Study On The Immunotherapeutic Effect Of Recombinant HIFN-α-2b-BCG Anti-bladder Tumor

Bladder cancer is the most common urological malignant disease in China.In recent years, a new schedule with concomitant administration of low-doseBCG plus IFN-αhas been proposed, and a number of pilot clinical trials haveshown that combination therapy is well tolerated and can yield a high completeresponse rate. The new schedule got a well result in orthotopic murine bladdercancer model also. Adding IFN-αto BCG bladder cancer immunotherapy couldlower BCG toxicity due to the reduced BCG dose while at the same timepreserving or enhancing BCG activity against tumours. Although the mechanismby which IFN-αenhancing BCG-mediated antitumor activity is poorlyunderstood, recent study shown some possible reasons:①IFN-alphaup-regulated BCG-induced IL-12 and TNF-alpha and down-regulatedBCG-induced IL-10;②Improved the cytokines of IL-6,IL-8,GM-CSF andTNF-α;③increased T cells;④IFN-alpha inhibited proliferation of bladdertumour cellBase on the current understanding of the combination therapy in bladder cancer,the study intended to make a new strain of BCG that constitutively expressescytokines or marker protein or antigen. With the development of molecularcloning technique, these schemes becoming into reality, the recombinant BCGcoming into a new time.A new topic coming out with the new recombinant BCG strain, for example thecharacter of growth, stable expressing of cytokines, ability of antitumor, impactto T lymphocyte, effect of immunotherapy and immune reaction in local and sys.The objective in this topic is exploring the mechanism of rBCG antitumor invitro and vivo.1. Immunoloregulation of hIFN-α-2b-BCG to Peripheral blood monocytes(PBMC)(1)Measure the IFN-α-2b content in supernatant of rBCG and the result shownthat nearly 5000IU/ml equivalently. (2) PBMC were abstracted and cultivated with rBCG, wild-type BCG (wBCG)and wBCG plus IFN-α-2b in different density in vitro. Measure proliferation ofPBMC with MTT test, and measure density of cytokines about hIFN-γ,hIL-12and hTNF-αwith ELISA, so we can compare the ability of activate PBMC indifferent strain. The result shown clearly that recombinant BCG can induce morecytokines that produced by PBMC than wild-type BCG does.(3)The activity of IFN-α-2b in supernatant with IFN-α-2b used in clinic isapproximate.2. Study about antitumor effect of recombinant BCG in vitro(1) PBMC were stimulated by recombinant BCG and transformed into bacilliCalmette-Guerin activated killer (BAK) cell, study the effect of anticancer ofBAK with LDH release assay. The result show that the antitumor activity ofBAK cells stimulated by Recombinant BCG was the highest in all groupsincluding wBCG group and wBCG with IFN-α-2b group.(2) Our observation revealed of wBCG and recombinant BCG all haveantiproliferative directly on bladder tumor cell growth in vitro, the recombinantBCG is most effective in all. After the recombinant BCG cultivated with bladdertumor cell together we find tumor cell become proliferate slowly, detach, quantitydecreasing and death under microscope, and various degree degeneration on mostof tumor cell, disorganization on organell, aggregation on chromatin, pycnosis onnucleolus, and some Cytoplasm Lysis were observed on tumor cell undertransmission electro microscopy. Cellular membrane bulged and some bubblesappearanced with AO staining under fluorescence microscope. Ho33258 assayalso indicated that a lots of tumor cells apoptosis.(3)DNA ladder was observed in DNA electrophoresis assay and confirm theapoptosis of bladder tumor cell.(4)Research the effect of recombinant BCG anti-tumor cell with MTT assay, theresult shown that rBCG inhibited the proliferation of bladder caner cell and moreactive than wBCG does. (5)Detect the apoptosis rate of MB49 after co-cultivated with rBCG or wBCG inflow cytometry. The apoptosis rate of rBCG group is 19.71％and 46.58％in24hours and 48hours respectively, more active than wBCG group which is10.81％and 20.94％respectively.(6) In vitro experiments on a panel of bladder tumor cell lines-MB49 using theflow cytometry analysis indicate that both of kind BCG could induce orup-regulate the expression of MHC-Ⅰand the recombinant BCG are mosteffective in all. These results suggest that the recombinant BCG have moreimmunomodulatory properties, anti-tumor effects and cytotoxicity in vitro, whichcould increase recognition of the tumor cell by leucocytes.3. Study the antitumor effect of recombinant BCG in vivo(1)At the base of successfully established the MB49 orthotopic murine bladdercancer model in C57BL/6 mice, the inhibition effect of recombinant BCG on thetumor bearing mice was studied by intravesical treatment. The result revealedthat the living time of rBCG treatment mice was longer than that of wBCG andwBCG with IFN-α, though the tumor weight in different groups were notsignificant difference which due to the variability of mero-different genus.(2)Analysis the change of lymphocyte subgroup in FCM and detect density ofcytokines about mTNF-αand mIL-12 with ELISA, which can reveal theimmunity of sys and local in mice. The result shown that the ratio of CD4+ Tlymphocyte was up-regulated, so the ratio of CD4+/CD8+ was up-regulated,retrieve the disproportion of lymphocyte subgroup in orthotopic murine bladdercancer mice. The ELISA assay shown that recombinant BCG significantup-regulate the level of mTNF-αand mIL-12 in blood-serum of orthotopicmurine bladder cancer mice. Both of researches indicated that rBCG reinforcedimmunity of mice bearing tumor.(3)Immunohistochemistry was used to detect the immune reaction in localbladder after intravesical with recombinant BCG or wild-type BCG, the CD3,CD4, CD8phenotype of immunocompetent cell numbers increased obviously in tumor tissues in rBCG group than that of wBCG.(4) Immunohistochemistry was used to detect the expressing of P53 and Fas inbladder tumour tissues. Expression of Fas transformed from negative or weaklypositive into positive after intravesical rBCG treatment; Expression of P53transformed from positive into weakly positive after intravesical treatment.In conclusion, we evaluated recombinant BCG immunosuppress activitieson bladder tumor in vitro and animal model. These results found a significantbasis for the further exploring biological agents to treat bladder tumor andprevent its recurrence, and reducing complications of BCG, base a firmfoundation for clinical application.