本文選題：尿激酶型纖溶酶原激活劑受體相關(guān)蛋白 + 成纖維細(xì)胞��； 參考：《中南大學(xué)》2008年博士論文

【摘要】： 研究背景與目的: 成纖維細(xì)胞是多種具有明顯異質(zhì)性和獨(dú)特表型的成纖維細(xì)胞亞群的總稱,不同的亞群具有不盡相同的生物學(xué)功能。成纖維細(xì)胞是維持膠原代謝平衡的主要細(xì)胞,在器官纖維化、創(chuàng)傷愈合中發(fā)揮關(guān)鍵作用。 膠原降解代謝有兩條途徑,即胞內(nèi)途徑和胞外途徑。成纖維細(xì)胞吞噬降解膠原是主要的胞內(nèi)途徑,也是組織器官更新和創(chuàng)傷修復(fù)的重要機(jī)制。一些纖維化性疾病,甚至腫瘤轉(zhuǎn)移均與膠原胞內(nèi)降解代謝紊亂有關(guān)。 在體外培養(yǎng)的牙齦成纖維細(xì)胞中,具有吞噬膠原能力的細(xì)胞亞群所占比例最多可達(dá)80%。但至今未見有關(guān)成纖維細(xì)胞膠原吞噬(Collagen Phagocytic Subpopulation of Fibroblast CPSF)和非吞噬亞群(non-Collagen Phagocytic Subpopulation of Fibroblast nCPSF)分子特征的研究報道,其難點(diǎn)在于尚無法將CPSF和nCPSF分離,既往有關(guān)成纖維細(xì)胞的大量研究均是以混合成纖維細(xì)胞群為研究對象,影響了結(jié)果的精確性。 本研究嘗試通過CPSF和nCPSF膠原吞噬功能的差異建立分離方法。通過生物芯片技術(shù)和real-time PCR篩選、驗證兩者差異表達(dá)基因,并采用免疫熒光技術(shù)檢測其蛋白在CPSF和nCPSF中的表達(dá),尋找CPSF和nCPSF具有特征性的分子標(biāo)志物。研究分子標(biāo)志物在成纖維細(xì)胞吞噬膠原過程中的作用,并以標(biāo)志物為靶分子,探討建立新的細(xì)胞分離方法的可行性,為進(jìn)一步研究成纖維細(xì)胞亞群奠定基礎(chǔ)。 方法: 1.取健康志愿者的正常牙齦,采用組織塊培養(yǎng)法獲得體外培養(yǎng)的成纖維細(xì)胞。將可溶性Ⅰ型鼠尾膠原蛋白或鼠IgG包被FITC-LB,按細(xì)胞:微球=1:4比例將已制備好的COL-FITC-LB或IgG-FITC-LB溶液分別加入各實驗組細(xì)胞內(nèi),檢測成纖維細(xì)胞0.5、1、2、4、6、12、24h對IgG-FITC-LB和COL-FITC-LB的吞噬率。 2.體外培養(yǎng)的成纖維細(xì)胞加入COL-FITC-LB 6h后,上流式細(xì)胞儀分選CPSF和nCPSF。體外培養(yǎng)分選后的CPSF和nCPSF,倒置顯微鏡下觀察其生長情況,每次傳代前檢測牙齦成纖維細(xì)胞4h膠原吞噬率。 3.將來自3位健康志愿者體外培養(yǎng)的牙齦成纖維細(xì)胞等量混合,轉(zhuǎn)至待測管內(nèi)。流式細(xì)胞儀分選CPSF和nCPSF。分別提取CPSF和nCPSF總RNA,通過人類基因組U133A 2.0芯片技術(shù)篩選獲得CPSF和nCPSF差異基因表達(dá)譜。 4.依據(jù)與細(xì)胞吞噬膠原過程的相關(guān)性,從差異基因表達(dá)譜中挑選出膠原吞噬功能相關(guān)基因,并采用Real-time PCR進(jìn)一步驗證。 5.通過半定量RT-PCR和免疫熒光定位技術(shù),分別檢測4健康志愿者的CPSF和nCPSF中整合素α_2、uPARAP的mRNA和蛋白表達(dá),比較研究整合素α_2和uPARAP在成纖維細(xì)胞膠原吞噬過程中的作用,探討以uPARAP為靶分子分離CPSF的可行性。 結(jié)果: 1.成纖維細(xì)胞在0.5h內(nèi)即開始吞噬膠原及IgG-FITC-LB,IgG-FITC-LB吞噬率在1h達(dá)到峰值后保持穩(wěn)定;而COL-FITC-LB吞噬率在4h接近峰值,隨后保持穩(wěn)定;6h之后成纖維細(xì)胞對COL-FITC-LB的吞噬率比IgG-FITC-LB吞噬率高10倍以上。 2.體外培養(yǎng)分選后的CPSF 24h,未見貼壁,逐漸死亡。nCPSF培養(yǎng)6h后,大部分細(xì)胞貼壁,并正常生長增殖、傳代。第1次傳代前4hCOL-FITC-LB吞噬率僅12.17%,第2次傳代前COL-FITC-LB吞噬率升至34.76%,第3次傳代前COL-FITC-LB吞噬率達(dá)60.65%,接近未經(jīng)分選的混合細(xì)胞群的COL-FITC-LB吞噬率。 3.從18400個轉(zhuǎn)錄本(代表14500個明晰的基因)中篩選出CPSF和nCPSF差異表達(dá)基因共17個,其中上調(diào)表達(dá)基因12個,下調(diào)表達(dá)基因有5個。 4.從12個上調(diào)表達(dá)基因中,挑選出與膠原吞噬功能可能相關(guān)的3個候選基因,即uPARAP、CYBB和HOOK1,通過Real-timePCR擴(kuò)增后比較其差異表達(dá)相對量。結(jié)果顯示,CPSF中uPARAP表達(dá)量為nCPSF的2788倍;CPSF中CYBB表達(dá)量為nCPSF的0.85倍;CPSF中HOOK1表達(dá)量為nCPSF的1.96倍。 5.體外培養(yǎng)的4例正常牙齦成纖維細(xì)胞均有同樣的表現(xiàn):CPSF中uPARAP mRNA表達(dá)顯著,而在nCPSF中uPARAP mRNA無表達(dá)。在CPSF和nCPSF中整合素α_2mRNA均有顯著的表達(dá)。 6.