厌氧菌联合介入治疗肝癌的实验研究
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摘要
第一部分Buffalo大鼠移植型肝癌模型的生物学特点及影像表现
目的评价一种新的大鼠原位肝细胞癌模型的磁共振、数字减影血管造影、生物学特点变化及病理特征,促进大批量小动物肝细胞癌介入治疗研究。
材料和方法将McA-RH7777细胞开腹直视下接种于30只Buffalo大鼠肝左或右叶。造模后第7和14天行MRI平扫及增强扫描并分别取荷瘤大鼠各3只行病理检查。14天后荷瘤鼠14只分别经胃十二指肠动脉(7只)或静脉(7只)插管行肝动脉和门静脉造影,了解移植瘤DSA血供特点。其余10只荷瘤鼠长期观察生存状况。
结果造模后第7天,MRI观察见30只大鼠均有肿瘤生长,肿瘤平均体积为19.53±15.65mm~3;14天后增大到400.33±242.34 mm~3。肿瘤在T1WI为低信号,T2WI为高信号,增强后呈结节状或明显不均匀强化。7例大鼠DSA显示肿瘤供血动脉均有不同程度增粗扭曲,实质期呈结节状染色3例或环状不均匀染色4例。7只大鼠门静脉造影均无明显供血。肿瘤包膜完整,中央可见坏死。免疫组化显示肿瘤甲胎蛋白呈强阳性表达。10只荷瘤大鼠平均生存期为50.80±4.44天。
结论Buffalo大鼠McA-RH7777肝细胞癌,具有同人类肝细胞肝癌相似的MRI和DSA表现,非常适于进行大批量的介入治疗实验研究。
第二部分厌氧条件下长双歧杆菌对体外培养的McA-RH7777肝癌细胞生长及VEGF表达的影响
目的:探索厌氧条件下长双歧杆菌对体外培养的大鼠McA-RH7777肝癌细胞的毒性及对其VEGF表达及分泌的影响。
材料和方法:采用细胞培养技术,以大鼠肝癌细胞McA-RH7777为靶细胞,将McA-RH7777肝癌细胞分为两组,A组(厌氧培养条件下加入长双歧杆菌)和B组(单纯厌氧培养)。处理后肝癌细胞6h、12h、24h、48h、72h后,利用WST-1比色法测定长双歧杆菌对癌细胞毒性(OD值)。应用ELISA测定上清液中0h、12h、24h、48h、72h VEGF蛋白含量,使用RT-PCR法分析厌氧培养72h后两组细胞VEGFb表达水平。
结果:将长双歧杆菌加入体外培养的McA-RH7777肝癌细胞中显示出细胞毒性和对该细胞系的抑制效应。作用12h后,细胞生长开始出现停滞呈球形;细胞浆内出现颗粒状物。72h后培养液内细胞碎片出现。厌氧培养条件下,细胞损伤随时间的延长逐渐加重,A组毒性更大,48小时后两组OD值具有显著差异。上清液中VEGF蛋白含量在0h、12h、24h、48h、72h时两组相比均无明显统计学差异。72小时后A组厌氧培养的细胞VEGF mRNA表达水平明显高于B组。
结论:在厌氧条件下长双歧杆菌对体外培养的McA-RH7777肝癌细胞具有明显毒性。癌细胞VEGF mRNA表达上调,但上清液中总VEGF含量在各时间点均未见显著增高。
第三部分MRI检测超顺磁性氧化铁纳米颗粒标记长双歧杆菌和C.novyi-NT的实验研究
目的探索超顺磁性氧化铁标记长双歧杆菌或C.novyi-NT及采用MRI检测的可行性。
材料与方法体外实验中四组不同组分的培养管厌氧条件下培养:(1)B.longum-SPIO组(n=6):PYG液态培养基+B.longum+SPIO;(2)Free-SPIO组(n=6):PYG液态培养基+SPIO;(3)B.longum组(n=6):PYG液态培养基+B.longum;(4)Medium组(n=6):PYG液态培养基。厌氧培养72小时后B.longum-SPIO和B.longum组取材行透射电镜和普鲁士蓝染色。各组培养管行T_2~* mapping和T_2 mapping MRI扫描,并重建出R_2~*mapping和R_2 mapping,测量R2*和R2值。同样的处理方法(RCM培养基)用来标记和检测C.novyi-NT。建立Baffulo大鼠肝癌皮下瘤模型及肝原位瘤模型各8只用于体内实验。大鼠皮下瘤直接瘤内注射B.longum-SPIO(28μg Fe/ml)及Free-SPIO(28μg Fe/ml)各l ml。大鼠种植性肝癌静脉注射B.longum-SPIO(28μg Fe/ml)各1 ml。15天后检查MRI信号改变及病理切片普鲁士蓝染色检测瘤内是否有铁颗粒存在。
