本文關(guān)鍵詞： 腺病毒 惡性黑素瘤 A375細(xì)胞 PPO 早期凋亡　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:惡性黑色素瘤(malignant melanoma,MM)是一類起源于神經(jīng)嵴的黑素細(xì)胞惡性腫瘤,發(fā)病率占皮膚惡性腫瘤的第三位(6.8%～20%)[1] ,是皮膚科領(lǐng)域最常引起死亡的惡性腫瘤。惡性黑色素瘤的惡性程度高發(fā)生轉(zhuǎn)移早,預(yù)后嚴(yán)重,治療的關(guān)鍵是早期診斷、正確分期;對(duì)于早期非侵襲性的病變,手術(shù)徹底清除病變?yōu)樽罴阎委熯x擇;對(duì)于晚期患者,主要治療為化療及免疫治療等�；熕幬锟蓡斡没蚵�(lián)合應(yīng)用[2],但目前的治療方法仍不理想,且免疫療法仍處在試驗(yàn)階段。 PPO (proliferation and phosphorylation oncogene)基因是劉思源[3]等利用克隆篩選技術(shù)找到的一個(gè)新的癌基因,該基因位于1p36區(qū),編號(hào)為KIAA0090, DNA長(zhǎng)度約36000bp,cDNA長(zhǎng)度6022bp。共有25個(gè)外顯子,編碼993個(gè)氨基酸。研究發(fā)現(xiàn)該基因在多種惡性腫瘤癌組織中都有高表達(dá),與細(xì)胞增殖和磷酸化關(guān)系密切,能夠使MAPK通路中的MEK1/2和ERK2磷酸化,因此把該基因命名為PPO (proliferation and phosphorylation oncogene)。RNA干擾技術(shù)是今年來發(fā)現(xiàn)的從基因水平治療腫瘤的新方法。RNA干擾(RNA interference RNAi)是一種由雙鏈RNA(double-standed RNA,dsRNA)誘發(fā)的基因沉默(gene sileneing)。RNA干擾技術(shù)是近年來發(fā)現(xiàn)的從基因水平治療腫瘤的新方法。某些惡性黑色素瘤演變后,對(duì)化療誘導(dǎo)的細(xì)胞凋亡、抑制癌基因的表達(dá)產(chǎn)生抵抗。RNA干擾技術(shù)為治療對(duì)化療產(chǎn)生耐藥性的惡性黑色素瘤開辟了新的途徑。 劉亞玲[4]等利用PPO基因的cDNA序列片段,免疫雌性BALB/c小鼠。取小鼠脾細(xì)胞進(jìn)行體外培養(yǎng),利用骨髓瘤細(xì)胞與脾細(xì)胞融合后,尋找雜交瘤抗體進(jìn)行克隆化。單克隆抗體用免疫擴(kuò)散的方法行Ig亞類測(cè)定�？寺』碾s交瘤細(xì)胞給小鼠腹腔注射,抽取腹水后使抗體純化,制作完成了PPO抗體。并用免疫印跡法檢測(cè)PPO基因在惡性黑色素瘤組織中表達(dá)情況,以及檢測(cè)PPO抗體。結(jié)果顯示,PPO蛋白印跡位于140×103附近,膜上無其他蛋白印跡,蛋白印跡清晰,表明PPO抗體具有很強(qiáng)的特異性,可以作為檢測(cè)惡性腫瘤的一種特異性抗體此前該課題組已經(jīng)進(jìn)行了PPO基因在A375細(xì)胞中的表達(dá)研究,發(fā)現(xiàn)PPO基因在細(xì)胞水平同樣存在過表達(dá)[5],但能否通過RNAi的方法沉默PPO基因及其表達(dá),抑制MAPK通路中PPO基因相關(guān)蛋白的表達(dá),從而在細(xì)胞水平抑制惡性黑色素瘤的生長(zhǎng)增殖,有待進(jìn)一步研究. 方法: 1細(xì)胞培養(yǎng)將A375細(xì)胞培養(yǎng)于含10%胎牛血清的DMEM培養(yǎng)基(含青霉素100U/ml,含鏈霉素100U/ml,PH7.2)中,置于37℃、飽和濕度、5%CO2的培養(yǎng)箱內(nèi)常規(guī)傳代培養(yǎng)。 2倒置相差顯微鏡觀察A375細(xì)胞細(xì)胞分化及形態(tài)。 3熒光顯微鏡下觀察不同MOI(病毒個(gè)數(shù)/細(xì)胞個(gè)數(shù))=5,10,20,50,100時(shí),腺病毒轉(zhuǎn)染A375細(xì)胞的效率及細(xì)胞分化、形態(tài)變化。 4取轉(zhuǎn)染后48小時(shí)細(xì)胞做RT-PCR,觀察A375細(xì)胞PPO的表達(dá)變化。 5 MTT比色分析法檢測(cè)MOI=50時(shí),在轉(zhuǎn)染后第1天、第3天、第5天、第7天對(duì)A375細(xì)胞增殖抑制的影響。 6采用流式細(xì)胞分析術(shù),Anexin-ⅴ-PI雙染色檢測(cè)MOI=50時(shí),在轉(zhuǎn)染后第1天、第3天、第5天、第7天A375細(xì)胞早期凋亡的變化,將實(shí)驗(yàn)分為陰性對(duì)照組(不加任何藥物),空轉(zhuǎn)錄病毒組(加入HK),實(shí)驗(yàn)組(加入同等量PPO) 7運(yùn)用統(tǒng)計(jì)軟件SPSS13.0對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)處理,采用單因素方差分析,以a=0.05為顯著性差異標(biāo)準(zhǔn)。 