携带肝细胞生长因子基因的软骨细胞生物学特性的实验研究

发布时间：2018-03-16 21:26

本文选题：软骨细胞　切入点：肝细胞生长因子　出处：《兰州大学》2012年硕士论文　论文类型：学位论文

【摘要】：耳廓在人体上仅仅占据很少数的总体表面积,但耳廓却是人体外部最复杂的三维立体结构之一。目前国内文献对先天性外中耳畸形发病率报道为1/7000,男女之比约2：1,双侧者占10%,且右侧畸形较多。目前小耳、耳畸形的重建及创伤后耳的重建仍然是耳鼻喉科医生面临的最大挑战。多年来耳廓软骨的最佳取代物为左冠状动脉前区域的肋软骨,1971年Tanzer提出运用肋软骨作为耳廓支架并分四个阶段进行耳廓重建手术,随着术式越来越标准化,现被大多数国家用于耳廓重建手术[2-7]。然而自体软骨移植后易吸收,同时手术也有着许多不可避免的问题,如手术瘢痕、创伤疼痛及气胸等情况。金属和有机材料支架经历从最初的铁铜合金到乳胶和塑料等,以及后来使用的硅胶,均因为无法满足对身体无不良反应、可塑性及生物相容性原则而未得到广泛的应用。随着组织工程技术的不断发展,通过生物组织工程软骨再塑耳廓成为耳廓重建的目标。这其中由于自身软骨组织取材少难以达到所需细胞数量,且体外培养的软骨细胞由于软骨细胞自身的性质易发生“去分化”不能达到组织工程软骨的需要,从而限制了软骨组织工程耳廓重建的发展。本研究将肝细胞生长因子(HGF)利用腺病毒表达载体转染于体外培养的新西兰大白兔耳廓软骨细胞,观察携带HGF基因的耳廓软骨细胞的生物学特性,为后期利用此类细胞应用于构建耳廓软骨组织工程的研究打下基础。第一章兔耳廓软骨细胞的分离及培养本部分的研究目的主要是探讨兔耳廓软骨细胞分离培养的方法和生物学特性。主要通过机械法及酶消化法从2周龄新西兰大白兔的耳廓软骨中分离获得软骨细胞并进行原代和传代培养。在倒置显微镜下观察细胞形态及其生长情况,并绘制生长曲线,使用甲苯胺蓝异染染色、免疫组化染色等了解其生物学特性。结果显示：兔耳廓软骨细胞体外原代培养形成单层细胞的时间为1周左右,传代培养时间约3至5天。原代细胞以类圆形或多角形形态为主,随着传代的进行,细胞形态向纤维样细胞改变并失去其表型特征。甲苯胺蓝染色证实细胞可合成蛋白多糖,异染反应主要集中在细胞集落样生长区,异染程度以前三代培养明显,随后逐渐减弱。机械法和酶消化法相结合分离获得的细胞活性率达85%以上,Ⅱ型胶原免疫组化染色可见细胞中Ⅱ型胶原含量随着体外培养传代而减少,到5代以后Ⅱ型胶原含量几乎完全丧失。研究结论：采用机械法与酶消化法结合体外培养兔耳廓软骨细胞可获得活性较高的软骨细胞,随着体外传代培养,耳廓软骨的Ⅱ型胶原含量逐渐减少,细胞形态改变,软骨组织的生物学特性丧失。第二章Ad-HGF对兔耳廓软骨细胞的转染表达及增殖效应本部分的研究目的主要是通过观察腺病毒携带的肝细胞生长因子(Ad-HGF)对兔耳廓软骨细胞的转染表达及增殖效应。实验以不同感染强度(50,100,200)的腺病毒携带的荧光蛋白(Ad-GFP)转染体外培养的新西兰大白兔耳廓软骨细胞,感染后72h用流式细胞仪检测转染效率。并以最佳感染强度的Ad-HGF转染兔耳廓软骨细胞,ELISA方法检测转染48h,72h后细胞上清中HGF的表达水平。MTT法绘制生长曲线,使用Ⅱ型胶原的免疫组化染色了解转染肝细胞生长因子基因的耳廓软骨细胞的生物学特性。实验结果示：Ad-GFP感染强度(MOI)为100时,转染效率达50%。以MOI=100的Ad-HGF转染细胞48h,72h上清中HGF的表达分别为(513±20.77)pg/ml和(312.7±23.37) pg/ml。Ad-HGF转染的三代细胞生长曲线较耳廓软骨三代细胞的曲线前移、抬高。Ⅱ型胶原的免疫组化阳性细胞比值在三代细胞,Ad-HGF转染的三代细胞中分别为(0.652±0.0278),(0.682±0.039),p0.05,无差异性；在兔耳廓软骨五代细胞,Ad-HGF转染的五代细胞中分别为(0.23±0.0469),(0.35±0.0367),p0.05。实验结果示：Ad-HGF可有效转染兔耳廓软骨细胞且持续表达HGF, HGF对体外培养的兔耳廓软骨细胞有促增殖、维持其生物学特性的作用。
[Abstract]:Only a handful of auricle occupy the overall surface area in the body, but one of the most complicated three-dimensional structure of the auricle is outside the human body. At present the literature on congenital external and middle ear malformation incidence reported for 1/7000, the ratio of men and women were 2:1, accounted for 10%, and more. The small right deformity of ear, ear reconstruction reconstruction and ear malformation after trauma is still the biggest challenge facing the doctor working in the Department of ENT. The best substitute for many years for the auricular cartilage of left coronary artery in the area of anterior costal cartilage, the 1971 Tanzer proposed the use of costal cartilage as auricle and four stages of auricle reconstruction surgery, with operation more and more standard, are most the state for auricle reconstruction surgery [2-7]. however after autologous cartilage transplantation is easy to absorb, and operation there are many unavoidable problems, such as surgical scar, wound pain situation and pneumothorax. Genus and organic material support from the original iron copper alloy to latex and plastic, and then use the silica gel, were unable to meet because no adverse reaction to the body, plasticity and biocompatibility principle has not been widely used. With the development of tissue engineering technology, through the biological tissue engineering cartilage remodeling auricle become of auricular reconstruction goal. This one because of its cartilage tissues were less difficult to reach the required number of cells, and in vitro cultured chondrocytes of cartilage cells due to the nature of their easy "dedifferentiated" can not meet the need of cartilage tissue engineering group, which limits the development of cartilage tissue engineering auricle reconstruction. The liver cell growth factor (HGF) by adenovirus expression vector was transfected into cultured auricle chondrocytes of New Zealand rabbits were observed, carrying HGF gene of auricle cartilage cells Biological characteristics are the basis for the later use of such cells in the construction of auricular cartilage tissue engineering.
