ABSTRACT
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.
ABSTRACT
Objective To compare the cellular immune responses induced by two different vectors,eukaryotic plasmid pcDN3.1(+) and attenuated typhia live vaccine Ty21a,-associated mimic epitopes of HCV hypervariable region 1,so to find a better way for vaccine immunization.Methods The DNA sequence based on the peptides' sequence of HCV hypervariable region 1 related consensus polymimotopes was synthesize and then inserted into plasmid pcDN3.1(+) to construct recombinant plasmid pcDN3.1(+)-SP,which then was transfected into live attenuated typhia vaccine Ty21a to construct Ty21a-SP.The mice were immunized orally with Ty21a-SP and intramuscularly with pcDN3.1(+)-SP,respectively,and then sacrificed by exsanguination.The splenocytes were separated and restimulated with pooled synthesized peptides,and then collected.Flow cytometry was employed to identify CD8+IFN-?+ T cells,and non-radioactive MTS method was adopted to test T cell proliferation,and non-radioactive LDH method was used to test cytotoxic T cytolytic reaction(CTL).Results Compared with control groups,the proliferation of splenocytes was apparently enhanced,the proportion of CD8+IFN-?+ cells obviously increased,and CTL responses also significantly increased after the spenocytes of mice immunized by pcDN3.1-SP and Ty21a-SP were restimulated with synthesized peptides.And the responses mentioned above in the mice immunized by Ty21a-SP were stronger than those mice immunized by pcDN3.1-SP.Conclusion Using attenuated typhia live vaccine Ty21a as DNA vector is an effective way to induce cellular immune responses.
ABSTRACT
Objective:To observe the effect on different mucosal sites and system immune sites after intranasal immunization with bivalent Shigella vaccines Methods:BALB/c mice were divided into three groups at random , 10 mice per group Mice were intranasally immunized respectively with FSM 2117or FS 5416 (4?10 7CFU) three doses with an interval of two weeks The NALT, NP, spleen, PP, MLN, lymphocytes were isolated on the seventh day after the last immunization to assay the change of the cell phenotype with FACS The nasal ?lung?intestine?genital tract lavage fluid and serum were taken to assay the specific IgA or IgG against F2a or Sonni LPS with ELISA Results:The specific IgA and IgG in different mucosal sites and serum increased significantly after intranasal immunization with two Shigella vaccines compared with the control (P