本文選題：血管內(nèi)皮細(xì)胞生長因子受體-2　切入點(diǎn)：毛囊　出處：《浙江大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景 血管內(nèi)皮生長因子(Vascular endothelial growth factor, VEGF)家族已經(jīng)被發(fā)現(xiàn)的成員包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E和PlGF(placenta growth factor,胎盤生長因子),是目前研究較為深入的血管生長因子家族。其中最主要的成員是VEGF-A,又稱VEGF、1-3在人類,主要包括幾種分子量不同的同種異構(gòu)體,如：VEGF121、VEGF145、VEGF165、VEGF189、VEGF206等1,其中VEGF165效應(yīng)性最強(qiáng)4。 血管內(nèi)皮細(xì)胞特異性的VEGF受體(VEGF receptor, VEGFR)介導(dǎo)VEGF家族成員對內(nèi)皮細(xì)胞增殖、微血管增生及增強(qiáng)血管通透性等的作用3一,其可分為兩大類：酪氨酸激酶受體(VEGFR-1, VEGFR-2, VEGFR-3),與非酪氨酸激酶受體(neuropilin-1(NRP-1), neuropilin-2(NRP-2)。對內(nèi)皮細(xì)胞的增殖、分化作用主要由VEGFR-2介導(dǎo)的6.7。近年來發(fā)現(xiàn)VEGFR-2除了表達(dá)在血管內(nèi)皮細(xì)胞外,而且還表達(dá)在一些非血管內(nèi)皮細(xì)胞上,如黑素細(xì)胞11、視網(wǎng)膜上皮細(xì)胞10、造血干細(xì)胞、神經(jīng)元細(xì)胞、血管平滑肌細(xì)胞9以及某些腫瘤細(xì)胞8。而且在正常人的皮膚角質(zhì)形成細(xì)胞、毛囊上皮細(xì)胞(包括毛囊隆突部細(xì)胞)、汗腺及皮脂腺細(xì)胞均發(fā)現(xiàn)VEGFR-2的表達(dá)。因此證實(shí)VEGF-2在毛囊上皮細(xì)胞中表達(dá)的生物學(xué)意義有助于解決毛囊生物學(xué)中的一些問題。 目的 明確VEGFR-2在毛囊上皮細(xì)胞中的表達(dá)情況,及與毛發(fā)周期的相關(guān)性。研究毛乳頭細(xì)胞分泌的VEGF除以旁分泌的形式發(fā)揮血管形成作用之外,是否還可以通過VEGFR-2直接作用于毛囊上皮細(xì)胞產(chǎn)生生理調(diào)節(jié)作用。重點(diǎn)研究VEGFR-2在毛囊隆突部細(xì)胞中的表達(dá)、VEGF對其表達(dá)的影響以及通過VEGFR-2介導(dǎo)的毛囊隆突部細(xì)胞的增殖、遷移和黏附,以及VEGFR-2下游信號表達(dá)的影響。 方法 第一部分：用免疫熒光法檢測VEGFR-2在小鼠不同毛發(fā)周期中的表達(dá)情況。利用免疫熒光雙染技術(shù),探討了在人頭皮毛囊中VEGFR-2與增殖、分化、凋亡指標(biāo)的免疫熒光共染情況。 第二部分：用VEGFR-2的中和性抗體MAB3572預(yù)處理,同時使用外源性VEGF165作為刺激因子刺激體外培養(yǎng)的人毛囊隆突部細(xì)胞,提取總RNA和蛋白質(zhì),以逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)檢測毛囊隆突部細(xì)胞VEGFR-2mRNA的表達(dá)水平；以Western-blot法檢測培養(yǎng)的隆突處細(xì)胞VEGFR-2蛋白質(zhì)的表達(dá)水平,觀察對角蛋白K10、K14、K16、K19以及β-catenin、integrinβ1、Lgr6、Artemis (Serine516)表達(dá)的影響。分別用MTT法、Trans well法觀察不同濃度VEGF165作用下體外培養(yǎng)的經(jīng)MAB3572預(yù)處理或未預(yù)處理的隆突部細(xì)胞增殖、粘附和遷移能力的變化。以25ng/ml VEGF165刺激MAB3572預(yù)處理或未預(yù)處理的隆突部細(xì)胞,以Western-blot法檢測觀察其對VEGFR-2、P38、C-Jun、ERK1/2磷酸化的影響,探討VEGFR-2介導(dǎo)的調(diào)節(jié)細(xì)胞活性的細(xì)胞內(nèi)信號傳導(dǎo)途徑。 第三部分：提取總RNA,以逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(Reverse transcriptpolymerase chain reaction, RT-PCR)檢測人毛囊Artemis mRNA的表達(dá)水平,用免疫熒光法檢測Artemis (Serine516)在人頭皮毛囊、皮脂腺、汗腺等處的表達(dá),以及在小鼠不同毛發(fā)周期毛囊中的表達(dá)情況。利用免疫熒光雙染技術(shù),探討了毛囊隆突部細(xì)胞Artemis (serine516)與增殖、分化、凋亡指標(biāo)的免疫熒光共染情況。 