體外培養(yǎng)的4例正常牙齦成纖細(xì)胞中,幾乎所有細(xì)胞均表達(dá)整合素α_2蛋白,而uPARAP蛋白只在部分細(xì)胞中表達(dá),小部分細(xì)胞不表達(dá)。uPARAP蛋白在CPSF中表達(dá),在nCPSF中基本不表達(dá),但整合素α_2蛋白在CPSF和nCPSF中均有明顯的表達(dá)。 結(jié)論: 1.利用CPSF和nCPSF在膠原吞噬功能上的差異,聯(lián)合應(yīng)用COL-FITC-LB吞噬技術(shù)和FCM細(xì)胞分離技術(shù)能夠?qū)烧叱晒Ψ蛛x,建立了分離CPSF和nCPSF的新方法,也為探討CPSF和nCPSF的生理和病理學(xué)意義提供了新的技術(shù)平臺。 2.CPSF和nCPSF最主要的差異表達(dá)基因是uPARAP。CPSF細(xì)胞表面表達(dá)uPARAP蛋白分子,而nCPSF不表達(dá),uPARAP是CPSF的功能分子標(biāo)志物,可用于鑒別或分離CPSF,為進(jìn)一步研究CPSF和nCPSF的表型特征、以及兩者的相互關(guān)系奠定了實驗基礎(chǔ)。 3.uPARAP是成纖維細(xì)胞的主要膠原受體,對成纖維細(xì)胞膠原吞噬功能具有重要影響,是治療膠原代謝疾病新的候選靶分子之一。
[Abstract]:Research background and purpose:
Fibroblasts are the general name of a variety of fibroblast subgroups with distinct heterogeneity and unique phenotype. Different subgroups have different biological functions. Fibroblasts are the main cells to maintain the balance of collagen metabolism and play a key role in organ fibrosis and wound healing.
There are two pathways for collagen degradation, namely, intracellular pathway and extracellular pathway. Phagocytosis and degradation of collagen in fibroblasts is the main intracellular pathway. It is also an important mechanism for tissue and organ regeneration and wound repair. Some fibrotic diseases, even tumor metastasis, are related to the degradation of collagen intracellular metabolism.
In the gingival fibroblasts cultured in vitro, the percentage of cells with the ability to phagocytiate collagenous is up to 80%., but up to now, there is no relation between the collagen phagocytosis of fibroblasts (Collagen Phagocytic Subpopulation of Fibroblast CPSF) and the non phagocytic subgroup (non-Collagen Phagocytic Subpopulation of Fibroblast). The difficulty of the study is that it is difficult to separate CPSF from nCPSF. A large number of previous studies on fibroblasts were studied with mixed fibroblast cell groups, affecting the accuracy of the results.
This study attempts to establish a separation method through the difference of CPSF and nCPSF collagen phagocytosis. Through biochip technology and real-time PCR screening, the differentially expressed genes are verified, and the expression of their protein in CPSF and nCPSF is detected by immunofluorescence technique, and the characteristic molecular markers of CPSF and nCPSF are found. Molecular markers are studied. In the process of fibroblast phagocytosis, and the marker as the target molecule, the feasibility of establishing a new cell separation method is discussed, which lays the foundation for further study of fibroblast subgroups.
