小鼠腫瘤模型建立

1、小鼠腫瘤模型的建立及鑒定 摘要：目的：建立小鼠荷瘤數(shù)據(jù)模型，探索腫瘤生長(zhǎng)的本質(zhì)及其發(fā)生發(fā)展的規(guī)律，尋求對(duì)腫瘤生長(zhǎng)及轉(zhuǎn)移有抑制作用的藥物及治療方法。方法：小鼠右腋下注射H22腫瘤細(xì)胞，使小鼠荷瘤，觀察腫瘤生長(zhǎng)，記錄體重?cái)?shù)據(jù)并建立動(dòng)物模型。結(jié)果：注射部位發(fā)生癌變，小鼠腫瘤生長(zhǎng)明顯，切片結(jié)果證明實(shí)驗(yàn)成功，模型構(gòu)建完成，為靈芝多糖抗腫瘤研究奠定了動(dòng)物實(shí)驗(yàn)基礎(chǔ)。關(guān)鍵詞：小鼠，腫瘤，體重?cái)?shù)據(jù)，動(dòng)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 引言腫瘤是由機(jī)體中某些細(xì)胞基因發(fā)生突變?yōu)樘卣鞯囊环N全身性疾病，它的危害不僅在于它損害身體的各器官，而且還會(huì)產(chǎn)生各種腫瘤并發(fā)癥，如轉(zhuǎn)移、積水、疼痛等，最終導(dǎo)致身體各器官功能衰竭而危及患者的生命。腫瘤細(xì)胞除了以浸潤(rùn)惡性生長(zhǎng)方式向周圍侵蝕傳播之外，還可以通過其他途徑擴(kuò)散到其它臟器和組織里邊去，使這些組織和臟器發(fā)生同樣的腫瘤，所以腫瘤的發(fā)現(xiàn)、診斷、治療要堅(jiān)持三早、避免三晚。近幾年來，腫瘤化療取得了相當(dāng)?shù)倪M(jìn)步，腫瘤患者生存時(shí)間明顯延長(zhǎng)，特別是對(duì)白血病、惡性淋巴瘤等的治療有了突破，但對(duì)危害人類生命健康最嚴(yán)重的、占惡性腫瘤90%以上的實(shí)體瘤的

5、治療未能達(dá)到滿意的效果。藥學(xué)家和腫瘤學(xué)家越來越深刻地認(rèn)識(shí)到：要提高腫瘤治療的療效，必須從腫瘤發(fā)生發(fā)展的機(jī)制著手，才能取得新的突破性進(jìn)展。近年來，分子腫瘤學(xué)、分子藥理學(xué)的發(fā)展使腫瘤本質(zhì)正在逐步闡明；大規(guī)?？焖俸Y選、組合化學(xué)、基因工程等先進(jìn)技術(shù)的發(fā)明和應(yīng)用加速了藥物開發(fā)進(jìn)程；抗腫瘤藥物的研究與開發(fā)已進(jìn)入一個(gè)嶄新的時(shí)代。當(dāng)今抗腫瘤藥物的發(fā)展戰(zhàn)略有以下幾點(diǎn)：以占惡性腫瘤90%以上的實(shí)體瘤為主攻對(duì)象；從天然產(chǎn)物中尋找活性成分；針對(duì)腫瘤發(fā)生發(fā)展的機(jī)制，尋找新的分子作用(酶、受體、基因)靶點(diǎn)；大規(guī)?？焖俸Y選(High-through put screening);新技術(shù)的導(dǎo)入和應(yīng)用：組合化學(xué)、結(jié)構(gòu)生物學(xué)、計(jì)

6、算機(jī)輔助設(shè)計(jì)、基因工程、DNA芯片、藥物基因組學(xué)(功能基因組學(xué)與藥理學(xué)結(jié)合)等。本實(shí)驗(yàn)構(gòu)建小鼠腫瘤的動(dòng)物模型，為靈芝多糖抗腫瘤藥物的研究提供動(dòng)物實(shí)驗(yàn)基礎(chǔ)。1材料與方法1.1實(shí)驗(yàn)材料 1.1.1動(dòng)物及分組：取20只昆明鼠隨機(jī)分成A組（10只），B組（5只），C組對(duì)照組（5只），編號(hào)飼養(yǎng)。1.1.2試劑：無菌水，生理鹽水，臺(tái)酚藍(lán)，2%冰醋酸，甲醛，乙醇，二甲苯，石蠟等。1.1.3儀器：離心機(jī)，顯微鏡，計(jì)數(shù)板，電子稱，超凈工作臺(tái)，溫箱，切片機(jī)等。1.2實(shí)驗(yàn)方法 1.2.1模型建立：取H22腫瘤細(xì)胞，3000轉(zhuǎn)離心分鐘，再用無菌生理鹽水洗滌腫瘤細(xì)胞3次，并做適當(dāng)稀釋，取40微升細(xì)胞懸液加入10微升0.

