| Bladder cancer is the most common urology malignant disease in China. It is important assistance means that intravesical Bacillus Calmette-Guerin (BCG) administeration for preventing and curing urine bladder tumor. In recent years,a new schedule with concomitant administration of iow-dose BCG plus INF-α has been proposed.But due to local and systemic complications of bladder makes the application of intravesical BCG therapy subject to certain limit. Recombinat human IFN-α-2b-BCG (Interferon-a-2b-BCG)that successfully manufactured by ourselves is a new germ bacterial vaccine,comparison with wild BCG, in addition to reserving basic characteristics of wild BCG, its advantage consist in keeping on secreting IFN-α-2b, BCG and IFN-α-2b simultaneous working can produce the best immunity effect, its anti-tumor effect is more obviously, side effect less takes place, more suitable for patients with long-term perfusion.Objective:The previous studies of our laboratory have recombinanted the human IFN-a-2b-BCG, which could secrete IFN-a-2b.To further study of recombinant human IFN-a-2b-BCG anti-tumor mechanisms, we conducted the following two studies:The first part mainly discussed the antitumor effect of recombinant BCG in vitro. In the second part:BCG resistants to multiple antibiotics, which instillation often cause adverse reactions such as urgency, urinary pain, sepsis.we detected the sensitivity of human recombinant IFN-α-2b-BCG for10common antibacterial drug, to explore safety and efficacy of human recombinant IFN-α-2b-BCG immunization by the appropriate use of antibacterial drugs. Experimental methods:Part I:(1)Peripheral blood monocytes (PBMC)were stimulated by recombinant BCG (rBCG) and transformed recombinant BCG activated killer (rBAK)cells,cultured with human bladder cancer t24cells,study the effect of antitumor of rBAK with MTT assay.(2)we cultured the recombinant BCG and human bladder cancer T24cells in vitro together, to study the antitumor effect of recombinant BCG with MTT, electron microscopy, fluorescence staining assayPart II:we detected sensitivity of human recombinant BCG vaccine and wild BCG for10common drugs by artificial marking medium and the radiometric BACTEC960method, to study the validity of the recombinant BCG infusion security, and correctly and effectively use of antimicrobial drugs, to decrease of antimicrobial agents against recombinant BCG strains antitumor effects.Results:Part I:The first part:(1), The result show that the anti-tumor activity of lymphocyte without was the lowest; lymphocytes by wild BCG stimulated,which began proliferation and secretion of cytokines, immune activity, antitumor effects enhanced obviously (26.39%-48.95); the anti-tumor activity of lymphocyte stimulated by continue secrete recombinant BCG was the highest in all groups(37.63-63.10) at different times.(2) Our observation revealed of wBCG and recombinant BCG all have antiproliferative directly on bladder tumor cell growth in vitro,the recombinant BCG is most effective in all.After recombinant BCG and bladder tumor (T24) cultured together, we find T24become proliferate slowly obviously, detach,quantity deceasing and death under microscope, and various degree degeneration on most of tumor cell, disorganization on organell, aggregation on chromatin, pycnosis on nucleolus, and some cytoplasm lysis were observed on tumor cell under transmission electro microscopy. Cellular membrane bulged and some bubbles appearance with Acridine orange staining under fluorescence microscope. We examined the antitumor effect of recombinant BCG in vitro. The results showed that the recombinant BCG can inhibit the growth of T24cells, and the inhibition rate were higher than wBCG.Part II:We determine the effects of modern antibacterial on recombinant BCG and wide BCG viability. BCG was assumed susceptible when the minimal inhibitory concentration determined for a drug did not exceed25%of the highest blood level achieved by the standard dose.Local drug susceptibility was assumed when the minimal inhibitory concentration was less than25%of the highest urinary level.Recombinant BCG was susceptible to rifampin, isoniazid, ethambutol in systemic infections but resistant to pyrazinamide of antituberculosis drugs tests, same as the wild BCG. Recombinant BCG was susceptible to amikacin, streptomycin but resistant to gentamicin and kanamycin in systemic infections, while wild BCG was resistant to gentamicin but showed good susceptibility toward the other aminoglycoside drugs tested. Both two BCG strains analyzed were susceptible to ofloxacin, ciprofloxacin of quinolones drugs tested. Since many drugs show high urinary recovery, the bladder level may far exceed the blood level.In partial infection, rBCG was susceptible to rifampin, isoniazid, ethambutol but resistant to pyrazinamide of antituberculosis drugs tests, same as the wild BCG. Both BCG and wBCG were susceptible to gentamicin, kanamycin, amikacin, streptomycin of aminoglycosides drugs tested and ofloxacin, ciprofloxacin of quinolone.Conclution:In conclution,we think that recombinant BCG have antitumor effects by stimulating lymphocyte and have antiproliferative directly on bladder tumor T24cell growth in vitro,and more active than wild type BCG dose. Recombinanted BCG was susceptible to toofloxacin, ciprofloxacin of quinolones but resistant gentamicin and kanamycin of aminoglycosides. It was resistant to pyrazinamide but showed good susceptibility toward the other antituberculosis drugs tested. The results of this study may contribute toward enhancing the therapeutic safety and efficacy of intravesical BCG immunotherapy by the appropriate use of antibacterial drugs. |