结果标记SPIO后B.longum活性没有受到明显影响。电镜见细菌体内形成较大铁颗粒,普鲁士蓝染色可见细菌铁染色明显。B.longum-SPIO R_2~*值明显高于Free-SPIO值(P＜0.001),而B.longum R_2*值同培养基R_2*值无显著差异(P＞0.05)。在R_2上Free-SPIO信号值明显高于B.longum-SPIO值(P＜0.001),而B.longum同培养基R_2值无明显差异(P＞0.05)。通过B.longum-SPIO和Free-SPIO比较发现在铁浓度相同情况下,B.longum-SPIO的R_2*值明显高于Free-SPIO R_2*值。相反,Free-SPIO R2值明显高于B.longum-SPIO R2值。B.longum-SPIO R2和R2*效应同细菌数呈线性关系。R2*效应斜率是R_2效应斜率的31.25倍。Free-SPIO浓度同R_2*效应亦呈线性关系,其R2*效应的斜率仅为R2效应斜率的1.99倍。C.novyi-NT也有类似的表现。大鼠肝癌皮下瘤显示注射B.longum-SPIO瘤体在R_2*上信号明显高于注射Free-SPIO的瘤体,而R_2信号则明显低于注射Free-SPIO的瘤体。尾静脉注射B.longum-SPIO后T_2WI显示瘤体内见散在低信号区。T_2*WI显示瘤体内低信号较T_2WI更加明显。铁染色后坏死区内广泛存在铁染色颗粒,有些区域见聚集的与细菌形态一致的杆状铁染色颗粒。
结论SPIO可以用来标记长双歧杆菌和C.novyi-NT并带来分布改变,同时引起相应的R_2和R_2*效应改变,并可以用来示踪瘤内长双歧杆菌或C.novyi-NT。
第四部分长双歧杆菌联合化疗及经皮无水酒精注射治疗Buffalo大鼠皮下移植肝癌
目的:探索长双歧杆菌治疗联合化疗及无水酒精瘤内注射治疗Buffalo大鼠皮下McA-RH7777肝细胞癌的疗效及可能机制。
材料和方法:建立40只Buffalo大鼠皮下肝癌动物模型,14天后随机分为四组:对照(Control)组(C组,n=10);长双歧杆菌溶瘤(B.longum)组(B组,n=10);经皮无水酒精注射和丝裂霉素化疗疗(Ethanol+MMC)组(EM组,n=10);长双歧杆菌溶瘤联合经皮无水酒精注射及丝裂霉素化疗(Ethanol+B.longum+MMC)组(EBM组,n=10)。在3、6、9、12、15天行磁共振检查计算瘤体积及相对瘤体积,比较瘤体增长情况。15天后处死全部大鼠行病理,免疫组化及细菌培养。
结果:40只Buffalo大鼠肿瘤模型全部建模成功。处理后C组和B组瘤体持续生长,而EM组和EBM组肿瘤生长明显受到抑制,瘤体积缩小。经两两比较后发现C组同B组之间相对瘤体积没有显著差异,同EM组或EBM组相比具有明显差异。EM的相对瘤体积同EBM的相对瘤体积也有显著差异。经EM处理后VEGF表达明显增强,加用B.longum处理后表达减弱,单独应用B.longum并未见VEGF表达明显减弱。6只EM组大鼠出现不同程度腹泻。EM组1例,EBM组2例出现皮肤坏死。各脏器尸检时均未见有脓肿形成。革兰氏染色显示长双歧杆菌可在肿瘤坏死区内聚集生长,呈片状,分布相对局限。处死后细菌培养见瘤组织内有大量细菌,而肝、肺、心、肾脏和脾脏均未见明显菌落形成。
结论:长双歧杆菌联合经皮无水酒精注射及化疗可有效抑制大鼠McA-RH7777肝细胞癌生长,长双歧杆菌在瘤内定植繁殖可能会减低局部VEGF表达。
第五部分肝动脉化疗栓塞联合无毒诺氏梭菌孢子瘤内注射治疗兔VX2肝癌
目的:探索无毒诺氏梭菌孢子(C.novyi-NT)联合动脉化疗栓塞治疗新西兰大白兔兔VX2肝癌的疗效。
