concluded that rabbit BM-MSCs were successfully isolated and culture-expanded. The biological characteristics of rabbit BM-MSCs are similar to those of human and rhesus BM-MSCs. IL-3 with low concentration can promote the proliferation of rBM-MSCs effectively, but high concentration of IL-3 can inhibit their proliferation.