成纤维细胞胶原吞噬亚群功能分子标志物（uPARAP）的实验研究

发布时间：2018-06-20 05:28

  本文选题：尿激酶型纤溶酶原激活剂受体相关蛋白 + 成纤维细胞　； 参考：《中南大学》2008年博士论文

【摘要】： 研究背景与目的: 成纤维细胞是多种具有明显异质性和独特表型的成纤维细胞亚群的总称,不同的亚群具有不尽相同的生物学功能。成纤维细胞是维持胶原代谢平衡的主要细胞,在器官纤维化、创伤愈合中发挥关键作用。 胶原降解代谢有两条途径,即胞内途径和胞外途径。成纤维细胞吞噬降解胶原是主要的胞内途径,也是组织器官更新和创伤修复的重要机制。一些纤维化性疾病,甚至肿瘤转移均与胶原胞内降解代谢紊乱有关。 在体外培养的牙龈成纤维细胞中,具有吞噬胶原能力的细胞亚群所占比例最多可达80%。但至今未见有关成纤维细胞胶原吞噬(Collagen Phagocytic Subpopulation of Fibroblast CPSF)和非吞噬亚群(non-Collagen Phagocytic Subpopulation of Fibroblast nCPSF)分子特征的研究报道,其难点在于尚无法将CPSF和nCPSF分离,既往有关成纤维细胞的大量研究均是以混合成纤维细胞群为研究对象,影响了结果的精确性。 本研究尝试通过CPSF和nCPSF胶原吞噬功能的差异建立分离方法。通过生物芯片技术和real-time PCR筛选、验证两者差异表达基因,并采用免疫荧光技术检测其蛋白在CPSF和nCPSF中的表达,寻找CPSF和nCPSF具有特征性的分子标志物。研究分子标志物在成纤维细胞吞噬胶原过程中的作用,并以标志物为靶分子,探讨建立新的细胞分离方法的可行性,为进一步研究成纤维细胞亚群奠定基础。 方法: 1.取健康志愿者的正常牙龈,采用组织块培养法获得体外培养的成纤维细胞。将可溶性Ⅰ型鼠尾胶原蛋白或鼠IgG包被FITC-LB,按细胞:微球=1:4比例将已制备好的COL-FITC-LB或IgG-FITC-LB溶液分别加入各实验组细胞内,检测成纤维细胞0.5、1、2、4、6、12、24h对IgG-FITC-LB和COL-FITC-LB的吞噬率。 2.体外培养的成纤维细胞加入COL-FITC-LB 6h后,上流式细胞仪分选CPSF和nCPSF。体外培养分选后的CPSF和nCPSF,倒置显微镜下观察其生长情况,每次传代前检测牙龈成纤维细胞4h胶原吞噬率。 3.将来自3位健康志愿者体外培养的牙龈成纤维细胞等量混合,转至待测管内。流式细胞仪分选CPSF和nCPSF。分别提取CPSF和nCPSF总RNA,通过人类基因组U133A 2.0芯片技术筛选获得CPSF和nCPSF差异基因表达谱。 4.依据与细胞吞噬胶原过程的相关性,从差异基因表达谱中挑选出胶原吞噬功能相关基因,并采用Real-time PCR进一步验证。 5.通过半定量RT-PCR和免疫荧光定位技术,分别检测4健康志愿者的CPSF和nCPSF中整合素α_2、uPARAP的mRNA和蛋白表达,比较研究整合素α_2和uPARAP在成纤维细胞胶原吞噬过程中的作用,探讨以uPARAP为靶分子分离CPSF的可行性。 结果: 1.成纤维细胞在0.5h内即开始吞噬胶原及IgG-FITC-LB,IgG-FITC-LB吞噬率在1h达到峰值后保持稳定;而COL-FITC-LB吞噬率在4h接近峰值,随后保持稳定;6h之后成纤维细胞对COL-FITC-LB的吞噬率比IgG-FITC-LB吞噬率高10倍以上。 2.体外培养分选后的CPSF 24h,未见贴壁,逐渐死亡。nCPSF培养6h后,大部分细胞贴壁,并正常生长增殖、传代。第1次传代前4hCOL-FITC-LB吞噬率仅12.17%,第2次传代前COL-FITC-LB吞噬率升至34.76%,第3次传代前COL-FITC-LB吞噬率达60.65%,接近未经分选的混合细胞群的COL-FITC-LB吞噬率。 3.从18400个转录本(代表14500个明晰的基因)中筛选出CPSF和nCPSF差异表达基因共17个,其中上调表达基因12个,下调表达基因有5个。 4.从12个上调表达基因中,挑选出与胶原吞噬功能可能相关的3个候选基因,即uPARAP、CYBB和HOOK1,通过Real-timePCR扩增后比较其差异表达相对量。结果显示,CPSF中uPARAP表达量为nCPSF的2788倍;CPSF中CYBB表达量为nCPSF的0.85倍;CPSF中HOOK1表达量为nCPSF的1.96倍。 5.体外培养的4例正常牙龈成纤维细胞均有同样的表现:CPSF中uPARAP mRNA表达显著,而在nCPSF中uPARAP mRNA无表达。在CPSF和nCPSF中整合素α_2mRNA均有显著的表达。 6.体外培养的4例正常牙龈成纤细胞中,几乎所有细胞均表达整合素α_2蛋白,而uPARAP蛋白只在部分细胞中表达,小部分细胞不表达。uPARAP蛋白在CPSF中表达,在nCPSF中基本不表达,但整合素α_2蛋白在CPSF和nCPSF中均有明显的表达。 结论: 1.利用CPSF和nCPSF在胶原吞噬功能上的差异,联合应用COL-FITC-LB吞噬技术和FCM细胞分离技术能够将两者成功分离,建立了分离CPSF和nCPSF的新方法,也为探讨CPSF和nCPSF的生理和病理学意义提供了新的技术平台。 2.CPSF和nCPSF最主要的差异表达基因是uPARAP。CPSF细胞表面表达uPARAP蛋白分子,而nCPSF不表达,uPARAP是CPSF的功能分子标志物,可用于鉴别或分离CPSF,为进一步研究CPSF和nCPSF的表型特征、以及两者的相互关系奠定了实验基础。 3.uPARAP是成纤维细胞的主要胶原受体,对成纤维细胞胶原吞噬功能具有重要影响,是治疗胶原代谢疾病新的候选靶分子之一。
[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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【参考文献】

相关期刊论文 前1条

1 唐小蕾,江一平;甘露糖受体家族[J];福建医科大学学报;2005年S1期

，

本文编号：2043143