血管内皮生长因子受体2在毛囊上皮细胞中的表达及功能研究

发布时间：2018-03-09 15:43

本文选题：血管内皮细胞生长因子受体-2　切入点：毛囊　出处：《浙江大学》2012年博士论文　论文类型：学位论文

【摘要】：研究背景血管内皮生长因子(Vascular endothelial growth factor, VEGF)家族已经被发现的成员包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E和PlGF(placenta growth factor,胎盘生长因子),是目前研究较为深入的血管生长因子家族。其中最主要的成员是VEGF-A,又称VEGF、1-3在人类,主要包括几种分子量不同的同种异构体,如：VEGF121、VEGF145、VEGF165、VEGF189、VEGF206等1,其中VEGF165效应性最强4。血管内皮细胞特异性的VEGF受体(VEGF receptor, VEGFR)介导VEGF家族成员对内皮细胞增殖、微血管增生及增强血管通透性等的作用3一,其可分为两大类：酪氨酸激酶受体(VEGFR-1, VEGFR-2, VEGFR-3),与非酪氨酸激酶受体(neuropilin-1(NRP-1), neuropilin-2(NRP-2)。对内皮细胞的增殖、分化作用主要由VEGFR-2介导的6.7。近年来发现VEGFR-2除了表达在血管内皮细胞外,而且还表达在一些非血管内皮细胞上,如黑素细胞11、视网膜上皮细胞10、造血干细胞、神经元细胞、血管平滑肌细胞9以及某些肿瘤细胞8。而且在正常人的皮肤角质形成细胞、毛囊上皮细胞(包括毛囊隆突部细胞)、汗腺及皮脂腺细胞均发现VEGFR-2的表达。因此证实VEGF-2在毛囊上皮细胞中表达的生物学意义有助于解决毛囊生物学中的一些问题。目的明确VEGFR-2在毛囊上皮细胞中的表达情况,及与毛发周期的相关性。研究毛乳头细胞分泌的VEGF除以旁分泌的形式发挥血管形成作用之外,是否还可以通过VEGFR-2直接作用于毛囊上皮细胞产生生理调节作用。重点研究VEGFR-2在毛囊隆突部细胞中的表达、VEGF对其表达的影响以及通过VEGFR-2介导的毛囊隆突部细胞的增殖、迁移和黏附,以及VEGFR-2下游信号表达的影响。方法第一部分：用免疫荧光法检测VEGFR-2在小鼠不同毛发周期中的表达情况。利用免疫荧光双染技术,探讨了在人头皮毛囊中VEGFR-2与增殖、分化、凋亡指标的免疫荧光共染情况。第二部分：用VEGFR-2的中和性抗体MAB3572预处理,同时使用外源性VEGF165作为刺激因子刺激体外培养的人毛囊隆突部细胞,提取总RNA和蛋白质,以逆转录-聚合酶链反应(RT-PCR)检测毛囊隆突部细胞VEGFR-2mRNA的表达水平；以Western-blot法检测培养的隆突处细胞VEGFR-2蛋白质的表达水平,观察对角蛋白K10、K14、K16、K19以及β-catenin、integrinβ1、Lgr6、Artemis (Serine516)表达的影响。分别用MTT法、Trans well法观察不同浓度VEGF165作用下体外培养的经MAB3572预处理或未预处理的隆突部细胞增殖、粘附和迁移能力的变化。以25ng/ml VEGF165刺激MAB3572预处理或未预处理的隆突部细胞,以Western-blot法检测观察其对VEGFR-2、P38、C-Jun、ERK1/2磷酸化的影响,探讨VEGFR-2介导的调节细胞活性的细胞内信号传导途径。第三部分：提取总RNA,以逆转录-聚合酶链反应(Reverse transcriptpolymerase chain reaction, RT-PCR)检测人毛囊Artemis mRNA的表达水平,用免疫荧光法检测Artemis (Serine516)在人头皮毛囊、皮脂腺、汗腺等处的表达,以及在小鼠不同毛发周期毛囊中的表达情况。利用免疫荧光双染技术,探讨了毛囊隆突部细胞Artemis (serine516)与增殖、分化、凋亡指标的免疫荧光共染情况。结果一、免疫组化发现人头皮毛囊表达VEGFR-2及磷酸化VEGFR-2,小鼠毛囊上VEGFR-2表达随毛发周期发生改变。二、VEGFR-2与K16、K14、p-P53、Bax、bcl-2、β-catenin、integrinβ1等荧光双染重叠良好。在髓质、皮质区域与K10、involucrin等荧光双染重叠良好。三、VEGFR-2培养的隆突部细胞的表达： 1) RT-PCR:体外培养的隆突部细胞表达VEGFR-2mRNA, VEGF165促进体外培养的隆突部细胞表达VEGFR-2mRNA, VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对体外培养的隆突部细胞表达VEGFR-2mRNA表达的上调。 2) Western-blot:体外培养的隆突部细胞在蛋白质水平表达VEGFR-2。VEGF162促进体外培养的隆突部细胞表达VEGFR-2, VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对体外培养的隆突部细胞表达VEGFR-2表达的上调。四、VEGF165促进体外培养的隆突部细胞的增殖、异质性粘附、迁移能力,抑制同质性粘附能力。五、VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对体外培养的隆突部细胞增殖、异质性粘附、迁移能力的促进及同质性粘附能力抑制。六、VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对体外培养的隆突部细胞促进表达整合素β1及Artemis (Serine516),抑制表达Lgr6。七、VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对体外培养的隆突部细胞表达K16、K19、K14等的促进作用。八、VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对体外培养的隆突部细胞表达K10的抑制作用。九、VEGF165促进体外培养的隆突部细胞VEGFR-2、c-Jun、ERK1/2、p38的磷酸化及P-catenin向细胞核内转位,VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对隆突部细胞VEGFR-2、c-Jun、ERK1/2、p38的磷酸化作用及β-catenin向细胞核内转位。十、VEGF165促进体外培养的隆突部细胞Artemis在serine516位点的磷酸化,VEGFR-2中和性抗体MAB3572能够拮抗VEGF165对隆突部细胞Artemis在serine516位点的磷酸化。十一Artemis (serine516)的表达方式随毛发周期改变。在人头皮生长期毛囊中的上半部分上皮细胞与表皮强阳性表达Artemis (serine516),在汗腺及皮脂腺也可见Artemis (serine516)表达。十二、Artemis (serine516)与K16荧光双染重叠良好,但是与K10、p-P53、 Bax、bcl-2、K14、Ki67等信号表达趋势相反；与c-myc、p21表达位置紧邻,信号表达趋势相一致。结论一、毛囊上皮包括隆突部细胞在mRNA和蛋白质水平上均表达VEGFR-2. 二、VEGFR-2参与介导毛囊隆突部细胞的增殖、迁移、粘附、分化。三、VEGFR-2参与毛囊的生长、分化过程及毛发周期调控；VEGFR-2对毛囊的调控可能与β-catenin相关。四、VEGFR-2发挥生物学效应部分通过Artemis的磷酸化,发挥对毛囊生物学活性的调节作用。