材料和方法:将无毒诺氏梭菌孢子经过至少14天厌氧培养产孢并纯化后备用。制作新西兰大白兔VX2肝癌模型40只,MRI确定肿瘤存在并测量肿瘤大小,随机分成4组,即假手术组(A组,n=8)、经动脉化疗栓塞(TACE)组(B组,n=8)、动脉化疗栓塞加瘤内注射C.novyi-NT组(C组,n=12)、瘤内注射C.novyi-NT组(D组,n=12)。C、D两组处理后1、3、7、14天各处死一只动物取材(每个肿瘤随机取3枚组织块)行细菌培养,1微升组织块匀浆厌氧培养检测瘤内定植的活菌。各组于治疗后21天时行MRI复查并各处死2只行病理检查及细菌培养。每组6只长期观察疗效及并发症。
结果:成功培养和纯化诺氏梭菌孢子。所有动物完成所有相应治疗操作。处理后21天四组相对肿瘤体积具有显著统计学差异(x~2=18.74,P＜0.001,Kruskal-Wallace H test)。四组间坏死比例相比C组＞B组＞D组＞A组,四组间具有显著统计学差异(P＜0.001,Kruskal-Wallace test)。A、B、C、D四组平均生存时间分别为30.83±3.98天、63.33±4.57天、86.50±2.93天和44.67±2.81天。Kaplan Meier生存分析曲线显示C组累计生存率明显比其它三组明显增高。C组和D组在瘤内注射C.novyi-NT孢子后24小时即可在瘤内检测出活菌,3天后活菌数明显增加,至14天时达到最高峰,随后活菌数稍有减少。TACE结合C.novyi-NT瘤内注射组肿瘤呈较硬结节,瘤组织切面呈淡黄色,镜下肿瘤坏死程度和范围明显较其它三组明显。
结论:肝动脉化疗栓塞联合C.novyi-NT瘤内注射显增加肿瘤坏死,缩小瘤体,延长荷瘤兔生存期。
结论
一、Buffalo大鼠McA-RH7777肝细胞癌具有同人类肝细胞肝癌相似的MRI和DSA表现,非常适于进行大批量的介入治疗实验研究。
二、在厌氧条件下,长双歧杆菌对体外培养的McA-RH7777肝癌细胞具有毒性作用,并可能对癌细胞VEGF表达产生影响。
三、超顺磁性氧化铁(SPIO)可以用来标记长双歧杆菌及诺氏梭菌孢子,并引起明显的磁共振信号的变化。
四、长双歧杆菌可在无水酒精制造的坏死区内大量繁殖,长双歧杆菌联合无水酒精和化疗可有效抑制大鼠McA-RH7777肝细胞肝癌生长,长双歧杆菌在瘤内生长繁殖可能通过破坏和杀灭瘤内乏氧细胞有效抑制由于乏氧引起的VEGF的表达和分泌。
五、TACE联合C.novyi-NT孢子治疗兔VX2肝癌可有效增加肿瘤坏死,缩小肿瘤体积,延长动物生存期。TACE可在短时间内增加C.novyi-NT孢子在肿瘤内的定制增殖。
创新点
一、在国内首次采用McA-RH7777细胞建立大鼠肝癌模型并对其MRI、DSA及病理特点进行描述。
二、首创采用超顺磁性氧化铁(SPIO)标记长双歧杆菌及诺氏梭菌孢子(C.novyi-NT),并描述了相应磁共振信号的变化。
三、首次采用厌氧菌(C.novyi-NT或B.longum)同介入治疗手段(TACE或PEI)结合治疗实验性肝癌。
四、厌氧菌对厌氧区乏氧肿瘤细胞的破坏可能导致乏氧引起的VEGF表达和分泌减少,这一作用可能是其抗肿瘤间接作用的机制之一。
PartⅠImaging Characteristics of Buffalo Rat Orthotopic Hepatocellular Carcinoma Model
PURPOSE 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 1.0×10~6 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 T_1WI and hyper-intensity signal on T_2WI.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.
PartⅡThe Effect on Cell Growth and VEGF Expression of McA-RH7777 Hepatocellular Carcinoma Treated by Bifidobacterium longum Under Anaerobic Condition
