Anerobic Bacteria In Combination With Interventional Therapy For Hepatic Carcinoma: An Experimental Study

Partâ… Imaging Characteristics of Buffalo Rat Orthotopic Hepatocellular Carcinoma ModelPURPOSE To facilitate interventional therapeutic studies of hepatocellular carcinoma(HCC) of small animal,we assessed magnetic resonance imaging(MRI), digital substraction angiographic(DSA) and pathologic characteristics in a novel HCC model.MATERIALS AND METHODS Rat hepatoma(McA-RH7777)cells were cultured with DMEM.Then 1.0×10⁶ cells were injected under direct visualization into the right or left lobe of the liver in 30 rats.MRI was performed at 7 and 14 days.MRI was used to measure the maximum tumor diameters.Three rats were killed at 7 and 14 days respectively,and tumors were harvested for pathologic study.After 14 days, hepatic artery or portal vein angiography were performed via the gastroduodenal artery and gastroduodenal vein respectively in 14 rats to assess the blood supply of the tumor on DSA.RESULTS Injection of tumor cells in the liver was successful in all 30 rats (100%).The tumor was detectable with MRI in all animals after seven days.The mean tumor volume was 19.53±15.65 mm3 at 7days and was increased to 400.33±242.34 mm3 at 14days on MRI.The tumor demonstrated well-demarcated hypo-intensity signal on T₁WI and hyper-intensity signal on T₂WI.After injection of the contrast agent,a nodular or uneven enhanced mass was detected.DSA demonstrated a mass with nodular or circular tumor staining and with enlarged and/or twisted feeding artery.The mass showed no obvious blood-supply on portal vein angiograph.Pathologically,the tumor was an approximately round or ellipse nodule accompanied with integrated pepios and central necrosis.The mean survival time was 50.80±4.44 days in the tumor-bearing rat group.CONCLUSION An orthotopic HCC Buffalo rat model has the similar MRI and DSA characteristics to that of human HCC.This novel rat model is suitable for high throughput interventional therapeutic studies. Partâ…¡The Effect on Cell Growth and VEGF Expression of McA-RH7777 Hepatocellular Carcinoma Treated by Bifidobacterium longum Under Anaerobic ConditionPurpose To explore the cytotoxicity and the effect on vascular endothelial growth factor(VEGF) expression and secretion after McA-RH7777 hepatocellular carcinoma cell were anaerobic incubated with Bifidobacterium longum in vitro.Materials and Methods The McA-RH7777 cells were used as target cells,and was divided into two groups,one(group A) was treated with anaerobic condition and Bifidobacterium longum(B.longum),the other(group B) was cultured only under anaerobic condition.The results(OD value) of cell cytotoxicity treated by B.longum were determined by WST-1 colorimetry after 6h,12h,24h,48h and 72h.The vascular endothelial growth factor(VEGF) protein containing in the supernatant was detected after 0h,12h,24h,48h and 72h using ELISA method.The expression level of VEGFb was analyzed after 72h anaerobic culture for these two groups.Results The cytotoxicity to McA-RH7777 hepatocellular carcinoma cells and suppressing effect on this cell line was shown significantly after the B.longum was added into cell cultural solution.After only 12 hr of contact,sensitivity of McA-RH7777 cells to B.longum was observed.Cell growth was arrested and,instead of elongating,remained spheroid;intracytoplasmic granules appeared.After 72hr, nearly all the cells became detached from the monolayer and some of them formed debris.As time went by,it reveals that the cells were injuried and aggravated gradually.The toxicity of cells in group A was more severe than that in group B,the OD value shows significant difference between the two groups after 48hr.The concentrations of VEGF in supernatant detected by ELISA method have no significant differences at 0h,12h,24h,48h and 72h,respectively,between group A and B.The expression level of VEGF mRNA was higher in group A than that of group B after 72h anaerobic incubation.Conclusion The B.longum had overt cytotoxicity to McA-RH7777 hepatocelluar carcinoma cells cultured in vitro under anaerobic condition.The VEGF mRNA level was up-regulated,but the VEGF concentrations in supernatant had no remarkable increase at different time. Partâ…¢MRI in detection of Bifidobacterium longum and C.novyi-NT labeled with superparamagnetic iron oxide nanoparticlesPURPOSE To explore the feasibility of Bifidobacterium longum and C.novyi-NT tagging with