小鼠肿瘤模型建立

1、小鼠肿瘤模型的建立及鉴定 摘要：目的：建立小鼠荷瘤数据模型，探索肿瘤生长的本质及其发生发展的规律，寻求对肿瘤生长及转移有抑制作用的药物及治疗方法。方法：小鼠右腋下注射H22肿瘤细胞，使小鼠荷瘤，观察肿瘤生长，记录体重数据并建立动物模型。结果：注射部位发生癌变，小鼠肿瘤生长明显，切片结果证明实验成功，模型构建完成，为灵芝多糖抗肿瘤研究奠定了动物实验基础。关键词：小鼠，肿瘤，体重数据，动物模型To Establish Mice Tumor Model and Identify CharacteristicsAbstract: Purposes: To establish mice tumor da

2、ta models, explore the essence of tumor growth, develop the law, and seek to an effect method of tumor growth and the transfer of drugs and treatments. Methods: Named right armpit injections H22 tumor cells to a mouse neoplasm observe tumor growth. To record weight data and establish animal models.

3、Results : Injection parts turn cancertization ,the mouse's tumors grow obviously, the consequence of slices prove the experiment successfully, and model established completely, for the anti-cancer research ganoderma lucidum polysaccharide lay a animal experiments foundation. Keywords: mouse, tum

4、ors, weight data, animal models0 引言肿瘤是由机体中某些细胞基因发生突变为特征的一种全身性疾病，它的危害不仅在于它损害身体的各器官，而且还会产生各种肿瘤并发症，如转移、积水、疼痛等，最终导致身体各器官功能衰竭而危及患者的生命。肿瘤细胞除了以浸润恶性生长方式向周围侵蚀传播之外，还可以通过其他途径扩散到其它脏器和组织里边去，使这些组织和脏器发生同样的肿瘤，所以肿瘤的发现、诊断、治疗要坚持三早、避免三晚。近几年来，肿瘤化疗取得了相当的进步，肿瘤患者生存时间明显延长，特别是对白血病、恶性淋巴瘤等的治疗有了突破，但对危害人类生命健康最严重的、占恶性肿瘤90%以上的实体瘤的

5、治疗未能达到满意的效果。药学家和肿瘤学家越来越深刻地认识到：要提高肿瘤治疗的疗效，必须从肿瘤发生发展的机制着手，才能取得新的突破性进展。近年来，分子肿瘤学、分子药理学的发展使肿瘤本质正在逐步阐明；大规模快速筛选、组合化学、基因工程等先进技术的发明和应用加速了药物开发进程；抗肿瘤药物的研究与开发已进入一个崭新的时代。当今抗肿瘤药物的发展战略有以下几点：以占恶性肿瘤90%以上的实体瘤为主攻对象；从天然产物中寻找活性成分；针对肿瘤发生发展的机制，寻找新的分子作用(酶、受体、基因)靶点；大规模快速筛选(High-through put screening);新技术的导入和应用：组合化学、结构生物学、计

6、算机辅助设计、基因工程、DNA芯片、药物基因组学(功能基因组学与药理学结合)等。本实验构建小鼠肿瘤的动物模型，为灵芝多糖抗肿瘤药物的研究提供动物实验基础。1材料与方法1.1实验材料 1.1.1动物及分组：取20只昆明鼠随机分成A组（10只），B组（5只），C组对照组（5只），编号饲养。1.1.2试剂：无菌水，生理盐水，台酚蓝，2%冰醋酸，甲醛，乙醇，二甲苯，石蜡等。1.1.3仪器：离心机，显微镜，计数板，电子称，超净工作台，温箱，切片机等。1.2实验方法 1.2.1模型建立：取H22肿瘤细胞，3000转离心分钟，再用无菌生理盐水洗涤肿瘤细胞3次，并做适当稀释，取40微升细胞悬液加入10微升0.