[Abstract]:Research background
Vascular endothelial growth factor (Vascular endothelial, growth factor, VEGF) family members have been found including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PlGF (placenta growth factor, placental growth factor), is currently the more in-depth study of vascular endothelial growth factor family members. The most important is VEGF-A, also called VEGF. 1-3 in humans, including several isoforms with different molecular weight, such as: VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 1, VEGF165 4. had the strongest effect
Endothelial cell specific receptor VEGF (VEGF receptor VEGFR) mediated by VEGF family members on endothelial cell proliferation, angiogenesis and vascular permeability enhancing the role of the 3 one, which can be divided into two categories: tyrosine kinase receptors (VEGFR-1, VEGFR-2, VEGFR-3), and non receptor tyrosine kinases (neuropilin-1 (NRP-1), neuropilin-2 (NRP-2) on endothelial cell proliferation, differentiation is mainly mediated by VEGFR-2 6.7. discovered in recent years, in addition to VEGFR-2 expression in vascular endothelial cells, but also expressed in some non endothelial cells, such as melanocytes 11, 10 retinal epithelial cells, hematopoietic stem cells, neuronal cells. Vascular smooth muscle cells and some tumor cells 8. and 9 in normal human skin keratinocytes, hair follicle epithelial cells (including hair follicle bulge cells), sweat glands and sebaceous gland cells were found in the expression of VEGFR-2. It is proved that the biological significance of VEGF-2 expression in the hair follicle epithelial cells can help to solve some problems in hair follicle biology.
objective
Clear expression of VEGFR-2 in hair follicle epithelium, and the correlation with the hair cycle. The form of secretion of dermal papilla cells VEGF by paracrine play role of vascular formation, whether can also through direct effects of VEGFR-2 on hair follicle epithelial cells to produce physiological regulating effect. The expression focuses on the study of VEGFR-2 in hair follicle bulge cells. And the effect of VEGF on the expression and proliferation of hair follicle bulge cells mediated by VEGFR-2, adhesion and migration, and the effect of the expression of VEGFR-2 downstream signal.
Method
Part I: the expression of VEGFR-2 in different hair cycles of mice was detected by immunofluorescence. The immunofluorescence CO staining between VEGFR-2 and proliferation, differentiation and apoptosis indexes in human hair follicle follicles was investigated by immunofluorescence double staining.