Purpose 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.
PartⅢMRI in detection of Bifidobacterium longum and C.novyi-NT labeled with superparamagnetic iron oxide nanoparticles
PURPOSE 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 R_2~* 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 R_2~* 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 R_2 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.
PartⅣBuffalo Rat Subcutaneous Transplanted Hepatocellular Carcinoma:Treated by Bifidobacterium longum Combined with Chemotherapy and Percutaneous Ethonal Injection
Purpose 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.
PartⅤTranshepatic Artery Chemoembolization Combined with Percutaneous C.novyi-NT Spore Injection for Rabbit VX2 Liver carcinom
Purpose 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(x~2=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.
目的评价一种新的大鼠原位肝细胞癌模型的磁共振、数字减影血管造影、生物学特点变化及病理特征,促进大批量小动物肝细胞癌介入治疗研究。
材料和方法将McA-RH7777细胞开腹直视下接种于30只Buffalo大鼠肝左或右叶。造模后第7和14天行MRI平扫及增强扫描并分别取荷瘤大鼠各3只行病理检查。14天后荷瘤鼠14只分别经胃十二指肠动脉(7只)或静脉(7只)插管行肝动脉和门静脉造影,了解移植瘤DSA血供特点。其余10只荷瘤鼠长期观察生存状况。
结果造模后第7天,MRI观察见30只大鼠均有肿瘤生长,肿瘤平均体积为19.53±15.65mm~3;14天后增大到400.33±242.34 mm~3。肿瘤在T1WI为低信号,T2WI为高信号,增强后呈结节状或明显不均匀强化。7例大鼠DSA显示肿瘤供血动脉均有不同程度增粗扭曲,实质期呈结节状染色3例或环状不均匀染色4例。7只大鼠门静脉造影均无明显供血。肿瘤包膜完整,中央可见坏死。免疫组化显示肿瘤甲胎蛋白呈强阳性表达。10只荷瘤大鼠平均生存期为50.80±4.44天。
结论Buffalo大鼠McA-RH7777肝细胞癌,具有同人类肝细胞肝癌相似的MRI和DSA表现,非常适于进行大批量的介入治疗实验研究。
第二部分厌氧条件下长双歧杆菌对体外培养的McA-RH7777肝癌细胞生长及VEGF表达的影响
目的:探索厌氧条件下长双歧杆菌对体外培养的大鼠McA-RH7777肝癌细胞的毒性及对其VEGF表达及分泌的影响。
材料和方法:采用细胞培养技术,以大鼠肝癌细胞McA-RH7777为靶细胞,将McA-RH7777肝癌细胞分为两组,A组(厌氧培养条件下加入长双歧杆菌)和B组(单纯厌氧培养)。处理后肝癌细胞6h、12h、24h、48h、72h后,利用WST-1比色法测定长双歧杆菌对癌细胞毒性(OD值)。应用ELISA测定上清液中0h、12h、24h、48h、72h VEGF蛋白含量,使用RT-PCR法分析厌氧培养72h后两组细胞VEGFb表达水平。