7、4%台酚蓝染色并镜检计数，制成浓度为3*106个/ml的肿瘤细胞悬液，每只小鼠右腋皮下接种肿瘤细胞悬液0.2毫升。1.2.2：细胞计数：细胞计数液2%冰醋酸溶液。取50微升细胞悬液加入450微升的细胞计数液中计数。1.3记录：接种完成后,逐日观察接种部位有无感染,肿瘤生长后有无自然消退,用游标卡尺，每周测量肿瘤长径a和短径b ,并计算平均直径,即r = (a + b) /2，每天称量小鼠体重和肿瘤并记录，绘制曲线。1.4制备组织切片：a处死小鼠，将肿瘤组织切成1*1*0.4cm的小块。b将切块的组织置于螺口瓶中，加入40%甲醛固定，过夜。c固定完成后，倒出固定液，立即换上新鲜的70%乙醇，洗脱

8、两次，室温放置20分钟，继而以75%，80%，85%，90%，95%，100%乙醇脱水各两次，每次20分钟。d从新鲜二甲苯除去乙醇，放置2次，每次10分钟。e将样品置于60温箱中融化石蜡4次，每次1.5小时。f将准备好的包埋工具，用一热玻璃吸管加入融化的石蜡，再用热镊子快速将样品移至充满石蜡的模具中，置于室温，使起完全凝结。g从包埋模具中取出石蜡块，放于室温干燥处，切片。2结果2.1肿瘤生长状况：接种当天全部动物右后肢外侧皮下接种部位均可见细胞悬液形成的液泡,半日后液体为组织所吸收,皮肤表面恢复平整。从A,B两组小鼠肿瘤生长状况看，前五天均无明显生长迹象，从第六天开始肿瘤迅速出现并增大，从肿瘤

9、曲线中可以发现，两组小鼠的肿瘤生长速度不尽相同，明显的分为三个档次：第一类生长缓慢，达到一定大小后既不再生长或生长缓慢；第二类先以较快的速度达到一定大小后，开始稳定的持续生长；第三类是少数个体以近似恒定的高速生长，曲线斜率基本达到1，生长迅速，效果明显。A组10号小鼠肿瘤于三周后萎缩消失，但生命力旺盛未见消瘦。4号和8号小鼠分别于第二十天，二十一天肿瘤处皮肤破裂，有积液流出。1号小鼠喉部于第二十三天出现另一肿瘤，大小约5*5mm，持续生长并于第二十七天与腋下肿瘤相连。7号小鼠肿瘤两周后扩散到右前肢，基本丧失行动能力，于第三周死亡。B组5号小鼠于第十七天在右颈部出现另一肿瘤，大小约1*2mm，一

10、直没有继续增大。C组小鼠在近一个月的时间里平均体重增幅为2g，增幅平缓。体重曲线与A,B组曲线对比明显。2.2 体重数据A组体重数据 单位/gA1A2A3A4A5A6A7A8A9A10平均值3.1131.9824.5728.828.1735.2225.4728.0429.9732.627.2829.213.1230.1124.4329.128.6634.6425.3528.6330.0633.0327.2929.133.1330.5424.829.428.8434.5226.9829.6730.1432.9828.0629.5933.1431.1325.4329.629.6634.9428.3

11、530.6330.634.0329.2930.3663.1531.5925.6429.5529.8734.5431.0532.0430.0433.9429.6530.7913.1632.1825.729.4330.7334.6832.4233.0727.0833.9230.4630.9673.1732.726.6830.9631.7836.1233.7332.4529.2633.7931.0731.8543.1832.1926.329.4530.8633.5932.3630.4528.7432.6730.8830.7493.1933.3926.1930.1130.932.433.1728.41

12、29.1732.6731.730.8113.233.6826.0130.5430.1532.8432.9828.9530.5432.8831.5131.0083.2134.2126.4131.8130.1933.1832.5929.5431.4832.9231.6731.43.2234.3826.9231.329.7433.6432.7330.932.6333.5831.7131.7533.2334.7827.331.1830.8534.3631.1831.0134.133.9232.4432.1123.2436.3328.1330.831.7135.7130.8834.6437.1834.9

13、132.333.2593.2537.8527.9330.4332.1336.730.0735.6839.1235.632.7833.8293.2637.4928.3630.8632.1935.833.5937.2440.2435.5533.6234.4943.2737.128.0131.6233.0737.2831.138.0841.736.1133.7434.7813.2835.6426.829.2231.2535.0731.4236.4740.2533.4431.733.1263.2937.9827.1732.7132.6337.6133.7532.8442.9234.8834.5834.

