Yongping Su

The efficacy of granulocyte colony-stimulating factor (G-CSF) in mobilizing mesenchymal stem cells (MSCs) into peripheral blood (PB) and the ability of PB-MSCs incorporated into injured brain were tested. Colony forming, cell phenotype and differentiation potential of mouse MSCs mobilized by G-CSF (40 μg/kg) were evaluated. Mortality and pathological changes in mice with serious craniocerebral trauma plus G-CSF treatment (40 μg/kg) were investigated. Bone marrow (BM) cells derived from GFP mice were fractionated into MSCs, hematopoietic stem cells (HSCs), and non-MSC/HSCs using magnetic beads and adherent culture. The resultant cell populations were transplanted into injured mice. The in vivo integration and differentiation of the transplanted cells were detected immunocytochemically. The expression of SDF-1 in injured area of brain was tested by Western blot. G-CSF was able to mobilize MSCs into PB (fourfold increase). PB-MSCs possessed similar characteristics as BM-MSCs in terms of colony formation, the expression pattern of CD73, 44, 90, 106, 31 and 45, and multipotential of differentiation. Accumulative total mice mortality was lower in TG group (5/14) than that in T group (7/14). It was MSCs, not HSCs or non-MSC/HSC cells integrated into the damaged cerebral tissue and differentiated into cells expressing neural markers. Increased SDF-1 expression in injured area of brain was confirmed, which could facilitate the homing of MSCs to brain. G-CSF can mobilize MSCs into PB and MSCs in PB can integrate into injured cerebral tissue and transdifferentiated into neural cells and may benefit the repair of trauma.

Human α-defensin 5 (HD₅), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD₅ through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD₅ mature peptide (rmHD₅) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD₅ by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD₅ into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD₅ vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD₅ nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD₅, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD₅. The results showed that about 165.0 mg/l of rmHD₅ with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD₅ was recovered after purification. The in vitro experiments revealed that rmHD₅ exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD₅ in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.

Zou, Z., Sun, H., Su, Y., Cheng, T. and Luo, C. Progress in Research on Radiation Combined Injury in China. Radiat. Res. 169, 722–729 (2008). The significant feature of radiation combined injury is the occurrence of a combined effect. For decades our institute has focused on studying the key complications of radiation-burn injury, including shock, suppression of hematopoiesis and immunity, gastrointestinal damage and local refractory wound healing. Here we summarize recent advancements in elucidating the mechanisms of and potential treatments for radiation combined injury. Concerning the suppression and regeneration of hematopoiesis in radiation combined injury, mechanisms of megakaryocyte damage have been elucidated and a new type of fusion protein stimulating thrombopoiesis has been developed and is being tested in animals. With regard to the damage and repair of intestinal epithelium, the important molecular mechanisms of radiation combined injury have been clarified, and new measures to preve...

DNA fragment containing human alpha-defensin 5 mature peptide (mHD-5) coding sequence with biased codons of E. coli was amplified by PCR, which was subsequently cloned into the plasmid pMAL-p2x in order to create pMAL-p2x-mHD-5 expression vector. The plasmid pMAL-p2x-mHD-5 was transferred into engineered strain BL21(DE3) to express heterogeneous fusion protein (MBP-mHD-5). The soluble MBP-mHD-5 targeted protein inducible expressed by IPTG was accounted for about 30% under optimized conditions. The recombinant mHD-5 (rmHD-5) peptide was successfully purified through a separation process including affinity chromatography, Factor Xa digestion and ion exchange chromatography. The bioactivity of rmHD-5 was examined by bacteria-inhibition tests in liquid culture. The growth of E. coli ATCC25922 was dramatically suppressed with an inhibition rate of 90%, with the presence of 62.5 microg/mL rmHD-5 in the media. These results indicate that the strategy of soluble expression of fusion protein in E. coli can be a useful and practical way to produce bioactive defensins.

Inducing mesenchymal stem cells (MSCs) differentiate to myoblasts with 5-azacytidine(5-Aza-CR), investigating the expression of Myf5 and the role of the signal transduction case of p38 in all the course of differentiation. Separating and purifying bone marrow-derived MSCs, inducing MSCs differentiation to myoblasts with 10 micromol/L 5-Aza-CR, assaying the gene expression time of Myf5 with RT-PCR method, the antigen expression of myosin with immunohistochemistry method and observing the changes of the activity of phosphorylation p38 before and after inhibited by SB203580 with Western-blot method. MSCs begin to express Myf5 delayed to the 9th day after inhibited by SB203580. Some of MSCs express myosin at the 7th day after induced; The phosphorylation p38 activity of MSCs enhanced after induced by 5-Aza-CR but obviously decreased after inhibited by SB203580. MSCs can express myogenic regulator factors and orientation differentiate to myoblasts after induced by 5-Aza-CR, p38 really have a positive signal transduction affection in this course.

To investigate therapeutics for and the pathological basis of combined radiation and burn injuries. Combined radiation and burn injuries on mice and rats were inflicted by gamma ray irradiation from a (60)Co source and thermal radiation from a 5 kW bromotungsten lamp. The dysfunction of myocardium played an important role in the development of early stage shock. Transfusion of irradiated (in vitro, 20 Gy) or stored (4 degrees C, 7 days) blood after irradiation was done to promote the success of allo-transplantation of bone marrow. Decrease of IL-4 mRNA expression was the molecular basis of depression of intestinal mucosa immune and intervention of IL-4 showed an antagonistic effect on enterogenic infection. A new lipid component extracted from burn eschar was documented for the first time and its toxic effects were elucidated. The survival rate of alloskin grafts after removal of burn eschar from the recipient animals was obviously increased in combined injury due to reduction of immune rejection activity by the radiation effect. In contrast, in animal models with simple burn, the alloskin grafts were all rejected within ten days after the procedure. A successful therapeutic result (survival rate: 92% for 30 days and 67% for 100 days) was obtained by comprehensive management of treated animals, while the untreated control animals all died within 3 - 7 days after injury. The pathogenesis of injury caused by simultaneous radiation and burn is extremely complicated and the treatment is very difficult. A comprehensive management program consisting of several therapeutic measures aimed at key links of the pathogenesis may achieve significantly improved results.