結(jié)果: 1未經(jīng)腺病毒轉(zhuǎn)染處理過的人A375細(xì)胞呈梭形或多角形,貼壁生長(zhǎng),分布均勻,大小基本一致,增殖旺盛,核固縮現(xiàn)象少見。 2轉(zhuǎn)染24小時(shí)后MOI=5,10,20,50,100時(shí)轉(zhuǎn)染效率分別為50%、67%,69%、70%、70%。 MOI=50時(shí)轉(zhuǎn)染效率達(dá)到最大且增大MOI值轉(zhuǎn)染效率不再變化,故以下轉(zhuǎn)染實(shí)驗(yàn)取MOI=50時(shí)轉(zhuǎn)染后不同時(shí)間對(duì)細(xì)胞的影響。A375出現(xiàn)不同程度的皺縮、破裂,細(xì)胞生長(zhǎng)狀態(tài)不佳,有的細(xì)胞變狹長(zhǎng)兩端出現(xiàn)偽足,漂浮細(xì)胞增多,隨時(shí)間延長(zhǎng),上述現(xiàn)象更加明顯,而陰性對(duì)照組、空轉(zhuǎn)錄病毒組細(xì)胞生長(zhǎng)旺盛,分布均勻,大小一致,僅有少數(shù)脫壁細(xì)胞。 3轉(zhuǎn)染A375細(xì)胞48小時(shí)后,用RT-PCR方法檢測(cè)PPO基因mRNA表達(dá),實(shí)驗(yàn)組較陰性對(duì)照組,空轉(zhuǎn)錄病毒組的mRNA表達(dá)強(qiáng)度減低,證明腺病毒已經(jīng)成功轉(zhuǎn)染A375細(xì)胞并部分沉默PPO基因。 4 MTT比色分析法檢測(cè)處理因素對(duì)人A375細(xì)胞增殖的影響。 轉(zhuǎn)染后第1天,第2天及第3天,陰性對(duì)照組,空轉(zhuǎn)錄病毒組,實(shí)驗(yàn)組間無顯著性差異(p0.05)。轉(zhuǎn)染后第5天,第7天陰性對(duì)照組與空轉(zhuǎn)錄病毒見無顯著性差異(p0.05),實(shí)驗(yàn)組與陰性對(duì)照組,實(shí)驗(yàn)組與空轉(zhuǎn)錄病毒組見均有顯著性差異(p0.05)。 5流式細(xì)胞儀,Anexin-ⅴ-PI雙染色檢測(cè)MOI=50轉(zhuǎn)染后不同時(shí)間各組對(duì)A375細(xì)胞早期凋亡率的變化與對(duì)照組有顯著性差異(p0.05) 結(jié)論: 1 PPO腺病毒載體成功轉(zhuǎn)染人A375惡性黑色素瘤細(xì)胞。 2經(jīng)腺病毒轉(zhuǎn)染后,能下調(diào)人A375惡性黑色素瘤細(xì)胞PPO基因mRNA的表達(dá)。 3下調(diào)對(duì)PPO基因的表達(dá)對(duì)A375細(xì)胞生長(zhǎng)有增殖抑制作用。 4 PPO腺病毒載體轉(zhuǎn)染人A375惡性黑色素瘤細(xì)胞能誘導(dǎo)A375細(xì)胞早期凋亡。
[Abstract]:Objective: malignant melanoma (malignant melanoma MM) is a kind of malignant tumor in cells derived from the neural crest of the black, the incidence of skin cancer accounted for third (6.8% ~ 20%) [1], the death of malignant tumor is most often caused by Department of dermatology. Malignant melanoma is a high degree of occurrence of evil? Early metastasis and prognosis of severe, treatment is the key to early diagnosis, accurate staging; for early noninvasive lesions, complete surgical removal of lesions was the best treatment options for patients with advanced primary treatment;, chemotherapy and immunotherapy. Chemotherapy drugs can be used alone or in combination with [2], but the treatment is not ideal, and immune therapy is still in the experimental stage.
