A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF.

Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.

Effect of hepatocyte growth factor on the promotion of in vitro spinal cord neurite regeneration / 中国组织工程研究

BACKGROUND: Hepatocyte growth factor (HGF) promotes neurite outgrowth from neocortical explants, and supports neuronal survival under serum-free condition. Thus, HGF can mediate neurotrophic function as a novel neurotrophic factor.OBJECTIVE: To establish an in vitro injury model with a semi-solid culture system for the purpose of improving the evaluation of neurite regeneration of transected spinal cord neurons from rat embryo, and investigate the effect of HGF on neurite regeneration.DESIGN: Randomized controlled study.SETTING: Hematology Laboratory of Radian Medical Institute of Academy of Military Medical Sciences of PLA.MATERIALS: The experiment was carried out at the Hematology Laboratory of Radian Medical Institute of Academy of Military Medical Sciences of PLA from August 2004 to May 2005. Wistar fetal rats of 14-16 days old were provided by Animal Center of Academy of Military Medical Sciences of PLA. Tail collagen was extracted from adult male Wistar rats with body mass of (250±50) g.METHODS: ① Rat tail type Ⅰ collagen substrate was prepared and spread on a culture dish, cut into about 0.5-1.0 mm3 slices, then spinal cord slices of 15-day-old fetal Wistar rat were explanted on the primary culture. Five days later, the outgrowing processes were severed, then a block of collagen, with the surface area of 2 mm2 and 200 μm away from the slice, was removed and the vacancy was replaced with a fresh collagen block of 2 μL after aspirating the medium. The fresh collagen block could be solidified and then fresh liquid medium was added as the secondary culture. The regeneration of neurite was observed by microscopy at 0, 1, 6,12 and 24 hours after severing. ② The medium was changed with 0.5% N3-conditioned medium. 10 μg/L HGF was added in the experimental group, and 0.5% N3-conditioned medium was added in the control group.The status of regeneration was evaluated by the average value of 3 longest regenerative neurites for each slice. There were 12 slices in each group.The status of neurite regeneration was calculated and was evaluated 24 hours later.MAIN OUTCOME MEASURES: ① neurite regeneration in situ; ②comparisons of neurite regeneration between control group and experimental group.RESULTS: ① Neurite regeneration in situ: The neurites disintegrated near the severing line immediately following the transection injury. This process persisted about 1-2 hours and the distance away from the severing line was about 20 μm. Then the proximal end of neurites would swell and thicken. At this time neurites stopped collapsing and neurite regeneration began. Their regenerating rate would quicken at 12 hours after severing. Regenerating neurites were more branching and curlier as compared with original neurites. ② Comparisons of neurite regeneration between control group and experimental group: The average length of regenerative neurites was more in the experimental group than that in the control group [(375±96) μm, (200±75) μm, P ＜ 0.05].CONCLUSION: ① We establish a simple, economic model to evaluate neurite regeneration. ② By this model, we prove that HGF can promote neurite regeneration.

HGF prolongs the survival period of primary cultured rat spinal neurons / 基础医学与临床

Objective To explore the protective effect of HGF on primary cultured spinal neurons in vitro.Methods Cultured neurons were transfected with Ad-GFP or Ad-HGF in different multiplicity of infection(MOI),then flow cytometer and PI-Hoechst double stains were used to assay transfer rate or to determine the status of cells respectively.ELISA was used to detect the expression of HGF in Ad-HGF transfected neurons,and the activity of neurons was judged by neutral red stain,MTT and NSE-ELISA methods.Results After transfection with Ad-GFP in MOI of 50,the transfer rate was high as detected by flow cytometer,and the status of cells was well as judged by PI-Hoechst double stain.The results of ELISA showed that HGF was expressed in medium.After transfection with Ad-HGF,the activity of neurons was better than neurons without treatment(P

