Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

BACKGROUND: Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. METHODS: In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. RESULTS: Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. CONCLUSIONS: This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

GDF11 enhances therapeutic functions of mesenchymal stem cells for angiogenesis.

BACKGROUND: The efficacy of stem cell therapy for ischemia repair has been limited by low cell retention rate. Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-ß super family, which has multiple effects on development, physiology and diseases. The objective of the study is to investigate whether GDF11 could affect the efficacy of stem cell transplantation. METHODS: We explored the effects of GDF11 on proangiogenic activities of mesenchymal stem cells (MSCs) for angiogenic therapy in vitro and in vivo. RESULTS: Mouse bone marrow-derived MSCs were transduced with lentiviral vector to overexpress GDF11 (MSCGDF11). After exposed to hypoxia and serum deprivation for 48 h, MSCGDF11 were significantly better in viability than control MSCs (MSCvector). MSCGDF11 also had higher mobility and better angiogenic paracrine effects. The cytokine antibody array showed more angiogenic cytokines in the conditioned medium of MSCGDF11 than that of MSCvector, such as epidermal growth factor, platelet-derived growth factor-BB, placenta growth factor. When MSCs (1 × 106 cells in 50 µl) were injected into ischemic hindlimb of mice after femoral artery ligation, MSCGDF11 had higher retention rate in the muscle than control MSCs. Injection of MSCGDF11 resulted in better blood reperfusion and limb salvage than that of control MSCs after 14 days. Significantly more CD31+ endothelial cells and α-SMA + smooth muscle cells were detected in the ischemic muscles that received MSCGDF11. The effects of GDF11 were through activating TGF-ß receptor and PI3K/Akt signaling pathway. CONCLUSION: Our study demonstrated an essential role of GDF11 in promoting therapeutic functions of MSCs for ischemic diseases by enhancing MSC viability, mobility, and angiogenic paracrine functions.

Effect of Ulinastatin Combined with Octreotide on Serum Endothelin, Endotoxin Levels and Immune Function in Acute Pancreatitis.

The aim of this study was to determine clinical efficacy of ulinastatin combined with octreotide in treatment of acute pancreatitis, and its effect on serum endothelin, endotoxin level and immune function. It was an analytical observational study carried out from September 2016 to March 2018. A total of 108 patients with acute pancreatitis were randomly divided into observation group and control group, 54 cases in each group. Control group was additionally treated with octreotide. Observation group was treated with ulinastatin. Therapeutic effects of two groups were compared. The total effective rate in observation group was higher than that in control group (p=0.046). After seven days of treatment, serum endothelin and endotoxin levels in observation group were lower than those in control group (both p<0.001); IgA, IgM and IgG levels in observation group were higher than those in control group (p=0.031, 0.007, and 0.001, respectively). Ulinastatin combined with octreotide can reduce level of endothelin and endotoxin and improve immune function.

α-Tocopherol, especially α-tocopherol phosphate, exerts antiapoptotic and angiogenic effects on rat bone marrow-derived endothelial progenitor cells under high-glucose and hypoxia conditions.

OBJECTIVE: Considering the poor efficacy of local intramuscular injections with endothelial progenitor cells (EPCs) for critical limb ischemia in patients with diabetes, the study aimed to investigate the effect of α-tocopherol (α-T) and α-tocopherol phosphate (α-TP) on apoptosis and angiogenesis in a rat model under oxidative stress conditions. METHODS: Primary EPCs from Sprague-Dawley rats were harvested and treated with α-T and α-TP for 24 hours. Gene transcription and protein expression were evaluated by real-time polymerase chain reaction and Western blot, respectively. Cell apoptosis, migration, and tube formation ability were detected by flow cytometry, Transwell assay (Chemicon International, Temecula, Calif), and Matrigel-based angiogenesis assay (Corning Inc, Corning, NY). The in vivo experiments were carried out using 30 single hind limb ischemic models of diabetic rats that were treated with allogeneic EPCs. Capillary density was evaluated by immunohistochemistry. RESULTS: α-T and α-TP attenuated high glucose/hypoxia-induced cell apoptosis by promoting Bcl-2 and Akt and inhibiting nuclear factor κB p65, JNK, Notch-1, and p38MAPK genes. Furthermore, α-T and α-TP promoted the transcription and expression of vascular endothelial growth factor receptor 2 and decreased the transcription and expression of Tie-2 and Notch-1 in EPCs under high-glucose/hypoxic conditions. Moreover, α-T and especially α-TP enhanced the migratory activity of EPCs under high-glucose/hypoxic conditions. Capillary density of ischemic hind limbs was increased on day 14 after administration of EPCs pretreated with α-T and α-TP. CONCLUSIONS: α-T, especially α-TP, possesses therapeutic potential in the inhibition of apoptosis and increases the migratory capacity of EPCs under high-glucose/hypoxic conditions. It promotes angiogenesis by upregulating Bcl-2, Akt, and vascular endothelial growth factor receptor 2 and decreasing nuclear factor κB p65, p38MAPK, Notch-1, JNK, and Tie-2.

[Endovenous holmium laser treatment for varicose veins].

OBJECTIVE: To discuss the technical points, advantages, follow-up results and mechanism of endovenous holmium laser treatment for varicose veins. METHODS: Endovenous holmium laser procedures were performed for 96 patients (99 legs) with primary varicosity of lower extremities. Perioperative Duplex was used for preoperative diagnosis, intraoperative guide and postoperative follow-up. The time of procedure and clinical results were observed. The mean follow-up time was 7 months. RESULTS: Sixty-seven in 99 legs with saphenous vein occluded immediately during operation. All saphenous veins were confirmed to be occluded 7 days after the procedure. There was no recanalization with Duplex finding during the follow-up period. No wound complications. Two cases were with minor skin burn. One case was with saphenous nerve injury. Three cases were with thigh ecchymosis. CONCLUSION: Preliminary results show endovenous holmium laser treatment for varicose veins is safe and effective in treating varicose veins with cosmetic appearance and quicker recovery.