7、4%臺(tái)酚藍(lán)染色并鏡檢計(jì)數(shù)，制成濃度為3*106個(gè)/ml的腫瘤細(xì)胞懸液，每只小鼠右腋皮下接種腫瘤細(xì)胞懸液0.2毫升。1.2.2：細(xì)胞計(jì)數(shù)：細(xì)胞計(jì)數(shù)液2%冰醋酸溶液。取50微升細(xì)胞懸液加入450微升的細(xì)胞計(jì)數(shù)液中計(jì)數(shù)。1.3記錄：接種完成后,逐日觀察接種部位有無感染,腫瘤生長(zhǎng)后有無自然消退,用游標(biāo)卡尺，每周測(cè)量腫瘤長(zhǎng)徑a和短徑b ,并計(jì)算平均直徑,即r = (a + b) /2，每天稱量小鼠體重和腫瘤并記錄，繪制曲線。1.4制備組織切片：a處死小鼠，將腫瘤組織切成1*1*0.4cm的小塊。b將切塊的組織置于螺口瓶中，加入40%甲醛固定，過夜。c固定完成后，倒出固定液，立即換上新鮮的70%乙醇，洗脫

8、兩次，室溫放置20分鐘，繼而以75%，80%，85%，90%，95%，100%乙醇脫水各兩次，每次20分鐘。d從新鮮二甲苯除去乙醇，放置2次，每次10分鐘。e將樣品置于60溫箱中融化石蠟4次，每次1.5小時(shí)。f將準(zhǔn)備好的包埋工具，用一熱玻璃吸管加入融化的石蠟，再用熱鑷子快速將樣品移至充滿石蠟的模具中，置于室溫，使起完全凝結(jié)。g從包埋模具中取出石蠟塊，放于室溫干燥處，切片。2結(jié)果2.1腫瘤生長(zhǎng)狀況：接種當(dāng)天全部動(dòng)物右后肢外側(cè)皮下接種部位均可見細(xì)胞懸液形成的液泡,半日后液體為組織所吸收,皮膚表面恢復(fù)平整。從A,B兩組小鼠腫瘤生長(zhǎng)狀況看，前五天均無明顯生長(zhǎng)跡象，從第六天開始腫瘤迅速出現(xiàn)并增大，從腫瘤

9、曲線中可以發(fā)現(xiàn)，兩組小鼠的腫瘤生長(zhǎng)速度不盡相同，明顯的分為三個(gè)檔次：第一類生長(zhǎng)緩慢，達(dá)到一定大小后既不再生長(zhǎng)或生長(zhǎng)緩慢；第二類先以較快的速度達(dá)到一定大小后，開始穩(wěn)定的持續(xù)生長(zhǎng)；第三類是少數(shù)個(gè)體以近似恒定的高速生長(zhǎng)，曲線斜率基本達(dá)到1，生長(zhǎng)迅速，效果明顯。A組10號(hào)小鼠腫瘤于三周后萎縮消失，但生命力旺盛未見消瘦。4號(hào)和8號(hào)小鼠分別于第二十天，二十一天腫瘤處皮膚破裂，有積液流出。1號(hào)小鼠喉部于第二十三天出現(xiàn)另一腫瘤，大小約5*5mm，持續(xù)生長(zhǎng)并于第二十七天與腋下腫瘤相連。7號(hào)小鼠腫瘤兩周后擴(kuò)散到右前肢，基本喪失行動(dòng)能力，于第三周死亡。B組5號(hào)小鼠于第十七天在右頸部出現(xiàn)另一腫瘤，大小約1*2mm，一

10、直沒有繼續(xù)增大。C組小鼠在近一個(gè)月的時(shí)間里平均體重增幅為2g，增幅平緩。體重曲線與A,B組曲線對(duì)比明顯。2.2 體重?cái)?shù)據(jù)A組體重?cái)?shù)據(jù) 單位/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組腫瘤數(shù)據(jù) 單位/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組體重?cái)?shù)據(jù) 單位/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組腫瘤數(shù)據(jù) 單位/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組體重?cái)?shù)據(jù) 單位/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