【作者】 徐建；

【导师】 王艳红；

【作者基本信息】 复旦大学 ， 内科学， 2011， 硕士

【摘要】 研究目的：血液中单核细胞进入外周组织后在不同刺激因素作用下可分化为经典激活的巨噬细胞(M1)和选择性激活的巨噬细胞(M2)。研究报道,肿瘤组织中巨噬细胞具有M2型巨噬细胞表型；原发性肝癌切除患者,其肿瘤组织中巨噬细胞浸润增多提示术后预后不良；在肝癌组织中,细胞毒性T细胞浸润增多是无病生存期和总生存期改善的独立预后因素。为了进一步观察和了解肿瘤微环境中巨噬细胞对细胞毒性T细胞的影响,从而进一步明确巨噬细胞在肿瘤发生发展中所起的作用,我们设计了该实验。研究方法：构建Wistar大鼠Walker-256肝癌模型。将大鼠分为3组：A组(腹腔注射氯磷酸盐脂质体)、B组(腹腔注射PBS脂质体)、C组(腹腔注射生理盐水)。干预12天后摘取肿瘤组织,测量肿瘤大小、观察腹腔肿瘤转移及腹水情况；应用免疫组化方法检测肿瘤组织、癌周组织及癌远端组织中浸润的CD68,CD163,CD8及GranzymeB P日性细胞个数(表达CD68、CD163的细胞为巨噬细胞,表达CD8,GranzymeB的细胞为T细胞)；应用CD8与Tunel双标方法检测肿瘤组织中T细胞的凋亡。研究结果：1 A、B、C三组的肿瘤体积分别为：(0.13±0.09)cm3、(0.27±0.14)cm3、(0.41±0.36)cm3；肿瘤重量分别为：(0.16±0.14)g、(0.31±0.13)g、(0.34±0.35)g；腹腔转移阳性率分别为0%,42.86%,33.33%；腹水阳性率分别为0%,42.86%,33.33%。三组间比较,差异无统计学意义(p>0.05)。2未进行干预的大鼠,其癌组织、癌旁肝组织及癌远端肝组织中,CD68阳性细胞个数分别为：(134.29±35.45)、(41.72±13.62)、(38.20±13.43),癌组织分别与癌旁肝组织、癌远端肝组织比较,差异有统计学意义(p<0.05)。CD163阳性细胞个数分别为：(47.84±1.61)、(32.55±31.23)、(31.66±17.68)；CD8阳性细胞个数分别为：(104.95±35.77)、(119.14±28.24)、(106.12±19.57)；GranzymeB阳性细胞个数分别为：(11.48±6.60)、(13.13±1.84)、(12.39±1.62)。CD163、CD8、GranzymeB阳性细胞数,三组之间差异无统计学意义(p>0.05)。3 A、B、C三组中,CD68阳性细胞个数分别为：(91.73±22.36)、(153.88±47.66)、(134.29±35.45),A、B组之间比较,差异有统计学意义(p<0.05)。CD163阳性细胞个数分别为：(34.43±9.32)、(53.33±1.45)、(47.84±1.61),A、B组之间比较,差异有统计学意义(p<0.05)。CD8阳性细胞个数分别为：(194.52±71.60)、(183.36±59.96)、(104.95±35.77),A、C组之间,B、C组之间,差异均有统计学意义(p<0.05)。Granzyme阳性细胞个数分别为(26.88±1.14)、(18.44±6.70)、(11.48±6.60),A、C组之间比较,差异有统计学意义(p<0.05)；其余各组间比较,差异无统计学意义(p>0.05)。4相关分析显示,肿瘤组织中CD68与CD8阳性细胞之间呈负相关(r=-0.221,p=0.030)；CD163与GranzymeB阳性细胞之间呈负相关(r=-0.464,p=0.030)；CD163与CD8阳性细胞之间无相关性(r=0.224,p=0.316)；CD68与GranzymeB阳性细胞之间无相关性(r=0.017,p=0.939)。5 A、B、C三组中T细胞(CD8)凋亡率分别为：(3.63±1.49)、(6.60±6.60)、(9.42±4.40),其中,A组和C组之间,差异有统计学意义(p<0.05)：其余各组间比较,差异无统计学意义(p>0.05)。结论：1氯磷酸盐脂质体组大鼠肝癌组织中巨噬细胞浸润明显低于PBS脂质体组和生理盐水对照组。提示,氯磷酸盐脂质体可有效清除肝癌组织中巨噬细胞。2氯磷酸盐脂质体组大鼠肝癌组织中细胞毒性T细胞浸润明显高于PBS脂质体组和生理盐水组。提示,巨噬细胞被清除后T细胞浸润增多；间接提示,巨噬细胞抑制T细胞的浸润。3氯磷酸盐脂质体组大鼠肝癌组织中细胞毒性T细胞凋亡明显低于PBS脂质体组和生理盐水组。提示,巨噬细胞被清除后T细胞凋亡减少；间接提示,巨噬细胞可促进T细胞凋亡。更多还原