结果:将长双歧杆菌加入体外培养的McA-RH7777肝癌细胞中显示出细胞毒性和对该细胞系的抑制效应。作用12h后,细胞生长开始出现停滞呈球形;细胞浆内出现颗粒状物。72h后培养液内细胞碎片出现。厌氧培养条件下,细胞损伤随时间的延长逐渐加重,A组毒性更大,48小时后两组OD值具有显著差异。上清液中VEGF蛋白含量在0h、12h、24h、48h、72h时两组相比均无明显统计学差异。72小时后A组厌氧培养的细胞VEGF mRNA表达水平明显高于B组。
结论:在厌氧条件下长双歧杆菌对体外培养的McA-RH7777肝癌细胞具有明显毒性。癌细胞VEGF mRNA表达上调,但上清液中总VEGF含量在各时间点均未见显著增高。
第三部分MRI检测超顺磁性氧化铁纳米颗粒标记长双歧杆菌和C.novyi-NT的实验研究
目的探索超顺磁性氧化铁标记长双歧杆菌或C.novyi-NT及采用MRI检测的可行性。
材料与方法体外实验中四组不同组分的培养管厌氧条件下培养:(1)B.longum-SPIO组(n=6):PYG液态培养基+B.longum+SPIO;(2)Free-SPIO组(n=6):PYG液态培养基+SPIO;(3)B.longum组(n=6):PYG液态培养基+B.longum;(4)Medium组(n=6):PYG液态培养基。厌氧培养72小时后B.longum-SPIO和B.longum组取材行透射电镜和普鲁士蓝染色。各组培养管行T_2~* mapping和T_2 mapping MRI扫描,并重建出R_2~*mapping和R_2 mapping,测量R2*和R2值。同样的处理方法(RCM培养基)用来标记和检测C.novyi-NT。建立Baffulo大鼠肝癌皮下瘤模型及肝原位瘤模型各8只用于体内实验。大鼠皮下瘤直接瘤内注射B.longum-SPIO(28μg Fe/ml)及Free-SPIO(28μg Fe/ml)各l ml。大鼠种植性肝癌静脉注射B.longum-SPIO(28μg Fe/ml)各1 ml。15天后检查MRI信号改变及病理切片普鲁士蓝染色检测瘤内是否有铁颗粒存在。
结果标记SPIO后B.longum活性没有受到明显影响。电镜见细菌体内形成较大铁颗粒,普鲁士蓝染色可见细菌铁染色明显。B.longum-SPIO R_2~*值明显高于Free-SPIO值(P＜0.001),而B.longum R_2*值同培养基R_2*值无显著差异(P＞0.05)。在R_2上Free-SPIO信号值明显高于B.longum-SPIO值(P＜0.001),而B.longum同培养基R_2值无明显差异(P＞0.05)。通过B.longum-SPIO和Free-SPIO比较发现在铁浓度相同情况下,B.longum-SPIO的R_2*值明显高于Free-SPIO R_2*值。相反,Free-SPIO R2值明显高于B.longum-SPIO R2值。B.longum-SPIO R2和R2*效应同细菌数呈线性关系。R2*效应斜率是R_2效应斜率的31.25倍。Free-SPIO浓度同R_2*效应亦呈线性关系,其R2*效应的斜率仅为R2效应斜率的1.99倍。C.novyi-NT也有类似的表现。大鼠肝癌皮下瘤显示注射B.longum-SPIO瘤体在R_2*上信号明显高于注射Free-SPIO的瘤体,而R_2信号则明显低于注射Free-SPIO的瘤体。尾静脉注射B.longum-SPIO后T_2WI显示瘤体内见散在低信号区。T_2*WI显示瘤体内低信号较T_2WI更加明显。铁染色后坏死区内广泛存在铁染色颗粒,有些区域见聚集的与细菌形态一致的杆状铁染色颗粒。
结论SPIO可以用来标记长双歧杆菌和C.novyi-NT并带来分布改变,同时引起相应的R_2和R_2*效应改变,并可以用来示踪瘤内长双歧杆菌或C.novyi-NT。
第四部分长双歧杆菌联合化疗及经皮无水酒精注射治疗Buffalo大鼠皮下移植肝癌
目的:探索长双歧杆菌治疗联合化疗及无水酒精瘤内注射治疗Buffalo大鼠皮下McA-RH7777肝细胞癌的疗效及可能机制。