Chapter 1 Isolation and culture of rabbit auricle cartilage cells
The research purpose of this part is mainly to explore the rabbit auricle cartilage cell separation methods and biological characteristics of culture. Mainly from the 2 week old New Zealand rabbit auricle cartilage isolated by mechanical method and enzyme digestion method to obtain cartilage cells and cultured. Cell morphology was observed under inverted microscope and the growth, and draw the growth curve, using toluidine blue staining, immunohistochemical staining and observe its biological characteristics. The results show that: the in vitro cultured rabbit auricle cartilage cells form a monolayer cell for about 1 weeks, subculture time is about 3 to 5 days. The primary cells with round or polygonal shape, with the passage of cell morphology of fibroblast like cells, to change and lose their phenotypic characteristics. Toluidine blue staining confirmed that cells can synthesize proteoglycan, metachromatic reaction mainly concentrated in the colony like Long before the three degree area, metachromatic cultured, then gradually decreased. The mechanical method and enzyme digestion method combined with the separation of cells the rate reached more than 85%, type II collagen immunohistochemical staining of type II cells in collagen content decreased with in vitro, to the 5 generation in type II collagen content almost completely lost. Conclusion: chondrocytes combined with in vitro cultured rabbit auricle cartilage cells can obtain higher activity by mechanical method and enzyme digestion method, with in vitro culture, type II collagen content of auricle cartilage decreased, cell morphological change, loss of biological characteristics of cartilage tissue.
The transfection expression and proliferation effect of second chapter Ad-HGF on rabbit auricular cartilage cells
The research purpose of this part is mainly through the observation of adenovirus carrying hepatocyte growth factor (Ad-HGF) expression and proliferation of rabbit auricle cartilage cells. Experiments with different intensity of infection (50100200) adenovirus carrying fluorescent protein (Ad-GFP) transfection in vitro rabbit auricle cartilage cells after 72h infection the transfection efficiency by flow cytometry. And with the best infection intensity of Ad-HGF transfected rabbit auricle cartilage cells, ELISA were detected by 48h, draw the growth curve of the expression level of.MTT 72h in HGF cell supernatant method, the biological characteristics of type II collagen by immunohistochemical staining to understand the transfection of hepatocyte growth factor gene of auricle cartilage cells. The experimental results showed that Ad-GFP infection intensity (MOI) is 100, the transfection efficiency of 50%. Ad-HGF 48h MOI=100 transfected cells, expression of 72h was HGF respectively (513. 20.77) and pg/ml (312.7 + 23.37) curve forward, pg/ml.Ad-HGF transfected cells of the three generation growth curve with auricular cartilage cells of the three generation. The elevation of type II collagen immunohistochemical positive cells ratio in the cells of the three generation, the three generation of cells transfected with Ad-HGF were (0.652 + 0.0278), (0.682 + 0.039) P0.05, there was no difference; in rabbit auricle cartilage cells of the five generation, the five generation of cells transfected with Ad-HGF were (0.23 + 0.0469), (0.35 + 0.0367) p0.05., experimental results showed that Ad-HGF can transfect rabbit auricle cartilage cells and the expression of HGF, HGF on proliferation of cultured rabbit auricle the cartilage cells maintain their biological characteristics.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764

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