Method:
1. the normal gingiva of healthy volunteers was obtained by tissue mass culture. The soluble type I rat tail collagen or rat IgG package was FITC-LB, and the prepared COL-FITC-LB or IgG-FITC-LB solution was added to the cells of each test group according to the =1:4 ratio of the microspheres, and the fibroblast cell 0.5,1,2,4,6,1 was detected. The phagocytosis rate of 2,24h to IgG-FITC-LB and COL-FITC-LB.
2. after COL-FITC-LB 6h was added to the cultured fibroblasts in vitro, CPSF and nCPSF were selected by CPSF and nCPSF. in vitro by upflow cytometer. The growth conditions were observed under the inverted microscope, and the rate of 4H collagen phagocytosis was detected in gingival fibroblasts before each passage.
3. the gingival fibroblasts derived from 3 healthy volunteers were mixed and transferred to the tube to be measured. The flow cytometry was selected to extract CPSF and nCPSF total RNA respectively by CPSF and nCPSF.. The differential gene expression profiles of CPSF and nCPSF were screened by the human genome U133A 2 chip technology.
4. according to the correlation with the process of cell phagocytosis, collagen phagocytosis related genes were selected from the differential gene expression profiles and further verified by Real-time PCR.
5. by semi quantitative RT-PCR and immunofluorescence localization technique, the expression of integrin alpha _2, uPARAP mRNA and protein in the CPSF and nCPSF of 4 healthy volunteers were detected respectively. The role of integrin alpha _2 and uPARAP in the process of collagen phagocytosis in fibroblasts was compared and the feasibility of uPARAP as a target for the separation of CPSF was discussed.
Result:
The 1. fibroblasts began to phagocytic collagen and IgG-FITC-LB in 0.5h, and the phagocytosis rate of IgG-FITC-LB remained stable after the peak of 1H, while the COL-FITC-LB phagocytosis rate was close to the peak value of 4H and then remained stable; after 6h, the phagocytosis rate of COL-FITC-LB was more than 10 times higher than the IgG-FITC-LB phagocytosis rate.
2. after the separation of CPSF 24h in vitro, no adherence was found, and after the gradual death of.NCPSF for 6h, most of the cells were adhered to the wall, and they grew and proliferate normally. The phagocytosis rate of 4hCOL-FITC-LB before first passages was only 12.17%, and the rate of COL-FITC-LB phagocytosis before 34.76% passages was 34.76%, and the rate of COL-FITC-LB phagocytosis before third passages was 60.65%, close to the unsorted mixture. The COL-FITC-LB phagocytosis of the cell group.
3. a total of 17 differentially expressed genes of CPSF and nCPSF were screened from 18400 transcriptional transcripts (representing 14500 distinct genes), of which 12 were up-regulated and 5 were down regulated.
4. of the 12 up-regulated genes, 3 candidate genes related to collagen phagocytosis were selected, uPARAP, CYBB and HOOK1, and their differential expression was compared by Real-timePCR amplification. The results showed that the uPARAP expression in CPSF was 2788 times of nCPSF, and the amount of CYBB in CPSF was 0.85 times of nCPSF; CPSF HOOK1 expressed the amount of HOOK1. 1.96 times that.
5. in 4 normal gingival fibroblasts in vitro, 4 normal gingival fibroblasts had the same performance: the expression of uPARAP mRNA in CPSF was significant, but in nCPSF, uPARAP mRNA was not expressed. The integrin _2mRNA all had a significant expression in CPSF and nCPSF.
6. in 4 normal gingival fibroblast cells in vitro, almost all of the normal gingival fibroblast cells expressed integrin alpha _2 protein, while uPARAP protein was expressed only in some cells. A small portion of the cells did not express.UPARAP protein in CPSF, and the expression of integrin alpha _2 protein was not expressed in nCPSF, but the integrin alpha _2 protein was expressed in CPSF and nCPSF.
Conclusion:
1. using the difference between CPSF and nCPSF in the function of collagen phagocytosis, the combination of COL-FITC-LB phagocytosis and FCM cell separation can separate the two successfully, and establish a new method for separating CPSF and nCPSF. It also provides a new technical platform for exploring the physiological and pathological significance of CPSF and nCPSF.
The main differentially expressed genes of 2.CPSF and nCPSF are the expression of uPARAP protein on the surface of uPARAP.CPSF cells, while nCPSF is not expressed. UPARAP is a functional marker of CPSF, which can be used to identify or separate CPSF, which provides an experimental basis for further study of the phenotypic characteristics of CPSF and nCPSF and the interrelationship between the two.
3.uPARAP is the main collagen receptor of fibroblasts, which has an important influence on the collagen phagocytosis of fibroblasts, and is one of the new candidate targets for the treatment of collagen metabolic diseases.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

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相關(guān)期刊論文 前1條

1 唐小蕾,江一平;甘露糖受體家族[J];福建醫(yī)科大學(xué)學(xué)報;2005年S1期

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本文編號：2043143