材料和方法:建立40只Buffalo大鼠皮下肝癌动物模型,14天后随机分为四组:对照(Control)组(C组,n=10);长双歧杆菌溶瘤(B.longum)组(B组,n=10);经皮无水酒精注射和丝裂霉素化疗疗(Ethanol+MMC)组(EM组,n=10);长双歧杆菌溶瘤联合经皮无水酒精注射及丝裂霉素化疗(Ethanol+B.longum+MMC)组(EBM组,n=10)。在3、6、9、12、15天行磁共振检查计算瘤体积及相对瘤体积,比较瘤体增长情况。15天后处死全部大鼠行病理,免疫组化及细菌培养。
结果:40只Buffalo大鼠肿瘤模型全部建模成功。处理后C组和B组瘤体持续生长,而EM组和EBM组肿瘤生长明显受到抑制,瘤体积缩小。经两两比较后发现C组同B组之间相对瘤体积没有显著差异,同EM组或EBM组相比具有明显差异。EM的相对瘤体积同EBM的相对瘤体积也有显著差异。经EM处理后VEGF表达明显增强,加用B.longum处理后表达减弱,单独应用B.longum并未见VEGF表达明显减弱。6只EM组大鼠出现不同程度腹泻。EM组1例,EBM组2例出现皮肤坏死。各脏器尸检时均未见有脓肿形成。革兰氏染色显示长双歧杆菌可在肿瘤坏死区内聚集生长,呈片状,分布相对局限。处死后细菌培养见瘤组织内有大量细菌,而肝、肺、心、肾脏和脾脏均未见明显菌落形成。
结论:长双歧杆菌联合经皮无水酒精注射及化疗可有效抑制大鼠McA-RH7777肝细胞癌生长,长双歧杆菌在瘤内定植繁殖可能会减低局部VEGF表达。
第五部分肝动脉化疗栓塞联合无毒诺氏梭菌孢子瘤内注射治疗兔VX2肝癌
目的:探索无毒诺氏梭菌孢子(C.novyi-NT)联合动脉化疗栓塞治疗新西兰大白兔兔VX2肝癌的疗效。
材料和方法:将无毒诺氏梭菌孢子经过至少14天厌氧培养产孢并纯化后备用。制作新西兰大白兔VX2肝癌模型40只,MRI确定肿瘤存在并测量肿瘤大小,随机分成4组,即假手术组(A组,n=8)、经动脉化疗栓塞(TACE)组(B组,n=8)、动脉化疗栓塞加瘤内注射C.novyi-NT组(C组,n=12)、瘤内注射C.novyi-NT组(D组,n=12)。C、D两组处理后1、3、7、14天各处死一只动物取材(每个肿瘤随机取3枚组织块)行细菌培养,1微升组织块匀浆厌氧培养检测瘤内定植的活菌。各组于治疗后21天时行MRI复查并各处死2只行病理检查及细菌培养。每组6只长期观察疗效及并发症。
结果:成功培养和纯化诺氏梭菌孢子。所有动物完成所有相应治疗操作。处理后21天四组相对肿瘤体积具有显著统计学差异(x~2=18.74,P＜0.001,Kruskal-Wallace H test)。四组间坏死比例相比C组＞B组＞D组＞A组,四组间具有显著统计学差异(P＜0.001,Kruskal-Wallace test)。A、B、C、D四组平均生存时间分别为30.83±3.98天、63.33±4.57天、86.50±2.93天和44.67±2.81天。Kaplan Meier生存分析曲线显示C组累计生存率明显比其它三组明显增高。C组和D组在瘤内注射C.novyi-NT孢子后24小时即可在瘤内检测出活菌,3天后活菌数明显增加,至14天时达到最高峰,随后活菌数稍有减少。TACE结合C.novyi-NT瘤内注射组肿瘤呈较硬结节,瘤组织切面呈淡黄色,镜下肿瘤坏死程度和范围明显较其它三组明显。
结论:肝动脉化疗栓塞联合C.novyi-NT瘤内注射显增加肿瘤坏死,缩小瘤体,延长荷瘤兔生存期。
结论
一、Buffalo大鼠McA-RH7777肝细胞癌具有同人类肝细胞肝癌相似的MRI和DSA表现,非常适于进行大批量的介入治疗实验研究。
二、在厌氧条件下,长双歧杆菌对体外培养的McA-RH7777肝癌细胞具有毒性作用,并可能对癌细胞VEGF表达产生影响。
三、超顺磁性氧化铁(SPIO)可以用来标记长双歧杆菌及诺氏梭菌孢子,并引起明显的磁共振信号的变化。
四、长双歧杆菌可在无水酒精制造的坏死区内大量繁殖,长双歧杆菌联合无水酒精和化疗可有效抑制大鼠McA-RH7777肝细胞肝癌生长,长双歧杆菌在瘤内生长繁殖可能通过破坏和杀灭瘤内乏氧细胞有效抑制由于乏氧引起的VEGF的表达和分泌。