PPO (proliferation and phosphorylation oncogene) gene is Liu Siyuan [3] cloned by screening of a new cancer gene technology to find the gene, located in 1p36 District, numbered KIAA0090, DNA length of about 36000bp, the length of cDNA 6022bp. consists of 25 exons, encoding 993 amino acids. The gene has high expression in a variety of malignant cancer tissues, relationship with cell proliferation and phosphorylation closely, can make MEK1/2 and ERK2 phosphorylation in the MAPK pathway, so the gene was named PPO (proliferation and phosphorylation oncogene).RNA interference is found from this year to a new method for gene therapy of tumor.RNA interference level (RNA interference RNAi) is a double stranded RNA (double-standed RNA dsRNA) gene silencing induced by.RNA (gene sileneing) interference is found in recent years from the new level of gene therapy of tumor After the development of some malignant melanoma, the.RNA interference technology, which is resistant to chemotherapy-induced apoptosis and inhibits the expression of oncogenes, opens up a new way for the treatment of malignant melanoma with chemotherapy resistance.
Liu Yaling [4], the cDNA sequence of PPO gene, immune female BALB/c mice. The spleen cells were cultured in vitro, using myeloma cells and spleen cells after fusion, then cloned the hybridoma antibody. Methods using immunodiffusion monoclonal antibodies Ig subclass determination. Hybridoma cells into the abdominal cavity of mice after the injection, ascites antibody purification, made PPO antibody. And expression in malignant melanoma tissue in PPO gene was detected by Western blotting, and PPO antibody detection. The results show that PPO is located in the Western blot 140 * 103 near the membrane without other Western blotting, Western blotting showed that the clear and specific strong PPO antibody can be used as a kind of specific antibody detection of malignant tumor after the group has been carried out on PPO gene expression in A375 cells, PPO gene was found at the cellular level There is also over expression of [5], but whether we can silence the PPO gene and its expression through RNAi, inhibit the expression of PPO related protein in MAPK pathway, and thus inhibit the growth and proliferation of malignant melanoma at cell level, we need further research.
Method:
In the 1 cell culture, A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (containing penicillin 100U/ml, containing streptomycin 100U/ml, PH7.2), and then cultured in 37 100U/ml, saturated humidity and 5%CO2 incubator.
2 inverted phase contrast microscope was used to observe the cell differentiation and morphology of A375 cells.
The efficiency of adenovirus transfection of A375 cells and the change of cell differentiation and morphology were observed under 3 fluorescence microscope with different MOI (number of virus number / cell number) =5,10,20,50100.
4 after 48 hours of transfection, the cells were RT-PCR, and the expression of PPO in A375 cells was observed.
The effect of 5 MTT colorimetric assay on the proliferation inhibition of A375 cells at first days, third days, fifth days and seventh days after MOI=50 assay was detected.
6 by flow cytometry, Anexin- V -PI double staining MOI=50, after transfection in first days, third days, fifth days, seventh days of early A375 cell apoptosis, the experiments were divided into negative control group (without any drugs), empty adenovirus group (HK), experimental group (adding the same amount of PPO)
7 using statistical software SPSS13.0 to carry out statistical processing, using single factor analysis of variance, a=0.05 as a significant difference standard.
Result:
1 human A375 cells without adenovirus transfection were spindle shaped or polygonal. The cell growth was uniform, the size was basically the same, the proliferation was strong, and the nuclear pyknosis was rare.
After 2 transfection, the transfection efficiency of MOI=5,10,20,50100 was 50%, 67%, 69%, 70%, 70%., respectively, after 24 hours of transfection.
MOI=50 transfection efficiency reached the maximum MOI value and increase the transfection efficiency does not change, so the following transfection experiments of MOI=50 transfected.A375 cells in different time varying degrees of shrinkage, rupture, cell growth condition, some cells become narrow ends of pseudo foot, floating cells increased with time prolonging, the phenomenon is more obviously, while the negative control group, empty adenovirus group cell growth exuberant, uniform distribution, the same size, only a few cells.
3 after transfection of A375 cells for 48 hours, the mRNA expression of PPO gene was detected by RT-PCR method. The mRNA expression intensity of the experimental group was lower than that of the negative control group and the empty transcription virus group, indicating that adenovirus has successfully transfected A375 cells and partly silenced PPO gene.
4 MTT colorimetric assay was used to detect the effect of treatment factors on the proliferation of human A375 cells.
First days after transfection, second days and 3 days, negative control group, empty adenovirus group, there was no significant difference between the experimental groups (P0.05). Fifth days after transfection, seventh days to see the negative control group had no significant difference with empty adenovirus (P0.05), the experimental group and negative control group and experimental group empty adenovirus group had significant difference (P0.05).
5 flow cytometry, Anexin- V -PI double staining MOI=50 after transfection of A375 cells in different time were the early changes of apoptosis rate was significantly higher than the control group (P0.05)
Conclusion:
1 PPO adenovirus vectors were successfully transfected into human A375 malignant melanoma cells.
2 after transfection by adenovirus can express A375, under the human malignant melanoma cell PPO gene mRNA.
3 down regulation of the expression of PPO gene could inhibit the proliferation of A375 cells.
The transfection of 4 PPO adenovirus vector to human A375 melanoma cells can induce early apoptosis of A375 cells.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.5

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