發(fā)布時(shí)間：2018-06-29 09:46

  本文選題：蛋白質(zhì)組學(xué) + 差示凝膠電泳��； 參考：《天津醫(yī)科大學(xué)》2010年博士論文

【摘要】： 一、研究目的： 以B16F10及其相應(yīng)肺轉(zhuǎn)移的移植瘤作為研究對(duì)象,應(yīng)用2D-DIGE的方法聯(lián)合質(zhì)譜技術(shù)鑒定黑色素瘤轉(zhuǎn)移相關(guān)的蛋白,并驗(yàn)證部分蛋白的功能,以期為臨床上找到預(yù)測(cè)黑色素瘤血道轉(zhuǎn)移的標(biāo)志物提供理論依據(jù)。 二、研究?jī)?nèi)容： 1.第一部分的研究?jī)?nèi)容主要是在小鼠B16F10的皮下移植瘤傳代過程中,發(fā)現(xiàn)若干小鼠出現(xiàn)自發(fā)性肺轉(zhuǎn)移,我們將具有黑色素瘤轉(zhuǎn)移的肺臟進(jìn)一步移植至小鼠的鼠蹊部,并進(jìn)行傳代,獲得了穩(wěn)定傳代的肺轉(zhuǎn)移移植瘤(B16M組),并比較了肺轉(zhuǎn)移移植瘤與B16F10移植瘤(B16組)之間的特性差異。 2.第二部分的研究?jī)?nèi)容主要是應(yīng)用差示凝膠電泳技術(shù)比較了B16組和B16M組之間的差異蛋白表達(dá),應(yīng)用質(zhì)譜技術(shù)鑒定了相關(guān)蛋白。 3.第三部分的研究?jī)?nèi)容主要是利用western blotting的方法驗(yàn)證vimentin的質(zhì)譜結(jié)果,并在70例有完整預(yù)后的惡性黑色素瘤臨床標(biāo)本上探究其臨床意義。 三、研究方法： 1.第一部分：50只C57BL／6J小鼠,雌雄各半分成兩組,其中25只C57BL／6J小鼠的鼠蹊部接種B16F10單細(xì)胞懸液(B16組),另外25只接種肺轉(zhuǎn)移移植瘤的單細(xì)胞懸液(B16M組),待腫瘤直徑約為1cm左右時(shí)處死小鼠,分離收集腫瘤組織,進(jìn)行HE染色。觀察HE切片,計(jì)數(shù)2組的血管生成擬態(tài)密度(VMD)及微血管密度(MVD),進(jìn)行MMP-2及MMP-9的免疫組織化學(xué)染色。 2.第二部分：分別提取8例B16組及8例B16M組凍存腫瘤組織的蛋白,將每組8例的蛋白提取物混合在一起,進(jìn)行差示凝膠電泳。在同一塊膠上分別應(yīng)用Cy3、Cy5標(biāo)記各組樣本及Cy2標(biāo)記2組的混合樣本作為內(nèi)參,在第二塊膠上進(jìn)行反向標(biāo)記,Cy2, Cy3, Cy5的激發(fā)波長(zhǎng)分別是488/520nm,532/580nm和633/670nm, DeCyder軟件進(jìn)行膠內(nèi)和膠與膠之間的差異分析,獲得的差異表達(dá)的蛋白點(diǎn)經(jīng)基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜(MALDI-TOF/TOF-MS)分析,獲得肽質(zhì)量指紋圖的數(shù)據(jù),利用MASCOT Peptide Mass Fingerprint查詢軟件搜索并鑒定差異蛋白質(zhì)。 3.第三部分：應(yīng)用western blotting的方法在上述提取的每組8例的蛋白標(biāo)本中驗(yàn)證質(zhì)譜結(jié)果,并收集隨訪完整的臨床病例資料,進(jìn)行免疫組織化學(xué)染色來進(jìn)一步探究差異蛋白的臨床意義。 四、研究結(jié)果 1.B16M組與B16組的特性差異 B16M組的腫瘤組織外觀顏色變淺,呈灰白色或灰黃色,質(zhì)地呈魚肉樣,B16組的腫瘤組織呈典型的黑色,質(zhì)地較軟。通過HE染色的觀察和MMP-2、MMP-9的免疫組織化學(xué)染色,發(fā)現(xiàn)B16M中存在血管生成擬態(tài)(VM),且VMD較B16組顯著增高,MMP-2、MMP-9的陽(yáng)性表達(dá)率也高于后者,且B16M組有明顯色素缺失的特征,類似于臨床上的無(wú)色素黑色素瘤的表現(xiàn),說明B16M具有高轉(zhuǎn)移高侵襲力的特質(zhì)。 2.B16M組與B16組的差示凝膠電泳和質(zhì)譜結(jié)果 選擇2D-DIGE來替代傳統(tǒng)的2-DE篩選轉(zhuǎn)移組與原發(fā)組的差異表達(dá)蛋白,并應(yīng)用MALDI-TOF/TOF MS來鑒定差異蛋白點(diǎn),共獲得13個(gè)轉(zhuǎn)移相關(guān)蛋白,其中11個(gè)在B16M組中上調(diào),2個(gè)為下調(diào),上調(diào)的蛋白有：vimentin、PGK1、enolase 1、TPI、Bip、laminin binding protein、GAPDH、myoglobin、proteasome activator reg alpha、β-actin和γ-actin,下調(diào)的為2個(gè)未命名蛋白。所示蛋白其功能涉及細(xì)胞骨架、糖代謝、分子伴侶和免疫調(diào)節(jié)等多方面。 3. vimentin的質(zhì)譜結(jié)果驗(yàn)證和臨床意義 應(yīng)用Western Blotting方法驗(yàn)證了質(zhì)譜中vimentin的鑒定結(jié)果,并在70例臨床隨訪完整的標(biāo)本中應(yīng)用免疫組化檢測(cè)vimentin的表達(dá),vimentin的陽(yáng)性表達(dá)為腫瘤細(xì)胞胞漿染成黃色,陽(yáng)性細(xì)胞數(shù)40%為高表達(dá)組,=40%為低表達(dá)組。采用卡方檢驗(yàn)檢測(cè)vimentin的高／低表達(dá)與患者各臨床病理資料之間的關(guān)系,可見vimentin的高表達(dá)與血道轉(zhuǎn)移有密切的聯(lián)系,而與年齡、性別、發(fā)病部位、TNM分期和淋巴結(jié)轉(zhuǎn)移之間無(wú)明顯的聯(lián)系,P值均大于0.05。 五、研究結(jié)論 Vimentin與黑色素瘤的轉(zhuǎn)移密切相關(guān),高表達(dá)的vimentin對(duì)于原發(fā)黑色素瘤來說,是血道轉(zhuǎn)移的預(yù)測(cè)因素之一。在臨床上,Vimentin不僅僅是確診黑色素瘤的標(biāo)志物,還是指導(dǎo)臨床預(yù)后的預(yù)測(cè)因素之一,有望成為今后預(yù)測(cè)黑色素瘤血道轉(zhuǎn)移的標(biāo)志物和治療的靶點(diǎn)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.5

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