本文關鍵詞：羧甲基殼聚糖膜聯(lián)合培養(yǎng)人表皮細胞的實驗研究　出處：《山東大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： 白癜風 角質形成細胞 黑素細胞 羧甲基殼聚糖

【摘要】：研究背景及目的白癜風是一種常見的獲得性色素脫失性疾病,是由多種內外因素聯(lián)合作用導致的黑素細胞的破壞。對于穩(wěn)定期的白癜風患者,外科治療已成為一種臨床常規(guī)治療方式,主要包括自體皮片移植、表皮移植、自體非培養(yǎng)表皮細胞懸液移植以及自體培養(yǎng)黑素細胞移植等,近幾年自體培養(yǎng)黑素細胞移植在臨床中逐漸得到廣泛應用,然而對于身體的彎曲部位往往出現(xiàn)復色不均勻及細胞丟失的情況。為此本研究希望找到一種可以適合表皮細胞定植的材料來作為細胞移植的載體。羧甲基殼聚糖是一種殼聚糖的衍生物,因其具有良好的水溶性、組織相容性、可吸收性以及無毒性。,在組織工程及制藥行業(yè)得到了廣泛應用。本實驗以羧甲基殼聚糖膜為載體,將體外培養(yǎng)的黑素細胞及角質形成細胞種植于膜的表面,同時選取含有不同濃度的羧甲基殼聚糖溶液的培養(yǎng)基進行培養(yǎng),觀察并檢測角質形成細胞及黑素細胞的形態(tài)及功能的變化,探討羧甲基殼聚糖對黑素細胞及角質形成細胞的影響。本研究旨在檢驗羧甲基殼聚糖膜是否適合人表皮細胞的生長,為細胞移植治療白癜風提供一種安全有效的細胞載體。方法采用KM角質細胞培養(yǎng)基和M254培養(yǎng)基分離培養(yǎng)正常人角質形成細胞及黑素細胞,采用抗人HMB45單克隆抗體通過免疫組化法對培養(yǎng)的黑素細胞進行鑒定；倒置顯微鏡觀察羧甲基殼聚糖膜對角質形成細胞及黑素細胞的形態(tài)的影響；MTT法和NaOH裂解法檢測羧甲基殼聚糖膜及不同濃度的羧甲基殼聚糖溶液對表皮細胞的增殖及黑素合成的影響；多巴氧化分析黑素細胞酪氨酸酶活性的變化；蛋白印跡法檢測羧甲基殼聚糖膜上黑素細胞的TRP-1及TRP-2的表達情況。結果1.角質形成細胞及黑素細胞種植于羧甲基殼聚糖膜6小時以后,貼壁細胞數(shù)量較普通培養(yǎng)皿組少；培養(yǎng)3天后觀察角質形成細胞及黑素細胞分布均勻,且黑素細胞樹枝狀突起伸展充分,與普通培養(yǎng)皿組無明顯差異。2.含不同濃度羧甲基殼聚糖溶液的細胞培養(yǎng)基(0.5%,0.25%,0.125%,0.0625%)培養(yǎng)表皮細胞,于第3天、7天、10天細胞的增殖情況較普通培養(yǎng)基組差異無統(tǒng)計學意義(P0.05)。3.羧甲基殼聚糖膜上的表皮細胞在第3天及第7天時,細胞的增殖情況、黑素細胞合成黑素能力較普通培養(yǎng)基組差異無統(tǒng)計學意義(P0.05)；于第10天時觀察由于羧甲基殼聚糖膜的溶解,細胞增殖開始出現(xiàn)脫落,MTT值明顯降低。4.羧甲基殼聚糖膜上的黑素細胞在第3天及第7天時,檢測黑素細胞的黑素含量及酪氨酸酶活性均與普通培養(yǎng)皿組比較差異無統(tǒng)計學意義(P0.05)。5.羧甲基殼聚糖膜上的黑素細胞正常表達TRP-1及TRP-2。結論羧甲基殼聚糖溶液及羧甲基殼聚糖膜與人表皮細胞具有較好的組織相容性,能很好地維持角質形成細胞及黑素細胞的生長及功能。
[Abstract]:Background and objective vitiligo is a common acquired disease of pigmented loss of pigment, which is the damage of melanocytes caused by a combination of various internal and external factors. For stable vitiligo patients, surgical treatment has become a routine clinical treatment, including autologous skin graft, skin transplantation, autologous noncultured epidermal cell suspension transplantation and autologous melanocyte transplantation in recent years, autologous melanocyte transplantation has been widely used in clinic, however for the bending parts of the body are often complex and uneven color loss of cells. Therefore, we hope to find a kind of material suitable for epidermal cell colonization as the carrier of cell transplantation. Carboxymethyl chitosan is a derivative of chitosan, which has good solubility in water, histocompatibility, absorbability and innocuity. It has been widely used in the organization engineering and the pharmaceutical industry. In the experiment of carboxymethyl chitosan as the carrier, the in vitro cultured melanocytes and keratinocytes grown in cell surface membrane, culture medium and selection of carboxymethyl chitosan solution containing different concentrations of the culture, to observe and detect changes in cell morphology and function of keratinocytes and melanocytes, to explore the influence of carboxymethyl chitosan to form cells on melanocytes and keratinocytes. The purpose of this study is to test whether carboxymethyl chitosan film is suitable for the growth of human epidermal cells, and provide a safe and effective cell carrier for cell transplantation for vitiligo. Methods KM and M254 were isolated and cultured keratinocytes cultured normal human keratinocytes and melanocytes culture medium, using anti human HMB45 monoclonal antibody on cultured human melanocytes were identified by immunohistochemical method; inverted microscope to form cells and influence the morphology of black pigment cells of carboxymethyl chitosan diagonal matrix; effect of proliferation and black carboxymethyl chitosan membrane was detected by MTT and NaOH lysis method and different concentrations of Carboxymethyl Chitosan on epidermal cell biosynthesis; DOPA oxidation changes of melanocyte tyrosinase activity of carboxymethyl chitosan; Western blot analysis on melanoma cells TRP-1 and TRP-2 expression. Results 1. keratinocytes and melanocytes grown in carboxymethyl chitosan after 6 hours, the number of adherent cells was less than ordinary Petri dish; after 3 days of culture on keratinocytes and melanocytes and melanocyte distribution, dendritic full extension has no obvious difference with ordinary Petri dish group. 2., cell culture medium (0.5%, 0.25%, 0.125%, 0.0625%) containing different concentrations of carboxymethyl chitosan solution cultured epidermal cells, on third days, 7 days, 10 days, the proliferation of cells was not significantly different from that in the normal media group (P0.05). 3. carboxymethyl chitosan membrane on epidermal cells in 7 days and third days, the proliferation of cells, melanocytes synthetic melanocyte ability than ordinary medium group had no significant difference (P0.05); on the tenth day observation due to the dissolution of carboxymethyl chitosan membrane, cell proliferation began to fall the MTT value decreased significantly. There was no significant difference in melanocyte content and tyrosinase activity between melanocytes in 4. carboxymethyl chitosan membrane on third days and 7 days, compared with those in Petri dish group (P0.05). The melanocytes on the 5. carboxymethyl chitosan membrane normally express TRP-1 and TRP-2. Conclusion carboxymethyl chitosan solution and carboxymethyl chitosan membrane have good histocompatibility with human epidermal cells, which can maintain the growth and function of keratinocytes and melanocytes.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R758.41

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本文編號：1338723