本文選題：軟骨細(xì)胞　切入點(diǎn)：肝細(xì)胞生長因子　出處：《蘭州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：耳廓在人體上僅僅占據(jù)很少數(shù)的總體表面積,但耳廓卻是人體外部最復(fù)雜的三維立體結(jié)構(gòu)之一。目前國內(nèi)文獻(xiàn)對(duì)先天性外中耳畸形發(fā)病率報(bào)道為1/7000,男女之比約2：1,雙側(cè)者占10%,且右側(cè)畸形較多。目前小耳、耳畸形的重建及創(chuàng)傷后耳的重建仍然是耳鼻喉科醫(yī)生面臨的最大挑戰(zhàn)。多年來耳廓軟骨的最佳取代物為左冠狀動(dòng)脈前區(qū)域的肋軟骨,1971年Tanzer提出運(yùn)用肋軟骨作為耳廓支架并分四個(gè)階段進(jìn)行耳廓重建手術(shù),隨著術(shù)式越來越標(biāo)準(zhǔn)化,現(xiàn)被大多數(shù)國家用于耳廓重建手術(shù)[2-7]。然而自體軟骨移植后易吸收,同時(shí)手術(shù)也有著許多不可避免的問題,如手術(shù)瘢痕、創(chuàng)傷疼痛及氣胸等情況。金屬和有機(jī)材料支架經(jīng)歷從最初的鐵銅合金到乳膠和塑料等,以及后來使用的硅膠,均因?yàn)闊o法滿足對(duì)身體無不良反應(yīng)、可塑性及生物相容性原則而未得到廣泛的應(yīng)用。隨著組織工程技術(shù)的不斷發(fā)展,通過生物組織工程軟骨再塑耳廓成為耳廓重建的目標(biāo)。這其中由于自身軟骨組織取材少難以達(dá)到所需細(xì)胞數(shù)量,且體外培養(yǎng)的軟骨細(xì)胞由于軟骨細(xì)胞自身的性質(zhì)易發(fā)生“去分化”不能達(dá)到組織工程軟骨的需要,從而限制了軟骨組織工程耳廓重建的發(fā)展。本研究將肝細(xì)胞生長因子(HGF)利用腺病毒表達(dá)載體轉(zhuǎn)染于體外培養(yǎng)的新西蘭大白兔耳廓軟骨細(xì)胞,觀察攜帶HGF基因的耳廓軟骨細(xì)胞的生物學(xué)特性,為后期利用此類細(xì)胞應(yīng)用于構(gòu)建耳廓軟骨組織工程的研究打下基礎(chǔ)。 第一章兔耳廓軟骨細(xì)胞的分離及培養(yǎng) 本部分的研究目的主要是探討兔耳廓軟骨細(xì)胞分離培養(yǎng)的方法和生物學(xué)特性。主要通過機(jī)械法及酶消化法從2周齡新西蘭大白兔的耳廓軟骨中分離獲得軟骨細(xì)胞并進(jìn)行原代和傳代培養(yǎng)。在倒置顯微鏡下觀察細(xì)胞形態(tài)及其生長情況,并繪制生長曲線,使用甲苯胺藍(lán)異染染色、免疫組化染色等了解其生物學(xué)特性。結(jié)果顯示：兔耳廓軟骨細(xì)胞體外原代培養(yǎng)形成單層細(xì)胞的時(shí)間為1周左右,傳代培養(yǎng)時(shí)間約3至5天。原代細(xì)胞以類圓形或多角形形態(tài)為主,隨著傳代的進(jìn)行,細(xì)胞形態(tài)向纖維樣細(xì)胞改變并失去其表型特征。甲苯胺藍(lán)染色證實(shí)細(xì)胞可合成蛋白多糖,異染反應(yīng)主要集中在細(xì)胞集落樣生長區(qū),異染程度以前三代培養(yǎng)明顯,隨后逐漸減弱。機(jī)械法和酶消化法相結(jié)合分離獲得的細(xì)胞活性率達(dá)85%以上,Ⅱ型膠原免疫組化染色可見細(xì)胞中Ⅱ型膠原含量隨著體外培養(yǎng)傳代而減少,到5代以后Ⅱ型膠原含量幾乎完全喪失。研究結(jié)論：采用機(jī)械法與酶消化法結(jié)合體外培養(yǎng)兔耳廓軟骨細(xì)胞可獲得活性較高的軟骨細(xì)胞,隨著體外傳代培養(yǎng),耳廓軟骨的Ⅱ型膠原含量逐漸減少,細(xì)胞形態(tài)改變,軟骨組織的生物學(xué)特性喪失。 第二章Ad-HGF對(duì)兔耳廓軟骨細(xì)胞的轉(zhuǎn)染表達(dá)及增殖效應(yīng) 本部分的研究目的主要是通過觀察腺病毒攜帶的肝細(xì)胞生長因子(Ad-HGF)對(duì)兔耳廓軟骨細(xì)胞的轉(zhuǎn)染表達(dá)及增殖效應(yīng)。實(shí)驗(yàn)以不同感染強(qiáng)度(50,100,200)的腺病毒攜帶的熒光蛋白(Ad-GFP)轉(zhuǎn)染體外培養(yǎng)的新西蘭大白兔耳廓軟骨細(xì)胞,感染后72h用流式細(xì)胞儀檢測轉(zhuǎn)染效率。并以最佳感染強(qiáng)度的Ad-HGF轉(zhuǎn)染兔耳廓軟骨細(xì)胞,ELISA方法檢測轉(zhuǎn)染48h,72h后細(xì)胞上清中HGF的表達(dá)水平。MTT法繪制生長曲線,使用Ⅱ型膠原的免疫組化染色了解轉(zhuǎn)染肝細(xì)胞生長因子基因的耳廓軟骨細(xì)胞的生物學(xué)特性。實(shí)驗(yàn)結(jié)果示：Ad-GFP感染強(qiáng)度(MOI)為100時(shí),轉(zhuǎn)染效率達(dá)50%。以MOI=100的Ad-HGF轉(zhuǎn)染細(xì)胞48h,72h上清中HGF的表達(dá)分別為(513±20.77)pg/ml和(312.7±23.37) pg/ml。Ad-HGF轉(zhuǎn)染的三代細(xì)胞生長曲線較耳廓軟骨三代細(xì)胞的曲線前移、抬高。Ⅱ型膠原的免疫組化陽性細(xì)胞比值在三代細(xì)胞,Ad-HGF轉(zhuǎn)染的三代細(xì)胞中分別為(0.652±0.0278),(0.682±0.039),p0.05,無差異性；在兔耳廓軟骨五代細(xì)胞,Ad-HGF轉(zhuǎn)染的五代細(xì)胞中分別為(0.23±0.0469),(0.35±0.0367),p0.05。實(shí)驗(yàn)結(jié)果示：Ad-HGF可有效轉(zhuǎn)染兔耳廓軟骨細(xì)胞且持續(xù)表達(dá)HGF, HGF對(duì)體外培養(yǎng)的兔耳廓軟骨細(xì)胞有促增殖、維持其生物學(xué)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.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764

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