本文選題：人早胚間充質(zhì)干細(xì)胞 + 毛囊��； 參考：《第二軍醫(yī)大學(xué)》2008年博士論文

【摘要】： 目的:建立誘導(dǎo)人早胚間充質(zhì)干細(xì)胞(hMSC)向毛囊上皮細(xì)胞分化的模型,初步實現(xiàn)裸鼠局部皮膚毛發(fā)的生成。分析hMSC向毛囊上皮細(xì)胞分化過程中連接黏附分子1(JAM1)、腫瘤細(xì)胞遷移因子(Tiam1)、非典型性磷酸化的蛋白激酶ζ(p-PKCζ)與極性蛋白3(PAR3)的關(guān)系,分析JAM1、Tiam1與細(xì)胞極性形成的關(guān)系。分析JAM1與毛囊生長方向和細(xì)胞極性形成的關(guān)系。 方法:1.復(fù)蘇hMSC和表皮干細(xì)胞,進行生物學(xué)特性和相關(guān)的安全性檢測,隨后進行大規(guī)模培養(yǎng)擴增。2.分離培養(yǎng)人毛乳頭細(xì)胞(DP)并進行鑒定。3.DP細(xì)胞誘導(dǎo)hMSC,RT-PCR方法檢測誘導(dǎo)后hMSC中DP細(xì)胞特異性分子SCF、ET-1和bFGF表達(dá)的變化。4.DP細(xì)胞誘導(dǎo)表皮干細(xì)胞,RT-PCR和Western-blotting方法檢測誘導(dǎo)后表皮干細(xì)胞中uPA的變化。5.將一定數(shù)量的hMSC以皮下或皮內(nèi)注射的方式移植到裸鼠皮膚組織中,H.E觀察不同時間點毛囊生成情況,免疫組化檢測HLA-1在不同時間點裸鼠新生毛囊中的表達(dá),免疫組化分析角蛋白(K)和SM(smooth muscle)-a-actin在裸鼠新生毛囊中的表達(dá)。采用RNA干擾的方式抑制JAM1在hMSC中的表達(dá),將干擾后的hMSC移植到裸鼠皮內(nèi),后續(xù)實驗方法同正常hMSC移植裸鼠。6.激光共聚焦和Western-Blotting方法檢測hMSC皮內(nèi)移植后不同時間點裸鼠新生毛囊中JAM1、Tiam1、p-PKCζ和PAR3的表達(dá)并初步分析這些分子表達(dá)的位置關(guān)系和時間順序。 結(jié)果:1.hMSC以皮內(nèi)注射的方式移植到裸鼠皮膚組織,可引起裸鼠皮膚局部毛發(fā)的生成。2.激光共聚焦結(jié)果顯示在毛囊生成過程中,JAM1、PAR3和p-PKCζ的表達(dá)有很強的方向性,Tiam1表達(dá)均勻。Western-blotting結(jié)果顯示,JAM1、PAR3、p-PKCζ和Tiam1的表達(dá)量逐漸增加。抑制JAM1在hMSC中的表達(dá),其在裸鼠體內(nèi)形成毛囊的能力下降。3.在成熟毛囊中,JAM1、PAR3和p-PKCζ分布在毛囊的根鞘處。4.DP細(xì)胞可誘導(dǎo)表皮干細(xì)胞往毛囊方向分化,可誘導(dǎo)hMSC表達(dá)DP細(xì)胞的特異性分子并表現(xiàn)DP細(xì)胞特有的凝集樣生長。 結(jié)論:hMSC可誘導(dǎo)并參與裸鼠毛發(fā)的生成。在毛發(fā)生成過程中,JAM1、PAR3、p-PKCζ的分布和位置關(guān)系與毛囊的生成方向和體積增加有密切關(guān)系,體現(xiàn)明顯的方向性。p-PKCζ介導(dǎo)PAR3與Tiam1的反應(yīng)。JAM1感受局部微環(huán)境中的信息而引發(fā)毛囊的生成和拓展,Tiam1作為極性相關(guān)分子的反應(yīng)平臺而維持毛囊上皮細(xì)胞的極性。
[Abstract]:Aim: to establish a model of inducing human early embryonic mesenchymal stem cells (hMSCs) to differentiate into hair follicle epithelial cells (HSCs), and to preliminarily realize the formation of local skin hair in nude mice. To analyze the relationship between hMSC and polar protein 3PAR3 during the differentiation of hMSC into hair follicle epithelial cells, such as adhesion molecule 1, tumor cell migration factor Tiam1, atypical phosphorylated protein kinase 味 -p-PKC 味 and polar protein 3PAR3, and the relationship between JAM1Tiam1 and the formation of cell polarity. The relationship between JAM1 and hair follicle growth direction and cell polarity formation was analyzed. Method 1: 1. HMSC and epidermal stem cells were resuscitated for biological characteristics and related safety tests, followed by large-scale culture and amplification. 2. 2. Isolation and culture of human dermal papilla cells (DPN) and identification of. 3. The expression of DP cell specific molecules SCFtoET-1 and bFGF in hMSC were detected by RT-PCR and Western-blotting respectively. 4. DP cells induced epidermal stem cells were detected by RT-PCR and Western-blotting. Changes of uPA in cells. A certain amount of hMSC was transplanted into the skin tissue of nude mice by subcutaneous or intradermal injection to observe the hair follicle formation at different time points, and the expression of HLA-1 in the newborn hair follicles of nude mice at different time points was detected by immunohistochemistry. The expression of keratin K) and SM(smooth muscle)-a-actin in newborn hair follicles of nude mice was analyzed by immunohistochemistry. The expression of JAM1 in hMSC was inhibited by RNA interference, and the interfering hMSC was transplanted into the skin of nude mice. Laser confocal and Western-Blotting methods were used to detect the expression of JAM1Tiam1 p-PKC 味 and PAR3 in the hair follicles of nude mice at different time points after intradermal hMSC transplantation. Results 1.hMSC transplanted into skin tissue of nude mice by intradermal injection could induce local hair formation in nude mice. The results of laser confocal analysis showed that the expression of PAR3 and p-PKC 味 in hair follicles showed a strong directionality. Western-blotting results showed that the expressions of PAR3p-PKC 味 and Tiam1 increased gradually. Inhibition of JAM1 expression in hMSC decreased its ability to form hair follicles in nude mice. In mature hair follicles, JAM1PAR3 and p-PKC 味 were distributed in the root sheath of hair follicles. 4. DP cells could induce epidermal stem cells to differentiate into hair follicles, induce hMSC to express specific molecules of DP cells and express the specific agglutinating growth of DP cells. ConclusionTwo h MSC can induce and participate in hair formation in nude mice. The distribution and location of PAR3 p-PKC 味 during hair formation are closely related to the direction and volume of hair follicle formation. It showed obvious directionality. P-PKC 味 mediated the reaction between PAR3 and Tiam1. JAM1 sensed the information in local microenvironment, which induced the formation of hair follicles and expanded Tiam1 as a reaction platform of polarity related molecules to maintain the polarity of hair follicle epithelial cells.
【學(xué)位授予單位】：第二軍醫(yī)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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