大鼠移植性肝癌肝移植模型的建立及紫杉醇抑制大鼠原位肝移植急性排斥反应的机制
发布时间：2019-06-18 14:51
【摘要】：肝癌根治性治疗主要包括肝切除和肝移植，因为肝癌病人多伴有严重的肝硬变或多发肿瘤，所以大多数的肝癌病人不是肝切除的手术适应症。同时，即使切除了肿瘤，术后大多数病人仍死于术后肿瘤复发转移。肝移植是唯一能同时处理肝硬变及肝肿瘤两种病变的治疗手段，可用来治疗不能切除的肝癌，由于彻底切除了肿瘤发生的“土壤”，其术后肿瘤复发转移率也明显低于肝切除，然而术后肿瘤复发转移仍然是治疗肝癌肝移植长期疗效的最重要的因素为评价肝移植术后肿瘤转移模式，因此有必要建立类似肝癌肝移植术后肿瘤复发转移的动物模型。 材料和方法 1．实验动物： 供体：清洁级雄性SD大鼠，50只，体重220-250g； 受体：肝癌模型鼠为SD雄性成年大鼠50只，体重220-250 g供体体重略小于或等于受体。 2．紫杉醇注射液(paclitaxel)：为北京四环医药科技股份有限公司产品，规格30 mg／5 ml，纯度98％(HPLC)，分子量为853192，临用前用1640稀释至所需浓度。 3．实验分组：共分为两组： A组(n=25)肝移植术后无干预组：肝癌模型鼠Walker256瘤组织块接种后14天，接受肝移植手术，术后第一天起腹腔内注射生理盐水0.5 ml／d~(-1)。B组(n=25)肝移植术后紫杉醇干预组：25只肝癌模型鼠Walker256瘤组织块接种后14天，，接受肝移植手术，术后第一天起腹腔内注射紫杉醇(Paclitaxel，0.5 mg·kg~(-1)·d~(-1))。两组疗程均为14天。 4．病理和生存观察：A，B组肝移植术后21天开始，每周每组处死5只大鼠，处死大鼠后，10％formalin由气道注入后切取肺脏，10％formalin中保存，光镜下肺转移灶计数，图象分析技术测量肺组织面积。结果=肺转移灶计数／单位肺组织面积，同时切除肝癌，液氮冻存，肝组织在10％formalin中固定，常规H．E染色。计算生存率，瘤块接种成瘤率及其肺转移发生率。 结果 1．A组肝移植3周存活率80％(20／25)，B组：肝移植3周存活率84％(21／25，由于大鼠肝移植后3周开始处死大鼠并检测标本，所以未进一步计算生存率。 2．本研究中，两组的瘤块接种成瘤率均为100％。A组：第3，4，5，6周的肺转移率为0(0／5)，20％(1／5)，60％(3／5)，60％(3／5)；B组：第3，4，5，6周的肺转移率为O(0／5)，0(0／5)，40％(2／5)，33％(2／6)。A组共有7只大鼠发生肺转移，B组4只大鼠发生肺转移，A组大鼠的肺转移灶计数，显著高于B组(Mann 2 Whitney U非参数检验，U=40，P=0.039)。 结果和讨论 在这一研究中我们成功的建立了大鼠移植性肝癌肝移植的模型，并且发现肝移植可延长肝癌大鼠的生存时间，肺脏是大鼠肝癌肝移植术后肿瘤转移的主要部位，同时我们发现紫杉醇可以显著降低肝癌肝移植大鼠的术后肺转移瘤的发生率，减少转移瘤的数量，缩小转移瘤的侵及范围。我们可以在此模型的基础上探讨肝移植术后肿瘤转移复发的机制，特点，可能有益的预测指标，及干预措施。并作为临床及基础的进一步深入研究的实验平台。 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应的机制 目的探讨紫杉醇抑制大鼠原位肝移植急性排斥反应的机制。方法建立大鼠原位肝移植模型(Lewis大鼠—BN大鼠)，将大鼠分为三组：生理盐水对照组，紫杉醇低剂量干预组，高剂量干预组，观察受体生存时间，检测肝脏功能，病理组织学改变，外周血单核细胞(PBMCs)中对供体特异的CD4~+Th前体细胞(Th-p)的比率，脾脏T淋巴细胞凋亡的比率，和外周血细胞因子IFN-γ／IL-4(代表Th1／Th2)水平。结果紫杉醇明显延长大鼠术后生存时间(对照组：10.6±1.9d vs，低剂量组：16.1±3.4d，log rank=9.06，P＜0.05；低剂量组：16.1±3.4d vs，高剂量组：25.9±4.1d，log rank=7.81，P＜0.05)，改善肝功能，减轻肝脏病理组织学改变(排斥活动评分指数对照组：5.2±0.8 vs，低剂量组：3.8±0.4和高剂量组：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法对照组：3.42±0.48 vs，低剂量组：1.55±0.58和高剂量组：1.14±0.52，t=8.9和11.8，P值均＜0.05)，促进脾CD4~+的淋巴细胞凋亡(对照组：10.7％vs低剂量组：43.7％和高剂量组：55.6％，x2=27.49和93.4 P值均＜0.05)，紫杉醇同时降低大鼠血清中的IFN-γ，升高IL-4，使Th1／Th2细胞平衡向Th2移动(INF-γ／IL-4对照组：448.7％±149.1％vs．低剂量组：107.4％±41.4％和高剂量组：51.4％±15.9％，t=4.93和5.92，P值均＜0.05)。结论紫杉醇能够有效减轻肝移植大鼠的急性排斥反应，其机制可能与诱导活化的CD4~+Th细胞凋亡，从而降低受鼠Th-p对供鼠细胞的反应，并促使Th1／Th2细胞平衡向Th2移动有关。
[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锛
本文编号：2501570
[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锛
本文编号：2501570
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2501570.html
最近更新
教材专著
