Aim To observe the effect of endomorphin-1 (EM-1 )on TLR2 and TLR4 expressions of dendritic cells (DC)from human peripheral blood.Methods Monocytes isolated from human peripheral blood mono-nuclear cells were cultured in medium containing re-combinant human interleukin-4 and recombinant hu-man granulocyte macrophage colony stimulating factor. After six days of culture,the immature dendritic cells (imDC ) were divided into four groups,the control group (BLA group),EM-1 group,LPS group and LPS+EM-1 group.After 2 days of culture,the expres-sions of TLR2 and TLR4 were determined by fluores-cence activated cell sorter(FACS).The expressions of TLR2 and TLR4 at mRNA level in DC were detected by RT-PCR.Results The FACS results showed that the expressions of TLR2 and TLR4 in imDC were high-er,and their expressions were decreased with the mat-uration of DC.Compared with BLA group,the expres-sions of TLR2 and TLR4 in DC were down-regulated in EM-1 group (P<0.05 ).Compared with LPS group, TLR2,TLR4 on DC surface were significantly lower in LPS +EM-1 group (P <0.01 ). RT-PCR results showed that compared with BLA group,EM-1 interven-tion induced TLR2 mRNA expression was down-regula-ted significantly (P<0.01),there were no significant changes of TLR4 mRNA expression (P>0.05 ).mR-NA expressions of TLR2 and TLR4 on DC in LPS +EM-1 group were lower than those in LPS group (P<0.05 ).Conclusions EM-1 enables the down-regula-tion of the expressions of TLR2 and TLR4 on DC sur-face,the effects of EM-1 on immune function may be associated with TLR2 and TLR4 expressions on DC surface.

CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E. coli BL21 (DE3). The target protein was soluble and accounted for about 40% of the total protein in the supernatant. Following an ultrasonic cytolysis step, the recombinant protein was purified by anion exchange, affinity and desalting chromatography and the purity of the final preparation reached 95%. Cytotoxicity tests showed that the IC₅₀ value of CRM197 was 2.1×10⁷ times the IC₅₀ value of diphtheria toxin, and 9.6 times the IC50 value of diphtheria toxoid, telling that the target protein is safe and non-toxic. Subsequently, we found that both the high dose (20 μg) and the low dose (2 μg) of CRM197 were equally efficient in inducing an immune response against diphtheria toxiod in mice, and the antibodies titer of mice after three immunizations with low dose could reach 1:409 600. In conclusion, our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E. coli, with good immunogenicity and safety.

TSCI have dyskinesia and sensory disturbance that can cause various life-threaten complications. The patients with traumatic spinal cord injury (TSCI), seriously affecting the quality of life of patients. Based on the epidemiology of TSCI and domestic and foreign literatures as well as expert investigations, this expert consensus reviews the definition, injury classification, rehabilitation assessment, rehabilitation strategies and rehabilitation measures of TSCI so as to provide early standardized rehabilitation treatment methods for TSCI.

OBJECTIVE：To establish the fingerprint of crude/vinegar-processed Corydalis yanhusuo decoction pieces and their dispensing granules，and to determine the contents of five alkaloids （protopine，tetrahydropalmatine，corydaline，berberine hydrochloride，palmatine hydrochloride ）. METHODS ：HPLC method was adopted. The determination was performed on Agilent TC-C18 column with mobile phase consisted of 0.1％ phosphoric acid solution-methanol （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelength was set at 280 nm，and the column temperature was 30 ℃. The sample size was 10 μL. Using palmatine hydrochloride as reference ， Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition） was used to establish the fingerprint of 11 059） batches of C. yanhusuo decoction pieces ，7 batches of crude . yanhusuo dispensing granules ， 12 batches of vinegar- processed C. yanhusuo decoction pieces and 11 batches of vinegar-processed C. yanhusuo dispensing granules. The same HPLC method was adopted to determine the contents of protopine， tetrahydropalmatine， corydaline， berberine hydrochloride and palmatine hydrochloride in 41 batches of crude/ vinegar-processed C. yanhusuo decoction pieces and their dispensing granules. RESULTS ：There were 12 and 20 common peaks for crude C. yanhusuo decoction pieces and its dispensing granules ，and 14 and 16 common peaks for vinegar-processed C. yanhusuo decoction pieces and its dispensing granules. The similarity of each batch of same type were 0.529-0.981，0.342-0.985， 0.711-0.999，0.437-0.998，respectively. The linear range of protopine ，tetrahydropalmatine，corydaline，berberine hydrochloride and palmatine hydrochloride were 1.9-38.0，2.0-40.0，2.2-44.0，2.6-52.0，2.3-46.0 μg/mL（R2＞0.999 0）. The recoveries were 100.12％-100.98％（RSD＝1.05％-1.90％，n＝9）. RSDs of precision ，reproducibility，stability（24 h）and durability tests were all lower than 2.0％. The average contents of five alkaloids in different batches of crude/vinegar-processed C. yanhusuo decoction pieces and its dispensing granules were 0.24-0.46，0.37-0.82，0.24-0.58，0.07-0.75，0.24-0.76 mg/g. RSDs were 12.27％-147.48％. CONCLUSIONS：The fingerprint of crude/vinegar-processed C. yanhusuo decoction pieces and its dispensing granules is established successfully. The similarities of fingerprint are different before and after processing with vinegar ，and the contents of five alkaloids in C. yanhusuo decoction pieces and its dispensing granules are greatly different.