The second part: neutralizing antibody MAB3572 pretreatment of VEGFR-2, while exogenous VEGF165 as human hair follicle bulge cell factor stimulation in vitro, extracted total RNA and protein by reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of hair follicle bulge cell VEGFR-2mRNA; to the expression level of VEGFR-2 protein bulge cells the training of the Western-blot assay, to observe the diagonal protein K10, K14, K16, K19 and beta -catenin, integrin beta 1, Lgr6, Artemis (Serine516) was studied. By using MTT method, Trans well method was used to observe the different concentrations of VEGF165 in vitro by MAB3572 pretreatment or no pretreatment of bulge cells changes of proliferation, adhesion and migration ability. To 25ng/ml VEGF165 stimulation of MAB3572 pretreatment or no pretreatment of the bulge cells, observe the VEGFR-2, P38 detected by Western-blot C-Jun. The effect of ERK1/2 phosphorylation on the intracellular signal transduction pathway mediated by VEGFR-2 to regulate cell activity.
The third part: total RNA extraction, reverse transcriptase polymerase chain reaction (Reverse transcriptpolymerase chain reaction, RT-PCR) to detect the expression of human hair follicle Artemis mRNA, detected by Artemis (Serine516) in the head of hair follicle, sebaceous glands, sweat glands, expression, and in different mice the expression of hair follicle hair cycle using double immunofluorescence staining technique to investigate hair follicle bulge cells Artemis (serine516) and the proliferation, differentiation, apoptosis index of immunofluorescence staining.
Result
First, the expression of VEGFR-2 and phosphorylated VEGFR-2 was found in the human scalp hair follicle by immunohistochemistry, and the expression of VEGFR-2 on the hair follicles of the mice changed with the hair cycle.
Two, VEGFR-2 overlapped with K16, K14, p-P53, Bax, Bcl-2, beta -catenin, integrin beta 1 and other fluorescent double staining. In the medullary cortex, the fluorescence double staining with K10 and involucrin overlapped well.
Three, the expression of the protuberant cells in the VEGFR-2 culture:
1) in vitro, cultured RT-PCR: cells expressed VEGFR-2mRNA, VEGF165 promoted VEGFR-2mRNA expression in cultured carina cells, and VEGFR-2 neutralizing antibody MAB3572 could antagonize upregulation of VEGFR-2mRNA expression in cultured carina cells in vitro.
2) in vitro, cultured Western-blot: cells expressed VEGFR-2.VEGF162 at protein level, and promoted VEGFR-2 expression in cultured carina cells. VEGFR-2 neutralizing antibody MAB3572 could antagonize upregulation of VEGFR-2 expression in cultured bulge cells by VEGF165.
Four, VEGF165 promotes the proliferation of protuberant cells in vitro, heterogeneity adhesion, migration ability, and inhibition of homogeneity adhesion.
Five, VEGFR-2 neutralizing antibody MAB3572 can antagonize VEGF165 proliferation, heterogeneous adhesion, migration ability and homogeneity adhesion inhibition of cultured carina cells in vitro.
Six, VEGFR-2 neutralizing antibody MAB3572 can antagonize VEGF165 to promote the expression of integrin beta 1 and Artemis (Serine516) and inhibit the expression of Lgr6. in cultured protuberant cells in vitro
Seven, VEGFR-2 neutralizing antibody MAB3572 can antagonize the promoting effect of VEGF165 on the expression of K16, K19, K14, and so on in cultured protuberant cells in vitro.
Eight, VEGFR-2 neutralizing antibody MAB3572 can antagonize the inhibitory effect of VEGF165 on the expression of K10 in the cultured protuberant cells in vitro.
Nine, VEGF165 promotes bulge cells in vitro VEGFR-2, c-Jun, ERK1/2, p38 phosphorylation and P-catenin translocation to the nucleus, VEGFR-2 neutralizing antibody MAB3572 can antagonize the VEGF165 of bulge cells VEGFR-2, c-Jun, ERK1/2, phosphorylation of p38 and beta -catenin to nuclear translocation.
Ten, VEGF165 promoted the phosphorylation of Artemis at serine516 site in vitro, and VEGFR-2 neutralizing antibody MAB3572 could antagonize VEGF165's phosphorylation of Artemis at serine516 site in bulge cells.
Eleven, the expression of Artemis (serine516) changed with the hair cycle. In the growth stage of human scalp, the epithelial cells in the upper part of hair follicle were strongly positive for Artemis (serine516), and the expression of Artemis (serine516) in sweat glands and sebaceous glands was also observed.
Twelve, Artemis (serine516) overlapped with K16 double fluorescence staining, but it was contrary to K10, p-P53, Bax, Bcl-2, K14, Ki67 and other signal expression trends. It was consistent with c-myc, p21 expression location and signal expression trend.
conclusion
1. The cells in the upper envelope of the hair follicle express VEGFR-2. at the level of mRNA and protein.
Two, VEGFR-2 is involved in mediating the proliferation, migration, adhesion and differentiation of the follicle protuberant cells.
Three, VEGFR-2 participates in the growth of hair follicles, the process of differentiation and the regulation of hair cycle, and the regulation of VEGFR-2 on hair follicles may be related to beta -catenin.
Four, VEGFR-2 plays the biological effect part through the phosphorylation of Artemis, and plays the role in regulating the biological activity of hair follicle.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.71

【共引文献】