本文選題：毛囊　切入點：間充質(zhì)干細胞　出處：《吉林大學》2014年博士論文

【摘要】：組織工程血管的構建是組織工程研究領域的重點和難點，其成功構建不僅為可供移植的材料提供了新的來源，更為臨床上諸多心血管疾病的患者帶來治愈的希望。本研究擬以人毛囊間充質(zhì)干細胞為種子細胞，以脫細胞胎兒臍動脈為支架材料，，體外構建組織工程血管，所得結果如下： （一）應用拔取成人毛發(fā)的方法，從中分離獲得人毛囊間充質(zhì)干細胞，通過顯微鏡觀察、流式細胞分析的方法，從形態(tài)學、分子標記物方面檢測一般特性；通過誘導成骨、成脂肪及成軟骨檢測其多向分化潛能的生物學行為；通過流式細胞分析、免疫熒光染色及實時定量PCR對人毛囊間充質(zhì)干細胞來源的平滑肌細胞進行鑒定，發(fā)現(xiàn)其表達成熟平滑肌細胞的特異性標志物。從而證明人毛囊間充質(zhì)干細胞可以作為構建組織工程血管的種子細胞。 （二）獲取在臨床上廢棄的胎兒臍動脈，經(jīng)胰蛋白酶和十二烷基硫酸鈉聯(lián)合處理去掉臍動脈的細胞成分作為組織工程血管的支架。組織學觀察發(fā)現(xiàn)該動脈細胞成分已經(jīng)完全去除，但膠原和彈力纖維的結構仍保持完好。掃描電鏡進一步確定纖維互相交織，為以后種子細胞的鋪展、遷徙、分化提供合適的生存環(huán)境。細胞化學及生物化學分析還發(fā)現(xiàn)脫細胞臍動脈細胞外基質(zhì)成分依然存在，膠原蛋白及彈力纖維的含量并未發(fā)生明顯變化。將脫細胞前后的臍動脈進行生物力學的比較發(fā)現(xiàn)脫細胞的臍動脈具有較好的生物力學性質(zhì)�？梢娎门R床廢棄組織，經(jīng)脫細胞處理后作為組織工程血管的支架，具有很好的應用前景。 （三）將人毛囊間充質(zhì)干細胞接種在脫細胞臍動脈支架上構建組織工程血管，組織學確定種子細胞黏附并遷移至血管的中膜，免疫組織化學染色檢測平滑肌標志物α-actin和calponin呈陽性表達。重要的是構建的組織工程血管已經(jīng)表現(xiàn)出對血管活性物質(zhì)如氯化鉀、去甲腎上腺素及硝普鈉的反應活性，從而成功構建了在組織結構和生理功能與天然血管極為接近的組織工程血管。 綜上所述，我們建立了高效的分離、純化、培養(yǎng)及擴增人毛囊間充質(zhì)干細胞的技術體系；制備了具有合理的空間結構及組織相容性的脫細胞動脈支架；成功構建了具有生物活性的組織工程血管，為開展無免疫排斥的個性化血管移植治療打下堅實的實驗基礎。
[Abstract]:The construction of tissue engineering blood vessels is a key and difficult point in the field of tissue engineering. The successful construction of tissue engineering vessels not only provides a new source for transplantable materials, but also brings the hope of cure for many patients with cardiovascular diseases.In this study, human hair follicle mesenchymal stem cells were used as seed cells and acellular fetal umbilical artery as scaffold material to construct tissue engineered blood vessels in vitro. The results were as follows:(1) Human hair follicle mesenchymal stem cells (HMSCs) were isolated from adult hair by the method of extraction of adult hair. The general characteristics of human hair follicle mesenchymal stem cells were detected from morphology and molecular markers through microscope observation and flow cytometry.The biological behaviors of adipogenic and chondrogenic multidirectional differentiation potential were detected, and the smooth muscle cells derived from human hair follicle mesenchymal stem cells were identified by flow cytometry, immunofluorescence staining and real-time quantitative PCR.A specific marker for the expression of mature smooth muscle cells was found.It is shown that human hair follicle mesenchymal stem cells can be used as seed cells to construct tissue engineered blood vessels.(2) the fetal umbilical artery abandoned in clinic was obtained and removed from the umbilical artery by trypsin and sodium dodecyl sulfate as stents for tissue engineering vessels.Histological observation showed that the cellular components of the artery had been completely removed, but the structure of collagen and elastic fibers remained intact.Scanning electron microscopy (SEM) further confirmed that the fibers intertwined with each other, providing a suitable living environment for seed cells to spread, migrate and differentiate in the future.Cytochemistry and biochemistry analysis also showed that extracellular matrix of acellular umbilical artery still existed, and the contents of collagen and elastic fiber did not change obviously.The biomechanical comparison of the umbilical artery before and after acellular showed that the acellular umbilical artery had better biomechanical properties.It can be seen that the scaffold of tissue engineering vessels using clinical discarded tissue and acellular treatment has a good application prospect.(3) Human hair follicle mesenchymal stem cells were inoculated on the scaffold of acellular umbilical artery to construct tissue engineered blood vessels.The expression of 偽 -actin and calponin was detected by immunohistochemical staining.It is important that the constructed tissue engineered blood vessels have shown reactive activity to vasoactive substances such as potassium chloride, norepinephrine and sodium nitroprusside.Thus, tissue engineered blood vessels which are very close to natural blood vessels in tissue structure and physiological function were successfully constructed.To sum up, we established a highly efficient technique system for isolation, purification, culture and amplification of human hair follicle mesenchymal stem cells, and prepared acellular artery stents with reasonable spatial structure and histocompatibility.A bioactive tissue engineering blood vessel was successfully constructed, which laid a solid experimental foundation for individualized vascular transplantation without immune rejection.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R318.08

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