superparamagnetic iron oxide(SPIO) nanoparticles and to evaluate its MR imaging in vitro and in vivo.Material and methods Four groups tubes with different formulation were incubated under anaerobic condition in vitro experiment:(1) Group B.longum-SPIO (n=6):B.longum and SPIO coculture in the PYG medium;(2)Group Free-SPIO(n=6): only SPIO in the PYG medium;(3) Group B.longum(n=6):B.longum incubating in the PYG medium;(4) Group Medium(n=6):PYG medium;Transmission electron microscope,Gram staining and Prussian blue staining were used to examine for demonstrating intracytoplastic nanoparticles after 72hr anaerobic incubation.MRI scanning were performed for all tubes using T2^* mapping and T2 mapping,and R2^* mapping and R2 mapping were reconstructed.The R2^* and R2 value were measured. The same treatment is applied for C.novyi-NT(cultured with RCM medium).Eight Baffulo rats hepatocellular carcinoma subcutaneous tumor models and 8 hepatic orthotopic tumor models were established for in vivo testing.One milliliter 28μg Fe/ml B.longum-SPIO and Free-SPIO were directly injected in rats subcutaneous tumors.For hepatic orthotopic tumors 1ml 28μg Fe/ml B.longum-SPIO was injected via the tail vein.MRI change and pathologic section prussian blue staining were performed to detect whether there are iron particles exiting in the tumors.Results The B.longum activity hasn't been inhibited by tagging SPIO.Electron microscopic observation revealed many larger iron particles in the bacteria.The bacteria iron staining was obviously shown by Prussian blue staining.The R₂^* value of B.longum-SPIO(273.25 sec-1±22.35) was significantly higher than that of Free-SPIO(125.63 sec^-1±2.19)(P＜0.O01),however,the R₂^* value of B.longum(5.83 sec^-1±0.75) had no significant difference with that of medium(5.00 sec^-1±0.64)(P＞0.05).On R₂ mapping,the Free-SPIO signal intensity(75.61 sec^-1±0.20) was significant higher than that of B.longum-SPIO(2.70 sec^-1±0.11)(P＜0.001),whereas the signal intensity of B.longum had no significant differences with that of medium(P＞0.05).At the same concentration of SPIO,on R2^* mapping,the B.longum-SPIO signal intensity is higher than that of Free-SPIO.Contrary to R2^* mapping,the signal-intensity of Free-SPIO is higher than that of B.longum-SPIO on R2 mapping. R2 and R2^* values were linearly correlated with B.longum-SPlO,number of SPIO-labelled bacteria,and content of Free-SPIO.For B.longum-SPIO,R2^* effects were significantly greater than R2 effects(P＜.01),and the linear slope of R2^* effects is 31.25 times that of R2 effects.For free SPIO,R2 and R2^* effects were similar,and the linear slope of R2^* effects is 1.99 times that of R2 effects.The signal-intensity of subcutaneous tumor injected with B.longum-SPIO on R2^* mapping is obviously higher than that of tumors injected with Free-SPIO,however,on R2 mapping the signal intensity is lower than tumors injected with Free-SPIO.The similar results are required from C.novyi-NT test.T2WI shows that scattered hypointensity in tumors after injection with B.longum-SPIO via tail vein.The hypointensity showed on T2^*WI is extended and is even lower than that showed on T2WI.Iron staining particles is presented widespread in hypoxia and/or necrosis areas of tumor,and in some areas rod shaped iron staining particles was aggregated which morphologic changes is consistent with B.longum.Conclusion B.longum(or C.novyi-NT) can be labeled by SPIO.The distribution of SPIO was changed and caused corresponding changes of R2^* and R2 effects which might be used to track the B.longum(or C.novyi-NT) anchored in the tumor. Partâ…£Buffalo Rat Subcutaneous Transplanted Hepatocellular Carcinoma:Treated by Bifidobacterium longum Combined with Chemotherapy and Percutaneous Ethonal InjectionPurpose To explore the therapeutic effect and potential mechanism of bifidobacterium longum in combination with chemotherapy and percutaneous ethonal injection(PEI) for Buffalo rat subcutaneous transplanted McA-RH7777 hepatocellular carcinoma.Material and methods Forty Buffalo rats subcutaneous transplanted McA-RH7777 hepatocellular carcinoma model were established and were divided into four groups after 14 days.