Objective: To prepare the conjugate of supcrantigen (SAg) staphylococcal enterotoxin A (SEA) and monoclonal antibody (McAb) against human hepatocellular carcinoma HAbl8 F(ab' )_(2) fragment and to investigate the anti-human hepatoma effect of peripheral blood mononuclear cells (PBMC) targeted by HAbl8 F(ab' )i-SEA. Methods: McAb HAbl8 was extracted and its F(ab' )_(2) fragment was prepared with papain; the conjugate HAblS F(ab' )_(2)-SEA was prepared with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP); eveny collected peak after purification was identified with gel chromatography; the activity of antibody in the conjugate was identified with immunohistocheinical ABC method; the anti-hepatoma effect of PBMC targeted by HAbl8 F(ab' )_(2)-SEA was observed with MTT method. Results: The conjugate HAbl8 F(ab' )_(2)-SEA was prepared successfully and it had obvious effect of targeting PBMC to kill hepatoma cells, and this effect is correlated positively with the dose of HAbl8 F(ab')_(2)-SEA. Control groups had no such effect. Conclusion: Targeting therapy of hepatoma with McAb-SAg conjugate might be a kind of hopeful model of hepatoma im-munotherapy.

Objective To study the expression of glycosylphosphatidylinositol(GPI) anchored protein in lymphocyte subsets of healthy individuals. Methods Flow cytometry and two/three color McAbs were used to detect CD59 expression in granulocytes, lymphocytes and subsets. Results In 50 samples, granulocytes mainly showed a single population of cells strongly positive for CD59, the percentage of CD59 expression was (98.6?1 5)%. Lymphocytes had a small population of weakly positive and negative for CD59, positive cells being (90 0? 14.9)%. CD3 +T cells had a similar profile as that of total lymphocytes, but there were more weakly positive /negative cells and less strong positive cells. As compared with CD3 +T cells, CD4 +T cells had a larger population of strongly positive for CD59, positive cells being (92.9?8.1)%; while CD8 +T cells had a smaller population of strongly positive, positive cells being (74.6?9.1)%. CD59 expression of activated T cells was between that of CD3 +and CD8 +T cells. CD45RA +and CD45RO +T cells both had a similar profile like that of CD3 +T cells. CD20 +B cells showed a similar profile to that of granulocytes, positive cells being (96.3?0 6)%. Conclusions Population of low or negative expression of CD59 among the lymphocytes of healthy controls was related to T cells, especially CD8 +T cells. B cells had uniform positive expression of CD59, and could be a candidate for detecting PNH.

Object To study the mechanism of triptolide solid lipid nanoparticle (TP-SLN) for decreasing liver toxicity of triptolide (TP). Methods With ig three doses of TP-SLN to mice for 60 d, the A LT, AST activities in serum and superoxide dismutase (SOD), glutathione peroxida se (GSH-Px) activites and malondialdehyde (MDA) contents in liver were determin ed. Results The activities of ALT, AST, SOD, GSH-Px and conten t of MDA between experimental group and blank group did not have remarkable diff e rence. However, the activites of ALT, AST, SOD, GSH-Px for the middle- (20 ?g /kg) and high-dose (30 ?g/kg) group were higher and the contents of MDA were lower than the experimental group. Conclusion TP-SLN can decrease the liver toxicity of TP.

To study the chemical constituents of the Entada phaseoloides (L.) Merr., seeds of Entada phaseoloides were extracted with 70% ethanol at room temperature. Isolation and purification were performed by silica gel, reversed-phase silica gel column chromatography and semi-preparative HPLC. Structures of the pure compounds were established on the basis of spectral analysis. Four sulfur-containing amide compounds were isolated from the n-BuOH-soluble fraction and identified as entadamide A-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranoside (1), entadamide A (2), entadamide A-beta-D-glucopyranoside (3) and clinacoside C (4). Compound 1 is a new compound. Compound 4 is isolated from the genus Entada for the first time.

To study the chemical constituents from the root of Berchemia lineata (L.) DC., nine compounds were isolated from the EtOAc extract by using silica gel, RP-C18 silica gel column chromatography and preparative HPLC. Based on the spectroscopic analysis, their structures were identified as 5-hydroxy-7-(2'-hydroxypropyl)-2-methyl-chromone (1), (-)-(1'R, 2'S)-erythro-5-hydroxy-7-(1', 2'-dihydroxypropyl)-2-methyl-chromone (2), naringenin (3), eriodictyol (4), (+)-aromadendrin (5), (+)-taxifolin (6), (+)-catechin (7), (+)-epigallocatechin (8) and quercetin (9). Among them, compound 2 is a new chromone derivative. Compound 1 is a known chromone derivative and isolated from this genus for the first time. Compounds 3-9 are known flavonoids and isolated from this plant for the first time.

To study the chemical constituents of aerial parts of Laggera pterodonta (DC.) Benth., the air-dried aerial parts of this plant were powered and extracted with boiling water and purified by silica gel column chromatography and recrystallized. Eleven compounds were obtained from L.pterodonta. They were identified as to be 6-O-β-D-glucopyranosyl-carvotanacetone (1), pterodontic acid (2), 1β-hydroxy pterondontic acid (3), pterodontoside A (4), pterodondiol (5), pterodontriol B (6), 5-hydroxy-3,4′,6,7-tetramethoxyflavone (7), artemitin (8), chrysosplenetin B (9), quercetin (10) and β-sitosterol (11). Compound 1 is a new monoterpene glucoside. Compounds 10 and 11 were isolated from this plant for the first time. Compounds 2 and 5 showed moderate activity against bacteria including Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Mycobacteium phlei and Bacillus circulans by paper disc diffusion method, while they both displayed no activity against Escherichia coli.

Objective To establish an HPLC method for determination of pterodontic acid in Lingdancao Soft Capsula. Methods Agilent Zorbax XDB-C18 column (250 mm?4.6 mm, 5 ?m) was used. Acetonitrile-water (60∶40) was used as mobile phase, the detection wavelength was 210 nm, flow rate was 1.0 mL/min, and column temperature was at 25 ℃. Results The linear range of pterodontic acid was 12.91—129.1 ?g/mL (r=0.999 9), the average recovery rate of pterodontic acid was 99.7 %, RSD was 2.0%. Conclusion The method is simple, accurate, and reliable, and it can be used to the quality control of this preparation effectively as a quantitative method.

Objective: To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability. Methods: Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was ?. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.