【作者】 张志培；

【导师】 施新猷；

【作者基本信息】 第四军医大学 ， 实验动物学， 2001， 硕士

【摘要】 本实验将人肝癌细胞系HHCC-9724-P株接种到3种不同免疫缺陷和免疫正常BALB/c小鼠的不同部位，观察肿瘤的生长特点，测定小鼠免疫细胞和抗体的变化趋势及脾淋巴细胞对肝癌细胞杀伤力，探讨肝癌在不同免疫小鼠体内生长的模型特点及肝癌生长、转移与免疫间的相互关系。 本实验首先在体外培养人肝癌细胞系（HHCC-9724-P），达到一定数量的对数生长期时进行消化收取，然后接种到免疫正常的BALB/c，B细胞缺陷的CBA/N，T细胞缺陷的BALB/c-nu（nude）和T、B细胞严重联合缺陷的SCID小鼠的皮下、腹腔和肝内及用BLAB/c和CBA/N外周血免疫重建的SCID（BALB/c-PBL-SCID和CBA/N-PBL-SCID）皮下，监测肿瘤生长和转移情况。结果显示，肝癌细胞系在BALB/c小鼠体内各接种组不生长；在小鼠CBA/N的皮下、腹腔内组不生长，肝内组出现了少量的癌结节，但皮下随接种数达到2×10~7/0.2ml时，有癌结节出现，但到3周后又逐渐消失。nude、SCID和CBA/N-PBL-SCID各皮下接种组都有癌结节生长，但3组间癌体积差异显著（P＜0.05）；BALB/c-PBL-SCID皮下接种后长出的癌体到第3周逐渐有失；nude、SCID的腹腔或肝内接种组有广泛转移，而SCID小鼠的癌体大小、转移量和转移范围明显超过nude。 第四军医大学硕士学位论文 一 另一方面，本实验在接种癌细胞后监测小鼠体内免疫的变化。接 种后每周测定有B细胞的小鼠抗体变化，分别用小鼠血清与肝癌细胞 系进行凝集反应对比试验一5周后，取实验鼠进懈细胞毒杀伤实验； 同时测定具有 T细胞的，J、鼠 CD4”、CDS”T细胞百分率。结果表明， 脾细胞对靶细胞杀伤力依次为荷瘤CB洲、B从B／c、 CBA／N－PBL－SCID卜鼠，对照组及SCID、nude没杀伤性。CBA／N和 BALB儿的 CD4”、CDS＊细胞百分率均发生变化，CD4”T细胞下降 明显，CDS”T细胞变Jt不大。CD4”八b例降到 l．00左右。实验eA BALB儿，J、鼠血清与癌细胞凝集反应较强烈，B从B儿FBLECID和 CdC 依次次之Z 所测的B ALALB儿小鼠免疫球蛋白有一上升到下降的趋势， 而BALB儿PBL七CID小鼠是持续上升的，每毫升能达到毫克量。 结论：1．肝癌细胞在比ID体内百分之百成瘤，而且癌体比＿d：长 得更快更大，肝内接种转移率更高、转移范围和量更大，所以，SCID 是建立肝癌模型的最佳模型动物。2．本实验用 8 X 106的细胞数进行免 疫重建 SCID是成功的，不仅体现了 2种，J、鼠排斥肿瘤的特点，k且出 现的GVHD并不严重，因此SCID是兔疫重建和肿瘤免疫研究十分 理想的模型动物。3．肝原位和腹腔接种易发生转移，但肝内原位接种转 移率及转移范围更广。4l细胞在排斥肝癌中起主要作用，B细胞在抗 肿瘤中也起很重要的作用；在一定条件下，存在非T细胞依赖的B细 胞抗肿瘤或非B细胞依赖的T细胞排斥肿瘤作用；免疫细胞间存在着 相互促进的机制，肿瘤免疫需要综合性免疫。5．肿瘤在不同接种部位所 产生的免疫影响是不完全一样的；免疫在排斥肿瘤的同时，也受到肿 瘤抑制或影响。6．本实验h正明T细胞的细胞毒和B细胞产生的抗体是 异种特异性的抗肿瘤作用。更多还原

【Abstract】 The human hepatocarcinoma cells (H1-JCC-9724-p) were inoculated to different sites of three different immunodeficient mice and the immune normal mice -BALB/c,then the characteristics of the tumor growing were observed, and the T cell percentage and antibodies in mice were assayed; also the lymphocytotoxities of spleens to the hepatocarcinoma(target)cells were detected,so the model characteristics of tumor growing in these mice,and the correlation of the growth and metastasis of the hepatocarcinoma with mice inlmunity could been explored. In this experiment,the HFICC-9724-P were maintained in RPMII 1640 medium at 37 c in a fully humidified atmosphere containing 5% co2 in vitro.The cultures approaching 90% confluence were harvested with 0.25%(w/v) trypsin containing 0.02%(wlv) EDTA in 10mM PBS,then were inoculated to the s.c,i.p,i.h of immune normal BALB/c,B lymphocyte deficient CBAIN,T lymphocyte deficient mude,T and B lymphocytes combined immunodefient SCID mice, and also inoculated to s.c of immunoreconstituted BALB/c-PBL-SCID andCBAIN-PBL-.SCJD. After that,the situations of hepatocarcinoma growth and metastasis were surveyed. The results demonstrated that there were no growth in any sites of in BALB/c;no growth in s.c and i.p groups of CBA/N,but a little node in liver of i.h group.However,the S.C of CBA/N once appeared carcinoma node ,then gradually disappeared after3 weeks as the inoculation cell numbers reached 2x 107/0.2m1. There were all carcinoma growing in s.c of nude,SCID, CBAIN-PBL-SCID,but,the volumes of which were significant difference.Although there were carcinoma growth in the s.c of BALB/c- PBL-SCJD, the tumor volume gradually disappeared after inoculating the tumor cells 3 weeks.In i.p and i.h groups of nude ,SCID mice,there were tumor growth and extensive metastasis ,while the volume and metastasis and range of tumor in SCID overpassed those in nude. The change of immunity in mice were surveyed after inoculation the carcinoma cells.First, the antibodies were detected in the mice with B lymphocytes every week and the agglutiration reaction of sera of the mice with the carcinoma cells were compared.Second,the spleen lymphocytes of mice were examined for direct cytotoxicity agaist the carcinoma (target) cells. Third,the CD4+and CD8+T cell percentage of experimental mice were assayed. The results showed that the spleen lymphcytotoxic potentiality to the target cells was CBAIN,BALB/c,BALB/c-PBL-SCID in turn,while the splees of control groups and SCID ,nude mice showed no cytotoxicity to target cells.The CD4+ and CD8+T cell percentage changed: CD4+T cell and CD4+/ CD8+ratio evidently decreased,when CD8 T cells percentage changed not much.Agglutination of sera of experimental BALB/c with target cells was strong,next was BALB/c-PBL-SCID and nude.The detected immunogiobulin of BALB/c had a tendency from increasing to decreasing,while that of BALB/c-PBL-SCID gradually increased, which reched mg/mi. Conclusions: 1 .There was 100% hepatocarcinoma growth in SCID and the tumor volume of SCID was larger than that of nude;the ratio and range and quantity of metastasis in i.p group of SCJD were also higher than that of nude.So,SCJD mice are the best model animal for establishing hepatocarcinoma animal model.2.The immunoreconstitution of SCID mice was successful with 8 x 1 O6lymphocyte numbers,Which not only embody the characteristics of two mice immunity agaist hepatocarcinoma ,but the appearing GVI-17D was not更多还原