黑色素瘤转移相关蛋白的差异蛋白质组学研究

发布时间：2018-06-29 09:46

本文选题：蛋白质组学 + 差示凝胶电泳　；参考：《天津医科大学》2010年博士论文

【摘要】： 一、研究目的：以B16F10及其相应肺转移的移植瘤作为研究对象,应用2D-DIGE的方法联合质谱技术鉴定黑色素瘤转移相关的蛋白,并验证部分蛋白的功能,以期为临床上找到预测黑色素瘤血道转移的标志物提供理论依据。二、研究内容： 1.第一部分的研究内容主要是在小鼠B16F10的皮下移植瘤传代过程中,发现若干小鼠出现自发性肺转移,我们将具有黑色素瘤转移的肺脏进一步移植至小鼠的鼠蹊部,并进行传代,获得了稳定传代的肺转移移植瘤(B16M组),并比较了肺转移移植瘤与B16F10移植瘤(B16组)之间的特性差异。 2.第二部分的研究内容主要是应用差示凝胶电泳技术比较了B16组和B16M组之间的差异蛋白表达,应用质谱技术鉴定了相关蛋白。 3.第三部分的研究内容主要是利用western blotting的方法验证vimentin的质谱结果,并在70例有完整预后的恶性黑色素瘤临床标本上探究其临床意义。三、研究方法： 1.第一部分：50只C57BL／6J小鼠,雌雄各半分成两组,其中25只C57BL／6J小鼠的鼠蹊部接种B16F10单细胞悬液(B16组),另外25只接种肺转移移植瘤的单细胞悬液(B16M组),待肿瘤直径约为1cm左右时处死小鼠,分离收集肿瘤组织,进行HE染色。观察HE切片,计数2组的血管生成拟态密度(VMD)及微血管密度(MVD),进行MMP-2及MMP-9的免疫组织化学染色。 2.第二部分：分别提取8例B16组及8例B16M组冻存肿瘤组织的蛋白,将每组8例的蛋白提取物混合在一起,进行差示凝胶电泳。在同一块胶上分别应用Cy3、Cy5标记各组样本及Cy2标记2组的混合样本作为内参,在第二块胶上进行反向标记,Cy2, Cy3, Cy5的激发波长分别是488/520nm,532/580nm和633/670nm, DeCyder软件进行胶内和胶与胶之间的差异分析,获得的差异表达的蛋白点经基质辅助激光解析电离飞行时间质谱(MALDI-TOF/TOF-MS)分析,获得肽质量指纹图的数据,利用MASCOT Peptide Mass Fingerprint查询软件搜索并鉴定差异蛋白质。 3.第三部分：应用western blotting的方法在上述提取的每组8例的蛋白标本中验证质谱结果,并收集随访完整的临床病例资料,进行免疫组织化学染色来进一步探究差异蛋白的临床意义。四、研究结果 1.B16M组与B16组的特性差异 B16M组的肿瘤组织外观颜色变浅,呈灰白色或灰黄色,质地呈鱼肉样,B16组的肿瘤组织呈典型的黑色,质地较软。通过HE染色的观察和MMP-2、MMP-9的免疫组织化学染色,发现B16M中存在血管生成拟态(VM),且VMD较B16组显著增高,MMP-2、MMP-9的阳性表达率也高于后者,且B16M组有明显色素缺失的特征,类似于临床上的无色素黑色素瘤的表现,说明B16M具有高转移高侵袭力的特质。 2.B16M组与B16组的差示凝胶电泳和质谱结果选择2D-DIGE来替代传统的2-DE筛选转移组与原发组的差异表达蛋白,并应用MALDI-TOF/TOF MS来鉴定差异蛋白点,共获得13个转移相关蛋白,其中11个在B16M组中上调,2个为下调,上调的蛋白有：vimentin、PGK1、enolase 1、TPI、Bip、laminin binding protein、GAPDH、myoglobin、proteasome activator reg alpha、β-actin和γ-actin,下调的为2个未命名蛋白。所示蛋白其功能涉及细胞骨架、糖代谢、分子伴侣和免疫调节等多方面。 3. vimentin的质谱结果验证和临床意义应用Western Blotting方法验证了质谱中vimentin的鉴定结果,并在70例临床随访完整的标本中应用免疫组化检测vimentin的表达,vimentin的阳性表达为肿瘤细胞胞浆染成黄色,阳性细胞数40%为高表达组,=40%为低表达组。采用卡方检验检测vimentin的高／低表达与患者各临床病理资料之间的关系,可见vimentin的高表达与血道转移有密切的联系,而与年龄、性别、发病部位、TNM分期和淋巴结转移之间无明显的联系,P值均大于0.05。五、研究结论 Vimentin与黑色素瘤的转移密切相关,高表达的vimentin对于原发黑色素瘤来说,是血道转移的预测因素之一。在临床上,Vimentin不仅仅是确诊黑色素瘤的标志物,还是指导临床预后的预测因素之一,有望成为今后预测黑色素瘤血道转移的标志物和治疗的靶点。
[Abstract]:First, the purpose of the study is:
B16F10 and its corresponding lung metastases were used as the research object. The proteins associated with melanoma metastasis were identified by the method of mass spectrometry combined with 2D-DIGE, and the function of some proteins was verified in order to provide a theoretical basis for finding the marker for predicting the hematoma metastasis of melanoma in clinical.
Two, research content:
1. the first part of the study was mainly about the spontaneous lung metastasis of several mice during the subcutaneous transplantation of the subcutaneous transplanted tumor of B16F10 in mice. We further transplanted the lungs with melanoma metastases to the groin of mice and passed the passage to obtain a stable transplanting lung transfer tumor (group B16M), and compared the lung metastases. The difference between transplanted tumor and B16F10 transplanted tumor (group B16).
The main content of the 2. second part is to compare the differential protein expression between group B16 and group B16M by differential gel electrophoresis (gels), and identify the related proteins by mass spectrometry.
The 3. and third part of the study was mainly to use Western blotting to verify the vimentin mass spectrum and to explore the clinical significance of 70 cases of malignant melanoma with complete prognosis.
Three, research methods:
1. first part: 50 C57BL / 6J mice were divided into two groups. Among them, the groin of 25 C57BL / 6J mice was inoculated with B16F10 single cell suspension (group B16), and the other 25 were inoculated with single cell suspension of lung transfer tumor (group B16M), and the tumor tissue was collected and stained with the tumor diameter about 1cm. HE staining was performed. HE cut was observed. The vasculogenic mimicry density (VMD) and microvessel density (MVD) of the 2 groups were counted, and immunohistochemical staining of MMP-2 and MMP-9 was performed.
