【摘要】：目的：利用結(jié)腸癌細(xì)胞懸液皮下接種BALB/C裸小鼠,構(gòu)建HCT-116人結(jié)腸癌裸鼠動(dòng)物模型,觀測(cè)時(shí)間與移植瘤大小變化,檢測(cè)分析接種后不同時(shí)間裸鼠體內(nèi)微量蛋白譜的變化,尋找腫瘤相關(guān)的差異蛋白,探索大腸癌的早期診斷指標(biāo)。 方法：復(fù)蘇培養(yǎng)HCT-116大腸癌細(xì)胞,收集第3-5代對(duì)數(shù)生長(zhǎng)期細(xì)胞,將細(xì)胞懸液調(diào)至密度1×107/ml,皮下接種于10只3-4周齡裸鼠背部皮下,接種劑量為0.1ml/只,每隔15天測(cè)量瘤體大小并記錄.對(duì)照組同時(shí)用無(wú)血清培養(yǎng)基接種(0.1ml/只)。實(shí)驗(yàn)組和對(duì)照組裸鼠分別在接種后20d、40d和60d取血,前兩次采用斷尾法取血0.1ml/只,最后一次采用摘眼球取血0.5ml/只后處死裸鼠并剝離瘤體,測(cè)量瘤體體積,剝離的瘤體立即送病理科進(jìn)行病理切片檢查。所取血液標(biāo)本立即4℃3000rpm離心5min,將分離的血清分裝3管,30μL/管并進(jìn)行編號(hào),利用SELDI-TOF-MS技術(shù)對(duì)裸鼠血清進(jìn)行微量蛋白分析,將獲得的微量蛋白質(zhì)譜圖用Ciphergen ProteinChip3.0和Ciphergen Biomaker Wizard3.1軟件進(jìn)行數(shù)據(jù)的采集、校正和分析,篩選與大腸癌相關(guān)的微量差異蛋白,并對(duì)篩選出的差異蛋白在蛋白數(shù)據(jù)庫(kù)中進(jìn)行比對(duì)。 結(jié)果：裸鼠皮下接種后15天長(zhǎng)出腫瘤,成瘤率100%。篩選出與腫瘤相關(guān)的具有規(guī)律性變化的5個(gè)差異蛋白(p0.01),其分子量分別為2357Da、2424Da、2637Da、8182Da、10133Da。其中2637Da、8182Da、10133Da三種微量蛋白在接種后裸鼠體內(nèi)表達(dá)逐漸增強(qiáng),2357Da,2424Da兩種微量蛋白在接種后裸鼠體內(nèi)表達(dá)逐漸減弱,并在Swiss-Prot蛋白庫(kù)中進(jìn)行數(shù)據(jù)檢索,初步鑒定為Reactive oxygen species modulator1、 Leukocyte-specific transcript1protein、Brevinin-2R、Prosalusin、Calcitonin。在腫瘤細(xì)胞的培養(yǎng)基里面篩選出了一個(gè)差異蛋白,其分子量為4160Da,鑒定為WW domain-containing oxidoreductase或者Peptide YY。 結(jié)論：1裸鼠皮下接種HCT-116細(xì)胞能成功構(gòu)建移植瘤動(dòng)物模型,此模型易于操作,成功率高,模型穩(wěn)定,可為研究大腸癌提供幫助。2SELDI-TOF-MS技術(shù)對(duì)結(jié)腸癌裸鼠移植瘤模型差異蛋白的檢測(cè)是有效的,能發(fā)現(xiàn)早期大腸癌的標(biāo)志蛋白。3癌細(xì)胞體外培養(yǎng)發(fā)現(xiàn)的新物質(zhì)證明了癌細(xì)胞的疾病進(jìn)程并不是孤立的而是和外界緊密關(guān)聯(lián)的。
[Abstract]:Objective: to establish a nude mouse model of HCT-116 human colon cancer by subcutaneously inoculating colon cancer cell suspension into BALB/C nude mice, observe the changes of time and size of transplanted tumor, and analyze the changes of microprotein spectrum in nude mice at different time after inoculation. To search for tumor related differential proteins and to explore the early diagnostic criteria of colorectal cancer. Methods: HCT-116 colorectal cancer cells were cultured after resuscitation. The cells of logarithmic growth phase were collected and the cell suspension was adjusted to 1 脳 107 / ml. The cells were subcutaneously inoculated subcutaneously on the back of 10 nude mice aged 3-4 weeks. The tumor size was measured and recorded every 15 days. The control group was inoculated with serum-free medium (0.1ml/ only). The nude mice in the experimental group and the control group were collected blood at 20 days after inoculation for 40 days and 60 days after inoculation respectively. The first two blood samples were taken from 0.1ml/ by severed tail method, the last one was taken out of eyeball to get blood 0.5ml/, and then the nude mice were killed and stripped off the tumor body to measure the volume of the tumor. The dissected tumor was immediately sent to the pathology department for pathological section examination. The blood samples were immediately centrifuged at 4 鈩，

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