PPO基因沉默对人恶性黑素瘤细胞A375增殖影响的实验研究

发布时间：2018-02-04 13:05

本文关键词： 腺病毒恶性黑素瘤 A375细胞 PPO 早期凋亡　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:恶性黑色素瘤(malignant melanoma,MM)是一类起源于神经嵴的黑素细胞恶性肿瘤,发病率占皮肤恶性肿瘤的第三位(6.8%～20%)[1] ,是皮肤科领域最常引起死亡的恶性肿瘤。恶性黑色素瘤的恶性程度高发生转移早,预后严重,治疗的关键是早期诊断、正确分期;对于早期非侵袭性的病变,手术彻底清除病变为最佳治疗选择;对于晚期患者,主要治疗为化疗及免疫治疗等。化疗药物可单用或联合应用[2],但目前的治疗方法仍不理想,且免疫疗法仍处在试验阶段。 PPO (proliferation and phosphorylation oncogene)基因是刘思源[3]等利用克隆筛选技术找到的一个新的癌基因,该基因位于1p36区,编号为KIAA0090, DNA长度约36000bp,cDNA长度6022bp。共有25个外显子,编码993个氨基酸。研究发现该基因在多种恶性肿瘤癌组织中都有高表达,与细胞增殖和磷酸化关系密切,能够使MAPK通路中的MEK1/2和ERK2磷酸化,因此把该基因命名为PPO (proliferation and phosphorylation oncogene)。RNA干扰技术是今年来发现的从基因水平治疗肿瘤的新方法。RNA干扰(RNA interference RNAi)是一种由双链RNA(double-standed RNA,dsRNA)诱发的基因沉默(gene sileneing)。RNA干扰技术是近年来发现的从基因水平治疗肿瘤的新方法。某些恶性黑色素瘤演变后,对化疗诱导的细胞凋亡、抑制癌基因的表达产生抵抗。RNA干扰技术为治疗对化疗产生耐药性的恶性黑色素瘤开辟了新的途径。刘亚玲[4]等利用PPO基因的cDNA序列片段,免疫雌性BALB/c小鼠。取小鼠脾细胞进行体外培养,利用骨髓瘤细胞与脾细胞融合后,寻找杂交瘤抗体进行克隆化。单克隆抗体用免疫扩散的方法行Ig亚类测定。克隆化的杂交瘤细胞给小鼠腹腔注射,抽取腹水后使抗体纯化,制作完成了PPO抗体。并用免疫印迹法检测PPO基因在恶性黑色素瘤组织中表达情况,以及检测PPO抗体。结果显示,PPO蛋白印迹位于140×103附近,膜上无其他蛋白印迹,蛋白印迹清晰,表明PPO抗体具有很强的特异性,可以作为检测恶性肿瘤的一种特异性抗体此前该课题组已经进行了PPO基因在A375细胞中的表达研究,发现PPO基因在细胞水平同样存在过表达[5],但能否通过RNAi的方法沉默PPO基因及其表达,抑制MAPK通路中PPO基因相关蛋白的表达,从而在细胞水平抑制恶性黑色素瘤的生长增殖,有待进一步研究. 方法: 1细胞培养将A375细胞培养于含10%胎牛血清的DMEM培养基(含青霉素100U/ml,含链霉素100U/ml,PH7.2)中,置于37℃、饱和湿度、5%CO2的培养箱内常规传代培养。 2倒置相差显微镜观察A375细胞细胞分化及形态。 3荧光显微镜下观察不同MOI(病毒个数/细胞个数)=5,10,20,50,100时,腺病毒转染A375细胞的效率及细胞分化、形态变化。 4取转染后48小时细胞做RT-PCR,观察A375细胞PPO的表达变化。 5 MTT比色分析法检测MOI=50时,在转染后第1天、第3天、第5天、第7天对A375细胞增殖抑制的影响。 6采用流式细胞分析术,Anexin-ⅴ-PI双染色检测MOI=50时,在转染后第1天、第3天、第5天、第7天A375细胞早期凋亡的变化,将实验分为阴性对照组(不加任何药物),空转录病毒组(加入HK),实验组(加入同等量PPO) 7运用统计软件SPSS13.0对数据进行统计处理,采用单因素方差分析,以a=0.05为显著性差异标准。结果: 1未经腺病毒转染处理过的人A375细胞呈梭形或多角形,贴壁生长,分布均匀,大小基本一致,增殖旺盛,核固缩现象少见。 2转染24小时后MOI=5,10,20,50,100时转染效率分别为50%、67%,69%、70%、70%。 MOI=50时转染效率达到最大且增大MOI值转染效率不再变化,故以下转染实验取MOI=50时转染后不同时间对细胞的影响。A375出现不同程度的皱缩、破裂,细胞生长状态不佳,有的细胞变狭长两端出现伪足,漂浮细胞增多,随时间延长,上述现象更加明显,而阴性对照组、空转录病毒组细胞生长旺盛,分布均匀,大小一致,仅有少数脱壁细胞。 3转染A375细胞48小时后,用RT-PCR方法检测PPO基因mRNA表达,实验组较阴性对照组,空转录病毒组的mRNA表达强度减低,证明腺病毒已经成功转染A375细胞并部分沉默PPO基因。 4 MTT比色分析法检测处理因素对人A375细胞增殖的影响。转染后第1天,第2天及第3天,阴性对照组,空转录病毒组,实验组间无显著性差异(p0.05)。转染后第5天,第7天阴性对照组与空转录病毒见无显著性差异(p0.05),实验组与阴性对照组,实验组与空转录病毒组见均有显著性差异(p0.05)。 5流式细胞仪,Anexin-ⅴ-PI双染色检测MOI=50转染后不同时间各组对A375细胞早期凋亡率的变化与对照组有显著性差异(p0.05) 结论: 1 PPO腺病毒载体成功转染人A375恶性黑色素瘤细胞。 2经腺病毒转染后,能下调人A375恶性黑色素瘤细胞PPO基因mRNA的表达。 3下调对PPO基因的表达对A375细胞生长有增殖抑制作用。 4 PPO腺病毒载体转染人A375恶性黑色素瘤细胞能诱导A375细胞早期凋亡。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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