【Abstract】 Purpose:Monocytes can differentiate into classically activated macrophages (M1) and alternatively activated macrophages (M2) under different stimulation when recruited into peripheral tissues from the circulation. Tumor associated macrophages (TAM) resemble M2-polarized macrophages. High TAM density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection. High intratumoral cytotoxic T lymphocytes (CTL) infiltration was an independent prognostic factor for improved overall survival and disease-free survival in primary liver cancer. To investigate the influence of TAM on CTL and the role of TAM in tumorigenesis and tumor development, we designed our experiment.Methods:Walker-256 Wistar rat hepatoma models were finished. Then rats were randomly divided into three groups:A group (clodronate-lipsomes by intraperitoneal injection), B group (PBS-lipsomes by intraperitoneal injection), and C group (normal saline by intraperitoneal injection). Animals were killed at the 12th day after treatment. The tumor volume, positive rate of peritoneal metastasis and positive rate of ascites were measured, and the number of positive cells of CD68, CD8, CD163, and Granzyme B in tumor tissue, peritumoral tissue and distal liver tissue were detected by immunohistochemical method, respectively.(the CD68, CD8 positive cell was macrophage; the CD8, GranzymeB positive cell was T cell). The apoptosis of CD8+ T cells in tumor tissues was tested by the method of double staining with immunohistochemistry and TUNEL.Results:1 For A, B and C group, the tumor volume was (0.13±0.09) cm3, (0.27±0.14) cm3, and (0.41±0.36) cm3; the tumor weight was (0.16±0.14)g, (0.31±0.13)g and (0.34±0.35)g; the positive rate of peritoneal metastasis was 0%,42.86% and 33.33%; the positive rate of ascites was 0%,42.86% and 33.33%; respectively. There was no significant difference among three groups (p>0.05).2 In tumor tissue, peritumoral tissue and distal liver tissue, the number of positive cells for CD68 was (134.29±35.45), (41.72±13.62) and (38.20±13.43); for CD163 was (47.84±1.61), (32.55±31.23) and (31.66±17.68); for CD8 was (104.95±35.77), (119.14±28.24) and (106.12±19.57); for Granzyme B was (11.48±6.60), (13.13±1.84) and (12.39±1.62); respectively. There was significant difference between tumor and peritumoral tissue, between tumor and distal liver tissue for CD68 positive cells (p<0.05). There was no significant difference between tumor, peritumoral and distal liver tissue for CD163, CD8 and GranzymeB positive cells(p>0.05).3 In A, B and C group, the positive cells number was (91.73±22.36), (153.88±47.66) and (134.29±35.45) for CD68; (34.43±9.32), (53.33±1.45) and (47.84±1.61) for CD163; (194.52±71.60), (183.36±59.96) and (104.95±35.77) for CD8; (26.88±1.14), (18.44±6.70) and (11.48±6.60) for Granzyme B;respectively. There was significant difference between A and B group for CD68 and CD163 (p<0.05); between A and C group for CD8 and Granzyme B (p<0.05); between B and C group for CD8 (p<0.05).4 There was a negative correlation not only between the positive cells of CD68 and CD8 (r=-0.221, p=0.030), also between CD163 and Granzyme B (r=-0.464, p=0.030). Furthermore, there was no correlation between the positive cells of CD68 and Granzyme B (r=0.017, p=0.939), as well as between the positive cells of CD163 and CD8 (r=0.224, p=0.316).5 The apoptotic rate of CD8 T cells was (3.63±1.49)%, (6.60±6.60)% and (9.42±4.40)% in A, B and C group, respectively. There was significant difference between A and C group(p<0.05).Conclusions:1 The number of TAM in A group was much less than B group and C group in rat liver cancer, it suggested that clodronate lipsome can deplete macrophages effectively.2 The number of CTL in A group was much more than B group and C group in rat liver cancer, it suggested that TAM can inhibit CTL infiltration. 3 The apoptotic rate of CTL in A group was much less than B group and C group in rat liver cancer, it suggested that TAM can promote CTL apoptosis.更多还原