(1) Control group[G(C),n=10];(2) B.longum oncolysis group[G(B),n=10];(3) PEI and chemotherapy with mitomycin C(MMC) [G(EM),n=10];(4) B.longum oncolysis in combination with PEI and chemotherapy with MMC[G(EBM),n=10].The tumor volume and relative tumor volume(RV) were calculated by data measured on MR imaging performed at 3,6,9,12,15 days after treatment.The tumor growth was compared between groups.All the rats were sacrificed and pathologic change,immunohistochemistry and bacterial culture were performed after 15 days.Results Forty Buffalo rats subcutaneous transplanted tumor model were successfully established.Tumors in group C and group B grown continuously, however,tumor growth inhibition was observed causing tumor volume decreased in group EM and group EBM.The paired comparison by SNK method demonstrated that,RV has no significant differences between group C and group B,however,when group C compared with group EM or group EBM,significant differences were discovered.RV in group EM has significant differences with that in group EBM.The VEGF expression in tumor demonstrated by immunohistochemistry method was increased in group EM and might be decreased by combining with B.longum.The odd phenomenon is that VEGF expression doesn't decrease when B.longum is used alone. Diarrhea occurred in six rats treated with EM.Cutaneous necrosis emerged in one rats treated with EM and two rats treated with EBM.There is no abscess-formation in each organ at autopsy.Gram staining shows that B.longum aggregated in the hypoxia and/or necrosis areas of tumor and distributed in a confined space.A great quantity of B.longum were cultured in tumor tissue,however,there is no colony formed in the flat plate containing hepatic,lung,heart,kidney and spleen tissue. Conclusion Bifidobacterium longum combined with percutaneous ethanol injection and chemotherapy could effectively inhibit the tumor growth of Buffalo rat McA-RH7777 hepatocellular carcinoma,the B.longum colonization and propagation might decrease local VEGF expression. Partâ…¤Transhepatic Artery Chemoembolization Combined with Percutaneous C.novyi-NT Spore Injection for Rabbit VX2 Liver carcinomPurpose To investigate the therapeutic effect of transartery chemoembolization in combination with Percutaneous C.novyi-NT Spore Injection for New Zealand white rabbits VX2 liver carcinoma.Material and methods C.novyi-NT spores were purified for future use after at least 14 days anaerobic culture and sporulation.Forty New Zealand white rabbits VX2 liver tumor model were successfully established.MRI was performed after 14 days to identify the tumor and to measure the size.Animals were randomly divided into four groups:sham operation group(group A,n=8);transartery chemoembolization(TACE) group(group B,n=8);TACE and percutaneous C.novyi-NT spores injection(group C,n=12);percutaneous C.novyi-NT spores injection(group D,n=12);Animals in group C and group D were sacrificed individially(three tissue pieces from different point of each rabbit tumor at different time point) at 1,3,7 and 14 days after treatment.One microlitre tissue pieces homogenates were cultured under anaerobic condition to detect the bacteria anchored in tumor.MRI was re-examined after 21 days.Two rabbits in each group were sacrificed and pathology and bacteria culture were performed.The long term therapeutic effect and complication were observed in six rabbits each group.Results C.novyi-NT spores were sporulated and purified successfully. Therapeutic procedures completed in each group.The relative tumor volume has significant statistic difference among four groups after 21 days(x²=18.74,P＜0.001, Kruskal-Wallace H test).The proportion of necrosis measured on enhanced MRI has significant difference among four groups(P＜0.001,Kruskal-Wallace test).The mean survival time for A,B,C and D group is 30.83±3.98 days,63.33±4.57 days,86.50±2.93 days and 44.67±2.81 days respectively.Kaplan Meier survival anlysis shows that the cumulative survival rates of group C is obviously longer than other three groups.In group C and group D,live bacteria could be detected in tumor after 24 hours injection and increased significantly after three days.The number of live C.novyi-NT continue increased and reached peak at 14 days,then decreased. Pathology showed that tumors treated by TACE in combination with C.novyi-NT spores injection became hard nodular with light yellow cross-section.Microscopy shows that tumor necrosis is predominant when compared with other three groups.Conclusion Transheptic artery chemoembolization combined with percutaneous C.novyi-NT spores injection could promote C.novyi-NT's growth causing tumor necrosis extend,inhibit tumor growth,and prolong rabbit bearing tumor survival time.