發(fā)布時間：2018-06-09 01:01

  本文選題：Buffalo大鼠 + 活體熒光成像�。� 參考：《中國人民解放軍醫(yī)學院》2015年碩士論文

【摘要】：目的：利用活體成像技術(shù),建立可供活體觀察的Buffalo大鼠原位肝癌模型。在此基礎(chǔ)上,觀察選擇性結(jié)扎植瘤肝葉的膽管和門靜脈(Bile Duct Ligation with Portal Vein Ligation, BPL),與單純結(jié)扎門靜脈(Portal Vein Ligation, PVL)比較.對肝再生及肝癌生長、轉(zhuǎn)移的影響。方法：以pCDH-puromycin-CMV為載體,將熒光素酶(Lucferase, Luc)基因轉(zhuǎn)染至大鼠McA-RH7777肝癌細胞,將其皮下接種Buffalo大鼠,取瘤塊異體種植于大鼠肝臟左外葉,應(yīng)用Luc活體熒光成像技術(shù),結(jié)合吲哚菁綠近紅外成像,觀察肝腫瘤生長和轉(zhuǎn)移情況。建模成功后,隨機將大鼠分為三組,即左外側(cè)葉和中葉膽管聯(lián)合門靜脈結(jié)扎組(BPL)、單純門靜脈結(jié)扎組(PVL)和假手術(shù)組(Sham)。分別于結(jié)扎術(shù)后1d、3d、5d、7d、14d取血后處死大鼠,解剖肝、肺,測量各肝葉重量、肝內(nèi)種植腫瘤體積變化,觀察有無肝內(nèi)外轉(zhuǎn)移。測定血清轉(zhuǎn)氨酶(ALT和AST)、膽汁酸、肝內(nèi)膽汁酸水平。取保留側(cè)肝組織,Western blot則定PCNA表達量.免疫組化檢測ki67陽性細胞數(shù)。結(jié)果：(1)Buffalo大鼠皮下和肝內(nèi)種植成瘤率均為100%。肝內(nèi)種植2w后,利用熒光素酶活體成像系統(tǒng),可觀察到接種肝葉內(nèi)的熒光信號,并隨時間推移信號范圍和強度增加;利用吲哚菁綠近紅外成像系統(tǒng),也可見種植部位的腫瘤顯影,其他肝葉未見腫瘤。4w時腫瘤仍呈局部生長,未見明顯的肝內(nèi)和肺轉(zhuǎn)移。(2)PVL與BPL均引起肝臟萎縮-增生綜合征(AHC, Atropy/hypertrophy complex),但BPL組發(fā)生得更快、更明顯。術(shù)后血清轉(zhuǎn)氨酶水平出現(xiàn)一過性升高。BPL和PVL組均顯示血清膽汁酸水平顯著高于Sham組,BPL組于術(shù)后1d升至峰值,為123.8±14.6μmol/L,而PVL組于術(shù)后3d升至峰值,為73.8±8.2μmol/L,Sham組為24.6±1.4μmol/L。類似血清膽汁酸變化,BPL組未結(jié)扎側(cè)肝組織膽汁酸含量于術(shù)后1d升至峰值,為0.83±0.074μmol/g,隨后迅速降低,而PVL組肝組織膽汁酸術(shù)后3d最高,為0.58±0.13μmol/g。結(jié)扎術(shù)后14d,血清轉(zhuǎn)氨酶及膽汁酸均降至正常水平。(3)PCNA表達量及ki-67陽性細胞數(shù),BPL組均比PVL組提前達峰值。(4)結(jié)扎術(shù)后7d,BPL和PVL組腫瘤體積明顯大于Sham組(P0.05),但兩組間未見明顯差異(P0.05)。然而術(shù)后14d,BPL組腫瘤體積明顯大于PVL組(P0.05),并且出現(xiàn)腫瘤肺部轉(zhuǎn)移,轉(zhuǎn)移率為100%(4/4)。結(jié)論：(1)原位接種Luc-McA-RH7777細胞能在Buffalo大鼠肝內(nèi)成瘤,并可通過Luc活體熒光成像系統(tǒng)觀察腫瘤生長情況,但與ICG肝腫瘤成像類似,其強度均不同程度地受動物體壁厚度的影響,經(jīng)皮觀察肝內(nèi)腫瘤,Luc熒光成像較ICG遠紅外成像敏感。(2)BPL在加速結(jié)扎肝葉萎縮的同時,比PVL更有效地促進非結(jié)扎側(cè)肝葉的再生。(3)BPL和PVL均顯示促腫瘤生長作用,隨時間延長,BPL作用更為顯著,并且引起腫瘤肝外轉(zhuǎn)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 鹵14.6 渭 mol / L), while in the PVL group on the 3rd day after operation, they rose to the peak (73.8 鹵8.2 渭 mol / L) and 24.6 鹵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 鹵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 鹵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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.7

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本文編號：1998065