結(jié)果 一、免疫組化發(fā)現(xiàn)人頭皮毛囊表達(dá)VEGFR-2及磷酸化VEGFR-2,小鼠毛囊上VEGFR-2表達(dá)隨毛發(fā)周期發(fā)生改變。 二、VEGFR-2與K16、K14、p-P53、Bax、bcl-2、β-catenin、integrinβ1等熒光雙染重疊良好。在髓質(zhì)、皮質(zhì)區(qū)域與K10、involucrin等熒光雙染重疊良好。 三、VEGFR-2培養(yǎng)的隆突部細(xì)胞的表達(dá)： 1) RT-PCR:體外培養(yǎng)的隆突部細(xì)胞表達(dá)VEGFR-2mRNA, VEGF165促進(jìn)體外培養(yǎng)的隆突部細(xì)胞表達(dá)VEGFR-2mRNA, VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對體外培養(yǎng)的隆突部細(xì)胞表達(dá)VEGFR-2mRNA表達(dá)的上調(diào)。 2) Western-blot:體外培養(yǎng)的隆突部細(xì)胞在蛋白質(zhì)水平表達(dá)VEGFR-2。VEGF162促進(jìn)體外培養(yǎng)的隆突部細(xì)胞表達(dá)VEGFR-2, VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對體外培養(yǎng)的隆突部細(xì)胞表達(dá)VEGFR-2表達(dá)的上調(diào)。 四、VEGF165促進(jìn)體外培養(yǎng)的隆突部細(xì)胞的增殖、異質(zhì)性粘附、遷移能力,抑制同質(zhì)性粘附能力。 五、VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對體外培養(yǎng)的隆突部細(xì)胞增殖、異質(zhì)性粘附、遷移能力的促進(jìn)及同質(zhì)性粘附能力抑制。 六、VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對體外培養(yǎng)的隆突部細(xì)胞促進(jìn)表達(dá)整合素β1及Artemis (Serine516),抑制表達(dá)Lgr6。 七、VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對體外培養(yǎng)的隆突部細(xì)胞表達(dá)K16、K19、K14等的促進(jìn)作用。 八、VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對體外培養(yǎng)的隆突部細(xì)胞表達(dá)K10的抑制作用。 九、VEGF165促進(jìn)體外培養(yǎng)的隆突部細(xì)胞VEGFR-2、c-Jun、ERK1/2、p38的磷酸化及P-catenin向細(xì)胞核內(nèi)轉(zhuǎn)位,VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對隆突部細(xì)胞VEGFR-2、c-Jun、ERK1/2、p38的磷酸化作用及β-catenin向細(xì)胞核內(nèi)轉(zhuǎn)位。 十、VEGF165促進(jìn)體外培養(yǎng)的隆突部細(xì)胞Artemis在serine516位點(diǎn)的磷酸化,VEGFR-2中和性抗體MAB3572能夠拮抗VEGF165對隆突部細(xì)胞Artemis在serine516位點(diǎn)的磷酸化。 十一Artemis (serine516)的表達(dá)方式隨毛發(fā)周期改變。在人頭皮生長期毛囊中的上半部分上皮細(xì)胞與表皮強(qiáng)陽性表達(dá)Artemis (serine516),在汗腺及皮脂腺也可見Artemis (serine516)表達(dá)。 十二、Artemis (serine516)與K16熒光雙染重疊良好,但是與K10、p-P53、 Bax、bcl-2、K14、Ki67等信號表達(dá)趨勢相反；與c-myc、p21表達(dá)位置緊鄰,信號表達(dá)趨勢相一致。 結(jié)論 一、毛囊上皮包括隆突部細(xì)胞在mRNA和蛋白質(zhì)水平上均表達(dá)VEGFR-2. 二、VEGFR-2參與介導(dǎo)毛囊隆突部細(xì)胞的增殖、遷移、粘附、分化。 三、VEGFR-2參與毛囊的生長、分化過程及毛發(fā)周期調(diào)控；VEGFR-2對毛囊的調(diào)控可能與β-catenin相關(guān)。 四、VEGFR-2發(fā)揮生物學(xué)效應(yīng)部分通過Artemis的磷酸化,發(fā)揮對毛囊生物學(xué)活性的調(diào)節(jié)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R758.71

【共引文獻(xiàn)】

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