2. the second part: the protein of 8 cases of B16 group and 8 cases of group B16M were extracted respectively. The protein extracts of 8 cases in each group were mixed together to carry out differential gel electrophoresis. On the same glue, Cy3, Cy5 markers and Cy2 markers 2 groups were used as internal parameters, and the reverse markers on second glue, Cy2, Cy3, were used as internal reference. The excitation wavelengths of Cy5 are 488/520nm, 532/580nm and 633/670nm respectively. The difference analysis between glue and glue and glue is carried out by DeCyder software. The differentially expressed protein points are analyzed by matrix assisted laser analytical ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS), and the data of peptide mass fingerprints are obtained, and MASCOT Peptide Mass Fingerprint is used. Query software search and identify differential proteins.
3. the third part: the method of Western blotting was used to verify the results of mass spectrometry in 8 cases of each group of protein extracted above, and to collect complete follow-up clinical case data and carry out immunohistochemical staining to further explore the clinical significance of the differential protein.
Four, the results of the study
Characteristics difference between 1.B16M group and B16 group
The color of the tumor tissue in group B16M was pale, gray or gray, and the texture was fish like. The tumor tissue in group B16 was typical black and soft. By the observation of HE staining and the immunohistochemical staining of MMP-2 and MMP-9, the presence of vasculogenic mimicry (VM) was found in B16M, and VMD was significantly higher than the B16 group, MMP-2, MMP-9 positive expression. The rate is higher than that of the latter, and the B16M group has the characteristics of obvious pigmentation, similar to the clinical manifestation of the pigmented melanoma, indicating that B16M has high metastasis and high invasiveness.
Differential gel electrophoresis and mass spectrometry results in group 2.B16M and group B16
2D-DIGE was selected to replace the differential expression protein of the traditional 2-DE screening transfer group and the original group, and used MALDI-TOF/TOF MS to identify the differential protein points. 13 transfer related proteins were obtained, of which 11 were up regulated in the B16M group and 2 were down, and the up regulated proteins were vimentin, PGK1, enolase 1, TPI, Bip, laminin binding. Lobin, proteasome activator reg alpha, beta -actin and gamma -actin are down regulated by 2 unnamed proteins. The protein shows its functions involving cytoskeleton, glycometabolism, molecular chaperone and immunoregulation.
The results of 3. vimentin mass spectrometry and its clinical significance
The Western Blotting method was used to verify the identification results of vimentin in the mass spectrum, and the expression of vimentin was detected by immunohistochemistry in 70 cases of complete clinical follow-up. The positive expression of vimentin was that the tumor cell cytoplasm was dyed yellow, the number of positive cells 40% was high expression group, and the =40% was low expression group. The chi square test was used to detect vimentin. The relationship between high / low expression and the clinicopathological data of the patients showed that the high expression of vimentin was closely related to the metastasis of the blood. There was no significant relationship between the age, sex, the site of the disease, the TNM stage and the lymph node metastasis, and the value of P was greater than that of 0.05..
Five, the conclusion of the study
Vimentin is closely related to the metastasis of melanoma. High expression of vimentin is one of the predictors of hematogenous melanoma. In clinical, Vimentin is not only a marker for diagnosis of melanoma, but also one of the predictors for guiding clinical prognosis. It is expected to be the standard for predicting the metastasis of melanoma in the future. Objects and targets for treatment.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5

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