Study of in vitro expansion and differentiation into neuron-like cells of human umbilical cord blood mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro.</p><p><b>METHODS</b>Human cord blood samples were obtained sterilely with 20 U/ml preservative-free heparin. MSCs were isolated by lymphocyte separation medium (density 1.077 g/ml), and purified and expanded with Mesencult trade mark medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron-like cells. Specific markers and structures were detected by immunohistochemistry and histochemistry methods.</p><p><b>RESULTS</b>The number of MSCs increased two- to three-fold with each expanded passage. 6.6 x 10(5) primary MSCs were expanded ten passages to reach a number of 9.9 x 10(8), and was increased about 1.5 x 10(3)-fold. Flow cytometry showed that MSCs did not express antigens CD(34), CD(11a) and CD(11b), but expressed strongly CD(29) and weakly CD(71), which was identical to human bone marrow-derived MSCs. 70% cells exhibited typical neuron-like phenotype after induction. Immunohistochemistry staining showed that all of the induced different-passage MSCs expressed neurofilament (NF) and neuron-specific enolase (NSE). Special Nissl body was found by histochemistry.</p><p><b>CONCLUSION</b>MSCs in human umbilical cord blood can expand in vitro and differentiate into non-mesenchymal cells.</p>

Selection of the exogenous signal peptide that conducts the secretion of hepatopoietin / 中国病理生理杂志

AIM:To look for a suitable signal peptide which may effectively conduct hepatopoietin(HPO)secretion,various recombinant eukaryotic expression vectors were constructed.METHODS:Different exogenous signal sequences were spliced with HPO cDNA by PCR,and the spliced genes were cloned into eukaryotic expression plasmids.The different recombinants were respectively tansfected into COS-7 cells by Lipofectamine 2000 method and the secretion of HPO was analyzed by Western blotting.RESULTS:Western blotting analysis indicated that the signal peptides from interleukin-1 receptor antagonist(IL-1ra)and an artifical signal peptide did not secret HPO directly and effectively,but the signal peptide from murine Ig kappa secreted HPO directly with great efficiency.The molecular weight of the secreted HPO was 30 kD,which means that the secreted HPO existed in homodimer.CONCLUSION:Secreted recombinant expression plasmid is successful constructed.The result may pave the way for the gene therapy of hepatic fibrosis.

Three coumarins from seed of Cnidium monnieri and their multidrug resistance reversal effects / 中草药

Object To isolate the active compounds on reversing multidrug resistance (MDR) of tumor cell from the ethanol extract in the seeds of Cnidium monnieri (L ) Cuss Methods The fractionation directed by bioactivity was carried out with silica gel chromatography and RP HPLC Results Three active coumarins were obtained: imperatorin (Ⅰ), edultin (Ⅱ) and 3′ isobutyryloxy O acetyl columbionetin (Ⅲ) Their sturctures were identified by spectroscopic analysis Conclusion These three compounds have a medium reversing MDR of KBV200 in vitro

Effects of new drug of sulfonylurea on the glucose uptake in SMMC7721 cells / 中国病理生理杂志

AIM:To explore the effects of the new drug of sulfonylurea(1-{4-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido)ethyl]-phenylsulfonyl}-3-(1,4-tetramethylene)-urea,BGW) on the glucose uptake and the activation of Akt/PKB in SMMC7721 cells.METHODS:Cultured SMMC7721 cells were divided into control group,glibenclamide group,insulin group,BGW group and BGW+insulin group.Scintillation was used to detect the glucose uptake in SMM7721 cells.The activation of Akt/PKB was tested by Western blotting.RESULTS:Compared to control cells,gibenclamide,insulin,BGW and BGW+insulin significantly increased the glucose uptake(P

COMPARATIVE STUDIES ON THE CHARACTERISTICS OF PROLIFERATION AND DIFFERENTIATION OF SPLEEN COLONY FORMING CELLS FROM DIFFERENT SOURCES / 解放军医学杂志

By using single spleen colony transplantation technique and sex chromosome typing as natural cytogenetic markers, we have been able to demonstrate that most spleen colony forming cells in the adult bone marrow or in foetal liver of inbred LACA or C57 mice possess the potential of re-establishment of myeloid and lymphoid lines of cells in lethally irradiated mice. However, most of the spleen colony forming cells in the peripheral [blood of normal mice possess weak or no potentiality to proliferate or re-establish hemopoiesis in lethally irradiated mice. Our results support the notion that the CFU-S population in mice is heterogeneous.From the nature of colony forming cells in the peripheral blood of normal mice, we are convinced that the assessment of CFU-S content relying upon the spleen colonies as the unique sign to indicate the self-renewal ability of a spleen colony forming cell is by no means valid inasmuch as some plu-ripotent . hemopoietic progenitors derived from pluripotent hemopoietic stem cells may have the same ability to form colonies in vivo.