胆管联合门静脉结扎对Buffalo大鼠肝再生及肝癌生长转移的影响

发布时间：2018-06-09 01:01

本文选题：Buffalo大鼠 + 活体荧光成像　；参考：《中国人民解放军医学院》2015年硕士论文

【摘要】：目的：利用活体成像技术,建立可供活体观察的Buffalo大鼠原位肝癌模型。在此基础上,观察选择性结扎植瘤肝叶的胆管和门静脉(Bile Duct Ligation with Portal Vein Ligation, BPL),与单纯结扎门静脉(Portal Vein Ligation, PVL)比较.对肝再生及肝癌生长、转移的影响。方法：以pCDH-puromycin-CMV为载体,将荧光素酶(Lucferase, Luc)基因转染至大鼠McA-RH7777肝癌细胞,将其皮下接种Buffalo大鼠,取瘤块异体种植于大鼠肝脏左外叶,应用Luc活体荧光成像技术,结合吲哚菁绿近红外成像,观察肝肿瘤生长和转移情况。建模成功后,随机将大鼠分为三组,即左外侧叶和中叶胆管联合门静脉结扎组(BPL)、单纯门静脉结扎组(PVL)和假手术组(Sham)。分别于结扎术后1d、3d、5d、7d、14d取血后处死大鼠,解剖肝、肺,测量各肝叶重量、肝内种植肿瘤体积变化,观察有无肝内外转移。测定血清转氨酶(ALT和AST)、胆汁酸、肝内胆汁酸水平。取保留侧肝组织,Western blot则定PCNA表达量.免疫组化检测ki67阳性细胞数。结果：(1)Buffalo大鼠皮下和肝内种植成瘤率均为100%。肝内种植2w后,利用荧光素酶活体成像系统,可观察到接种肝叶内的荧光信号,并随时间推移信号范围和强度增加;利用吲哚菁绿近红外成像系统,也可见种植部位的肿瘤显影,其他肝叶未见肿瘤。4w时肿瘤仍呈局部生长,未见明显的肝内和肺转移。(2)PVL与BPL均引起肝脏萎缩-增生综合征(AHC, Atropy/hypertrophy complex),但BPL组发生得更快、更明显。术后血清转氨酶水平出现一过性升高。BPL和PVL组均显示血清胆汁酸水平显著高于Sham组,BPL组于术后1d升至峰值,为123.8±14.6μmol/L,而PVL组于术后3d升至峰值,为73.8±8.2μmol/L,Sham组为24.6±1.4μmol/L。类似血清胆汁酸变化,BPL组未结扎侧肝组织胆汁酸含量于术后1d升至峰值,为0.83±0.074μmol/g,随后迅速降低,而PVL组肝组织胆汁酸术后3d最高,为0.58±0.13μmol/g。结扎术后14d,血清转氨酶及胆汁酸均降至正常水平。(3)PCNA表达量及ki-67阳性细胞数,BPL组均比PVL组提前达峰值。(4)结扎术后7d,BPL和PVL组肿瘤体积明显大于Sham组(P0.05),但两组间未见明显差异(P0.05)。然而术后14d,BPL组肿瘤体积明显大于PVL组(P0.05),并且出现肿瘤肺部转移,转移率为100%(4/4)。结论：(1)原位接种Luc-McA-RH7777细胞能在Buffalo大鼠肝内成瘤,并可通过Luc活体荧光成像系统观察肿瘤生长情况,但与ICG肝肿瘤成像类似,其强度均不同程度地受动物体壁厚度的影响,经皮观察肝内肿瘤,Luc荧光成像较ICG远红外成像敏感。(2)BPL在加速结扎肝叶萎缩的同时,比PVL更有效地促进非结扎侧肝叶的再生。(3)BPL和PVL均显示促肿瘤生长作用,随时间延长,BPL作用更为显著,并且引起肿瘤肝外转移。
[Abstract]:Aim: to establish an in situ liver cancer model of Buffalo rats by using in vivo imaging technique. On this basis, the bile duct and portal vein Duct Ligation with portal ligination (BPLL) in the liver lobe of the tumor implanted with selective ligation were compared with those in the portal vein ligation alone. Effects on liver regeneration, growth and metastasis of liver cancer. Methods: luciferase (Lucferase) gene was transfected into rat McA-RH7777 hepatoma cell line with pCDH-puromycin-CMV as vector, and subcutaneously inoculated into Buffalo rat. Combined with indocyanine green near infrared imaging, liver tumor growth and metastasis were observed. After modeling successfully, the rats were randomly divided into three groups: left lateral lobe and middle lobe bile duct combined with portal vein ligation group (BPLL), simple portal vein ligation group (PVL) and sham-operated group. The rats were sacrificed at 1 day, 3 days, 5 days, 7 days and 14 days after ligation. The liver and lung were dissected, the weight of each liver lobe was measured, the volume of tumor implanted in the liver was measured, and the metastasis in and out of the liver was observed. Serum levels of alt, AST, bile acid and intrahepatic bile acid were measured. The expression of PCNA was determined by Western blot in the reserved liver tissue. The number of ki67 positive cells was detected by immunohistochemistry. Results the rate of tumor formation in subcutaneous and intrahepatic implantation was 100. After being planted in liver for 2 weeks, the fluorescence signal of liver inoculated with luciferase in vivo was observed, and the range and intensity of the signal increased over time, and the indocyanine green near infrared imaging system was used. There was no obvious intrahepatic and pulmonary metastasis. Both PVL and BPLs could induce AHC and Atropy/hypertrophy complexation of liver atrophy and hyperplasia syndrome, but the occurrence of AHC was faster and more obvious in BPLs group. The levels of serum aminotransferase in both the BPL and PVL groups were significantly higher than those in the Sham group on the 1st day after operation (123.8 卤14.6 渭 mol / L), while in the PVL group on the 3rd day after operation, they rose to the peak (73.8 卤8.2 渭 mol / L) and 24.6 卤1.4 渭 mol / L 路L ~ (-1). The level of bile acid in the unligated liver tissue in the BPL group increased to a peak of 0.83 卤0.074 渭 mol / g on the 1st day after operation, and then decreased rapidly. The highest level of bile acid in the liver tissue was 0.58 卤0.13 渭 mol / g on the 3rd day after operation in the BPL group. On the 14th day after ligation, the expression of serum aminotransferase and bile acid decreased to the normal level. The expression of PCNA and the number of ki-67 positive cells reached the peak value in BPL group than in PVL group. The tumor volume in BPL and PVL group was significantly larger than that in Sham group on the 7th day after ligation, but there was no significant difference between the two groups. However, the tumor volume in the BPL group was significantly larger than that in the PVL group on the 14th day after operation (P 0.05), and the lung metastasis rate was 100% 4 / 4%. Conclusion in situ inoculation of Luc-McA-RH7777 cells in the liver of Buffalo rats can be used to observe the tumor growth through Luc in vivo fluorescence imaging system. But similar to ICG liver tumor imaging, its intensity is affected by the thickness of animal body wall in varying degrees. Compared with ICG far-infrared imaging, the fluorescence imaging of Luc in intrahepatic neoplasms was more sensitive than ICG in accelerating ligation of liver lobe atrophy, and in promoting the regeneration of non-ligated liver lobes more effectively than PVL. Both of them could promote tumor growth. The effect of BPL was more obvious with time, and caused extrahepatic metastasis of tumor.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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