ABSTRACT
Objective To obtain a mouse CD40-specific single-chain antibody (scFv) with high purity and to investigate its in vivo and in vitro agonistic activity on natural killer(NK) cells against tumor. Methods Agonistic anti-mouse CD40 scFv with high purity was obtained by genetic engineering. Mouse dendritic cells (DCs) were treated with different strategies including anti-CD40 monoclonal antibody (McAb),anti-CD40 scFv and negative control. Expression of IL-12 in the supernatants of DC cultures was measured by ELISA. Then DCs and NK cells were co-cultured to obtain activated NK cells,which were co-cultured with T6-17 cells. WST-8 was used to test the cytotoxic effects of NK cells on T6-17 cells. A mouse tumor model was established by injecting BALB/c nude mice with T6-17 cells. Anti-CD40 scFv was injected into tumor to evaluate its inhibitory effect on tumor growth. Levels of IL-12 and IFN-γ in the tumor microen-vironment were detected by ELISA. Changes in the percentages of tumor-infiltrating NK cells and the expres-sion of NKG2D protein(natural-killer group 2,member D) were detected by flow cytometry and immunohis-tochemistry,respectively. Results The recombinant plasmid CD40 scFv-pET28a was confirmed to be con-structed correctly. Results of SDS-page and His-tag Western blot revealed that anti-CD40 scFv could be ex-pressed successfully with a relative molecular mass of 27×103. The level of IL-12 in the supernatant of DC culture of anti-CD40 scFv group was (555.86 ±40.48) pg/ml, which was significantly higher than that of negative control group (P<0.05). The killing ability of NK cells in anti-CD40 scFv group was (72.23 ± 3.99)%,which was significantly higher than that in negative control group (P<0.05). Anti-CD40 scFv significantly inhibited the tumor growth in BALB/c nude mice as compared with negative control group(P<0.05). The levels of IL-12 and IFN-γ in tumor microenvironment of anti-CD40 scFv group were(188.801± 32.718) pg/ml and(121.428±30.994) pg/ml,respectively,which were significantly higher than those in normal saline(NS) group(P<0.05). The positive rate of NKG2D protein and the percentage of CD3-DX5+cells in anti-CD40 scFv group were respectively(8.18±2.01)% and(19.15±2.24)%,which were signifi-cantly higher than those in NS group (P<0.05). Conclusion Agonistic anti-mouse CD40 scFv could en-hance the anti-tumor ability of NK cells by activating DCs.
ABSTRACT
Objective:Probing the clinical characteristic,ARF caused by MM.Method:Reviewing the diagnostic process and evolution of 16 MM patients in recent 10 years,which reflected as ARF at first and be diagnosed as MM at last.Result:ARF could be the prime symptom.Bence-Jones proteinuria,protein electrophoresis,X-ray to the bone have low positive finding in MM diagnose,pathological examination is directly diagnostic method.Conclusion:Diagnosing ARF patients,whose cause of disease is unclear,we should think of MM,and pathological examination is necessary,so we could cure these patients better.
ABSTRACT
A segment of the diaphysis was sawed off from both radii of 57 adult male rabbits under anesthetic and antiseptic conditions.During healing of the bone,the pituitary bodies of these animals and 10 normal controls were removed and fixed in formol- HgCl_2 solution.Serial horizontal sections were prepared and stained with Adams' per- formic acid—Alcian blue—PAS (periodic acid—Schiff)—Orange G method for differentia- tion of the alpha-,S-,R-,and C-cells in the anterior lobe.The number of the 4 cell- types were counted under high power magnification.A total number of 2—3,000 cells in 6 fields selected from each gland were counted and classified and the percentage of each cell-type was calculated. Three days after the fracture,there is in the anterior pituitary,a significant increase in number of the scattered small-type S-cells (7.20?0.86% to 17.85?0.78%) and their cytoplasm shows an increase in stainability and an accumulation of granules,while the large-type S-cells in the anterior region show a decrease in number and loss of granules and stainability.In the light of the current results,the diffused small-type S-cells are probably concerned with the secretion of ACTH,and the large-type S-cells with the secretion of TSH. Two weeks after the fracture,the number of the alpha-cells increases (37.93? 1.83% to 57.44?4.15%) and their cytoplasm shows an increase of granules in- dicating that they are in a higher functional activity.This change seems to be related to the increased secretion of the STH during the latter period of healing of the bone fracture. The changes of R-cells and their significance await elucidation by further work.