Study On The Effect Of Bifidobacterium With Tumstatin Gene On Treating With Large Bowel Carcinoma

Objectives:To explore the effect of Bifidobacterium with tumstatin gene on treating with large bowel carcinoma.Methods:The pre-constructed plasmid PBADs-BPP-EGFP was amplified in E.coli,then extracted,electrotransformed into Bifidobacterium longum,and induced the expression of EGFP by L-arabinose.The protein expression of EGFP gene was observed by using laser scanning conocal microscopy and detected by using western blot.The pre-constructed plasmid PBADs-BPP-tumstatin was amplifed in E.coli,then extracted,electrotransformed into Bifidobacteria longum too.The positive clonal Bifidobacteria were screened.The expression of tumstatin gene was detected by using western blot.The model of large bowel carcinoma was established by injecting CT26 cells into mouse's left axil subcutaneously.Bifidobacteria with tumstatin gene and Bifidobacteria with EGFP gene were injected into tumor-beating mice by gavaging passage,tumor injection and mouse tail vein injection respectively.A total of 15 times injection was acomplished.The tumor volume and the adverse effects were observed.All mice were sacrificed and tumors were excised for routine histological examination after one month. The rate of tumor inhibition was calculated.The tissue structure was observed by HE staining.Gram stain confirmed that whether the Bifidobacteria existed in the tumor or not.The microvessel density was detected by employing immunohistochemistry.The apoptosis of large bowel carcinoma and its vascular endothelium was detected by using TUNEL assay.Results:(1)Green fluorescence was observed by using laser scanning conocal microscopy in the EGFP engineered Bifidobacterium.(2) A 27 Kilo-Dolton protein band was detected by western blot in the Bifidobacteria with EGFP gene.(3) A 29 Kilo-Dolton protein band was detected by western blot in the Bifidobacteria with tumstatin gene.(4) The mouse model of large bowel carcinoma was constructed successfully.(5) The rate of tumor inhibition was 38.56 %,75.21%and 64.63%respectively in Bifidobacteria with human tumstatin gene gavaging group,tumor injection group and mouse tail vein injection group.(6) Gram stain confirmed that many Bifidobacteria exited in large bowel carcinoma.(7) Immunohistochemistry assay showed that the microvessel density in treatment group was significantly lower than that in control group. (P＜0.01)(8) TUNEL assay showed that more apoptotic cells were detected in large bowel carcinoma and its vascular endothelium in treatment group when compared with control group. Conclusions:(1) the vector that we had constructed could express exogenous gene EGFP.(2) Bifidobacteria with human tumstatin gene were constructed and identified.(3) Bifidobacteria with human tumstatin could effectively inhibit the growth of experimental large bowel carcinoma.(4) Bifidobacteria with human tumstatin gene could effectively inhibit angiogenesis of large bowel carcinoma.(5) Bifidobacterium with human tumstatin gene could effectively induce apoptosis of tumor cells and vascular endothelial cells of large bowel carcinoma.