【摘要】：肝癌根治性治療主要包括肝切除和肝移植，因?yàn)楦伟┎∪硕喟橛袊?yán)重的肝硬變或多發(fā)腫瘤，所以大多數(shù)的肝癌病人不是肝切除的手術(shù)適應(yīng)癥。同時(shí)，即使切除了腫瘤，術(shù)后大多數(shù)病人仍死于術(shù)后腫瘤復(fù)發(fā)轉(zhuǎn)移。肝移植是唯一能同時(shí)處理肝硬變及肝腫瘤兩種病變的治療手段，可用來(lái)治療不能切除的肝癌，由于徹底切除了腫瘤發(fā)生的“土壤”，其術(shù)后腫瘤復(fù)發(fā)轉(zhuǎn)移率也明顯低于肝切除，然而術(shù)后腫瘤復(fù)發(fā)轉(zhuǎn)移仍然是治療肝癌肝移植長(zhǎng)期療效的最重要的因素為評(píng)價(jià)肝移植術(shù)后腫瘤轉(zhuǎn)移模式，因此有必要建立類似肝癌肝移植術(shù)后腫瘤復(fù)發(fā)轉(zhuǎn)移的動(dòng)物模型。 材料和方法 1．實(shí)驗(yàn)動(dòng)物： 供體：清潔級(jí)雄性SD大鼠，50只，體重220-250g； 受體：肝癌模型鼠為SD雄性成年大鼠50只，體重220-250 g供體體重略小于或等于受體。 2．紫杉醇注射液(paclitaxel)：為北京四環(huán)醫(yī)藥科技股份有限公司產(chǎn)品，規(guī)格30 mg／5 ml，純度98％(HPLC)，分子量為853192，臨用前用1640稀釋至所需濃度。 3．實(shí)驗(yàn)分組：共分為兩組： A組(n=25)肝移植術(shù)后無(wú)干預(yù)組：肝癌模型鼠Walker256瘤組織塊接種后14天，接受肝移植手術(shù)，術(shù)后第一天起腹腔內(nèi)注射生理鹽水0.5 ml／d~(-1)。B組(n=25)肝移植術(shù)后紫杉醇干預(yù)組：25只肝癌模型鼠Walker256瘤組織塊接種后14天，，接受肝移植手術(shù)，術(shù)后第一天起腹腔內(nèi)注射紫杉醇(Paclitaxel，0.5 mg·kg~(-1)·d~(-1))。兩組療程均為14天。 4．病理和生存觀察：A，B組肝移植術(shù)后21天開(kāi)始，每周每組處死5只大鼠，處死大鼠后，10％formalin由氣道注入后切取肺臟，10％formalin中保存，光鏡下肺轉(zhuǎn)移灶計(jì)數(shù)，圖象分析技術(shù)測(cè)量肺組織面積。結(jié)果=肺轉(zhuǎn)移灶計(jì)數(shù)／單位肺組織面積，同時(shí)切除肝癌，液氮凍存，肝組織在10％formalin中固定，常規(guī)H．E染色。計(jì)算生存率，瘤塊接種成瘤率及其肺轉(zhuǎn)移發(fā)生率。 結(jié)果 1．A組肝移植3周存活率80％(20／25)，B組：肝移植3周存活率84％(21／25，由于大鼠肝移植后3周開(kāi)始處死大鼠并檢測(cè)標(biāo)本，所以未進(jìn)一步計(jì)算生存率。 2．本研究中，兩組的瘤塊接種成瘤率均為100％。A組：第3，4，5，6周的肺轉(zhuǎn)移率為0(0／5)，20％(1／5)，60％(3／5)，60％(3／5)；B組：第3，4，5，6周的肺轉(zhuǎn)移率為O(0／5)，0(0／5)，40％(2／5)，33％(2／6)。A組共有7只大鼠發(fā)生肺轉(zhuǎn)移，B組4只大鼠發(fā)生肺轉(zhuǎn)移，A組大鼠的肺轉(zhuǎn)移灶計(jì)數(shù)，顯著高于B組(Mann 2 Whitney U非參數(shù)檢驗(yàn)，U=40，P=0.039)。 結(jié)果和討論 在這一研究中我們成功的建立了大鼠移植性肝癌肝移植的模型，并且發(fā)現(xiàn)肝移植可延長(zhǎng)肝癌大鼠的生存時(shí)間，肺臟是大鼠肝癌肝移植術(shù)后腫瘤轉(zhuǎn)移的主要部位，同時(shí)我們發(fā)現(xiàn)紫杉醇可以顯著降低肝癌肝移植大鼠的術(shù)后肺轉(zhuǎn)移瘤的發(fā)生率，減少轉(zhuǎn)移瘤的數(shù)量，縮小轉(zhuǎn)移瘤的侵及范圍。我們可以在此模型的基礎(chǔ)上探討肝移植術(shù)后腫瘤轉(zhuǎn)移復(fù)發(fā)的機(jī)制，特點(diǎn)，可能有益的預(yù)測(cè)指標(biāo)，及干預(yù)措施。并作為臨床及基礎(chǔ)的進(jìn)一步深入研究的實(shí)驗(yàn)平臺(tái)。 第一部分：大鼠移植性肝癌肝移植模型的建立 第二部分：紫杉醇抑制大鼠原位肝移植急性排斥反應(yīng)的機(jī)制 目的探討紫杉醇抑制大鼠原位肝移植急性排斥反應(yīng)的機(jī)制。方法建立大鼠原位肝移植模型(Lewis大鼠—BN大鼠)，將大鼠分為三組：生理鹽水對(duì)照組，紫杉醇低劑量干預(yù)組，高劑量干預(yù)組，觀察受體生存時(shí)間，檢測(cè)肝臟功能，病理組織學(xué)改變，外周血單核細(xì)胞(PBMCs)中對(duì)供體特異的CD4~+Th前體細(xì)胞(Th-p)的比率，脾臟T淋巴細(xì)胞凋亡的比率，和外周血細(xì)胞因子IFN-γ／IL-4(代表Th1／Th2)水平。結(jié)果紫杉醇明顯延長(zhǎng)大鼠術(shù)后生存時(shí)間(對(duì)照組：10.6±1.9d vs，低劑量組：16.1±3.4d，log rank=9.06，P＜0.05；低劑量組：16.1±3.4d vs，高劑量組：25.9±4.1d，log rank=7.81，P＜0.05)，改善肝功能，減輕肝臟病理組織學(xué)改變(排斥活動(dòng)評(píng)分指數(shù)對(duì)照組：5.2±0.8 vs，低劑量組：3.8±0.4和高劑量組：3.6±0.5，U=23.0和23.5，P值均＜0.05)，紫杉醇能降低受鼠PBMCs的Th-p比率(logarithmic法對(duì)照組：3.42±0.48 vs，低劑量組：1.55±0.58和高劑量組：1.14±0.52，t=8.9和11.8，P值均＜0.05)，促進(jìn)脾CD4~+的淋巴細(xì)胞凋亡(對(duì)照組：10.7％vs低劑量組：43.7％和高劑量組：55.6％，x2=27.49和93.4 P值均＜0.05)，紫杉醇同時(shí)降低大鼠血清中的IFN-γ，升高IL-4，使Th1／Th2細(xì)胞平衡向Th2移動(dòng)(INF-γ／IL-4對(duì)照組：448.7％±149.1％vs．低劑量組：107.4％±41.4％和高劑量組：51.4％±15.9％，t=4.93和5.92，P值均＜0.05)。結(jié)論紫杉醇能夠有效減輕肝移植大鼠的急性排斥反應(yīng)，其機(jī)制可能與誘導(dǎo)活化的CD4~+Th細(xì)胞凋亡，從而降低受鼠Th-p對(duì)供鼠細(xì)胞的反應(yīng)，并促使Th1／Th2細(xì)胞平衡向Th2移動(dòng)有關(guān)。