14、7073.338.0626.7132.633.3838.3134.06死亡44.4635.8934.6835.353.3138.827.2331.933.538.7234.16死亡45.2536.4135.1835.683334.138.4427.332.0834.6539.835.07死亡48.3936.8336.2136.534.240.0227.532.5734.4140.9934.15死亡49.5237.2136.9437.034444.342.427.9332.4231.5641.9335.44死亡43.0135.7142.0236.935564.442.6628.5532.2530

15、.78死亡35.48死亡死亡36.3844.2935.774.545.228.4633.2630.79死亡37.38死亡死亡36.8546.9236.984.645.7828.6432.8130.08死亡36.31死亡死亡35.6448.9936.892864.748.5228.8831.0529.92死亡36.58死亡死亡34.6851.3837.28714A组肿瘤数据 单位/mmA1A2A3A4A5A6A7A8A9A103.1100000000003.1200000000003.1300010022223.1422122154333.153324217.57.5433.1656253210

16、.5953.53.1787375314.510.573.53.1897464516118.543.19108474519.5111143.2118484.5520.512.511.573.2113.585.5106.552216127.53.221486127523.5171283.2315751112623.5191283.24178411.51362422.512.57.53.2517.5941115527.522137.53.2618541314527.527.51483.27186.561216728.530.51583.2818561316727.529.51473.29205514

17、16728.5331573.322661316.510死亡341573.312256142010.5死亡36.5177.54.12266192111死亡36.51764.22267171911死亡3318消失4.3224.56162210.5死亡3819消失4.422.55716死亡8.5死亡3820消失4.524.55618.5死亡9死亡死亡20消失4.6245821.5死亡7.5死亡死亡23.5消失4.725.54.5820死亡8死亡死亡23.5消失B组体重数据 单位/gB1B2B3B4B5平均值3.1626.4929.3427.0723.8325.0326.3523.1726.3428.

18、5627.3124.6525.9826.5683.1826.4528.4726.8825.3926.1526.6683.1926.1228.8426.4526.7726.4526.9263.226.5529.0127.2128.4926.7227.5963.2125.629.2226.6529.1827.527.633.2227.1628.3233.0228.2528.5929.0683.2326.3329.5932.5829.8629.2329.5183.2426.8929.713329.6230.4429.9323.2527.2930.3332.3729.4730.4329.9783.26

19、25.1928.7932.328.0930.0728.8883.2728.130.8634.2730.5431.1230.9783.2827.663034.383030.4130.493.292831.1634.8531.731.3731.4163.328.131.4734.9331.330.6931.2983.3128.3931.4135.0233.1531.3331.864.128.3230.8734.5632.2630.9431.394.228.7231.2133.2732.1131.131.2824.329.9432.4634.7833.9831.7432.584.430.8532.7

20、734.734.5231.932.9484.531.231.534.0535.732.2232.9344.631.6332.4434.6834.7333.1733.334.730.9432.6934.134.532.3132.908B组肿瘤数据 单位/mmB1B2B3B4B53.16000003.17000003.18000003.19000003.2000003.21001003.2201.5111.53.2332.52223.243.52.5524.53.2572.55253.2672.55253.277.535.5263.287.536.5273.2983.57.5263.3105828

21、3.31123.57.52.574.11157374.2115849.54.3124.586104.410.55.56.56.5104.5135.56.58.59.54.613.55.5699C组体重数据 单位/gC1C2C3C4C5平均值3.1627.0226.5428.6127.4525.9327.113.1727.326.6428.4527.6125.8427.1683.1827.6126.8328.6427.8725.7527.343.1927.8426.7528.8727.9125.9327.463.227.6126.6428.9428.1626.1627.5023.2127.942

22、6.7228.6428.3626.7127.6743.2227.5426.9328.7628.7726.2627.6523.2327.6126.9728.3328.4525.9427.463.2428.8227.2128.9428.3726.7728.0223.2528.9627.1629.2128.4526.4528.0463.2627.0626.5928.0727.1625.1826.8123.2728.7927.5129.5428.6726.6128.2243.2828.5427.4829.3128.5226.0827.9863.2928.7427.5929.6528.3726.6128.1923.329.1327.9429.4528.6426.4828.3283.3129.1328.0629.9128.9226.6828.544.129.2428.1529.6729.0626.8628.5964.229.428.2230.1529.3726.9128.814.329.5128.4130.6829.4827.0329.0224.429.3328.3930.4529.6327.6129.0824.529.4228.363