羧甲基壳聚糖膜联合培养人表皮细胞的实验研究

发布时间：2017-12-26 20:23

本文关键词：羧甲基壳聚糖膜联合培养人表皮细胞的实验研究　出处：《山东大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的白癜风是一种常见的获得性色素脱失性疾病,是由多种内外因素联合作用导致的黑素细胞的破坏。对于稳定期的白癜风患者,外科治疗已成为一种临床常规治疗方式,主要包括自体皮片移植、表皮移植、自体非培养表皮细胞悬液移植以及自体培养黑素细胞移植等,近几年自体培养黑素细胞移植在临床中逐渐得到广泛应用,然而对于身体的弯曲部位往往出现复色不均匀及细胞丢失的情况。为此本研究希望找到一种可以适合表皮细胞定植的材料来作为细胞移植的载体。羧甲基壳聚糖是一种壳聚糖的衍生物,因其具有良好的水溶性、组织相容性、可吸收性以及无毒性。,在组织工程及制药行业得到了广泛应用。本实验以羧甲基壳聚糖膜为载体,将体外培养的黑素细胞及角质形成细胞种植于膜的表面,同时选取含有不同浓度的羧甲基壳聚糖溶液的培养基进行培养,观察并检测角质形成细胞及黑素细胞的形态及功能的变化,探讨羧甲基壳聚糖对黑素细胞及角质形成细胞的影响。本研究旨在检验羧甲基壳聚糖膜是否适合人表皮细胞的生长,为细胞移植治疗白癜风提供一种安全有效的细胞载体。方法采用KM角质细胞培养基和M254培养基分离培养正常人角质形成细胞及黑素细胞,采用抗人HMB45单克隆抗体通过免疫组化法对培养的黑素细胞进行鉴定；倒置显微镜观察羧甲基壳聚糖膜对角质形成细胞及黑素细胞的形态的影响；MTT法和NaOH裂解法检测羧甲基壳聚糖膜及不同浓度的羧甲基壳聚糖溶液对表皮细胞的增殖及黑素合成的影响；多巴氧化分析黑素细胞酪氨酸酶活性的变化；蛋白印迹法检测羧甲基壳聚糖膜上黑素细胞的TRP-1及TRP-2的表达情况。结果1.角质形成细胞及黑素细胞种植于羧甲基壳聚糖膜6小时以后,贴壁细胞数量较普通培养皿组少；培养3天后观察角质形成细胞及黑素细胞分布均匀,且黑素细胞树枝状突起伸展充分,与普通培养皿组无明显差异。2.含不同浓度羧甲基壳聚糖溶液的细胞培养基(0.5%,0.25%,0.125%,0.0625%)培养表皮细胞,于第3天、7天、10天细胞的增殖情况较普通培养基组差异无统计学意义(P0.05)。3.羧甲基壳聚糖膜上的表皮细胞在第3天及第7天时,细胞的增殖情况、黑素细胞合成黑素能力较普通培养基组差异无统计学意义(P0.05)；于第10天时观察由于羧甲基壳聚糖膜的溶解,细胞增殖开始出现脱落,MTT值明显降低。4.羧甲基壳聚糖膜上的黑素细胞在第3天及第7天时,检测黑素细胞的黑素含量及酪氨酸酶活性均与普通培养皿组比较差异无统计学意义(P0.05)。5.羧甲基壳聚糖膜上的黑素细胞正常表达TRP-1及TRP-2。结论羧甲基壳聚糖溶液及羧甲基壳聚糖膜与人表皮细胞具有较好的组织相容性,能很好地维持角质形成细胞及黑素细胞的生长及功能。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R758.41

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