[Abstract]:The radical treatment of the liver cancer mainly includes liver resection and liver transplantation, because the liver cancer patients are accompanied by severe liver cirrhosis or multiple tumors, so most of the liver cancer patients are not the surgical indications for hepatectomy. At the same time, most of the patients died of post-operative tumor recurrence, even if the tumor was removed. Liver transplantation is the only means of treatment for both liver cirrhosis and liver tumor, which can be used to treat non-resectable liver cancer. However, the most important factor in the long-term efficacy of liver transplantation for liver transplantation is to evaluate the model of tumor metastasis after liver transplantation. Therefore, it is necessary to establish an animal model of the recurrence and metastasis of the tumor after liver transplantation. material and method 1. experimental animals: Donor: clean-grade male SD rat,50 rats, body weight 220-250 g; receptor: liver the cancer model rat is 50 rats of the SD male adult rat, the body weight of the donor body is 220-250g, the weight of the donor is slightly less than or equal to the receptor, and the paclitaxel injection is a product of the Beijing Sihuan Medical Science and Technology Co., Ltd., Size:30 mg/5 ml, purity 98% (HPLC) with a molecular weight of 853192, diluted with 1640 to the desired concentration prior to use. The group was divided into two groups: A group (n = 25) and no intervention group after liver transplantation:14 days after the inoculation of the Walker256 tumor tissue mass of the liver cancer model, and the first day after the operation, 0.5 ml/ d to (-1) of normal saline was injected into the abdominal cavity, and the group B (n = 25) after the liver transplantation was treated with the paclitaxel intervention group:25 liver cancer model mice Walker. After the 256-tumor tissue block is inoculated, Paclitaxel (Paclitaxel, 0.5 mg 路 kg ~ (-1) 路 d ~ (-1)) was injected intraperitoneally on the first day of the operation for 14 days. From the beginning,5 rats were sacrificed each week, and after the rats were sacrificed,10% of formalin was injected into the lung after injection,10% of formin was preserved, and the light The measurement of lung tissue area by microscopic lung metastases, image analysis and image analysis . Junction Fruit = lung metastases/ unit lung tissue area, simultaneous removal of liver cancer, liquid nitrogen cryopreservation, liver tissue fixation in 10% formin, conventional H. E staining. The survival rate, the tumor rate and the incidence of lung metastasis were calculated. Results 1. The survival rate of 3-week survival in group A was 80% (20/25), and the survival rate of group B:3-week survival rate was 84% (21/25). The lung metastasis rate was 0 (0/5),20% (1/5),60% (3/5) and 60% (3/5) in group A. O(0錛

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