五、TACE联合C.novyi-NT孢子治疗兔VX2肝癌可有效增加肿瘤坏死,缩小肿瘤体积,延长动物生存期。TACE可在短时间内增加C.novyi-NT孢子在肿瘤内的定制增殖。
创新点
一、在国内首次采用McA-RH7777细胞建立大鼠肝癌模型并对其MRI、DSA及病理特点进行描述。
二、首创采用超顺磁性氧化铁(SPIO)标记长双歧杆菌及诺氏梭菌孢子(C.novyi-NT),并描述了相应磁共振信号的变化。
三、首次采用厌氧菌(C.novyi-NT或B.longum)同介入治疗手段(TACE或PEI)结合治疗实验性肝癌。
四、厌氧菌对厌氧区乏氧肿瘤细胞的破坏可能导致乏氧引起的VEGF表达和分泌减少,这一作用可能是其抗肿瘤间接作用的机制之一。
PartⅠImaging Characteristics of Buffalo Rat Orthotopic Hepatocellular Carcinoma Model
PURPOSE 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 1.0×10~6 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 T_1WI and hyper-intensity signal on T_2WI.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.
PartⅡThe Effect on Cell Growth and VEGF Expression of McA-RH7777 Hepatocellular Carcinoma Treated by Bifidobacterium longum Under Anaerobic Condition
Purpose 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.
PartⅢMRI in detection of Bifidobacterium longum and C.novyi-NT labeled with superparamagnetic iron oxide nanoparticles
PURPOSE 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 R_2~* 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 R_2~* 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 R_2 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.
PartⅣBuffalo Rat Subcutaneous Transplanted Hepatocellular Carcinoma:Treated by Bifidobacterium longum Combined with Chemotherapy and Percutaneous Ethonal Injection
Purpose 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.
PartⅤTranshepatic Artery Chemoembolization Combined with Percutaneous C.novyi-NT Spore Injection for Rabbit VX2 Liver carcinom
Purpose 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(x~2=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.
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