發(fā)布時(shí)間：2018-05-27 19:35

  本文選題：細(xì)胞片 + CDMP1��； 參考：《遼寧醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的 構(gòu)建軟骨源性形態(tài)發(fā)生蛋白-1（cartilage derived morphogeneticprotein-1,CDMP1）轉(zhuǎn)基因的細(xì)胞片，并對(duì)其生物活性進(jìn)行鑒定，運(yùn)用細(xì)胞膜片技術(shù)（cell sheet technology，CST）與組織工程技術(shù)相結(jié)合的方法探討新型無(wú)支架組織工程細(xì)胞片在兔甲狀軟骨缺損中的修復(fù)效果，以期為軟骨重建及再生醫(yī)學(xué)提供一個(gè)新的思路和方法。 方法 取1月齡左右的新西蘭白兔，無(wú)菌條件下分離兔雙側(cè)股骨，優(yōu)化方案分離純化BMSCs，體外擴(kuò)增培養(yǎng)傳代。取第4代兔BMSCs分別給予以下三種處理：（A）轉(zhuǎn)染Ad-CMV-hCDMP1-IRES-eGFP組；（B）轉(zhuǎn)染Ad-CMV-eGFP組；（C）BMSCs組，行MTT檢測(cè)細(xì)胞增殖活性，連續(xù)培養(yǎng)14d后接種至溫度敏感性培養(yǎng)皿制備細(xì)胞片，行Real time PCR、Western blot及組織學(xué)方法檢測(cè)轉(zhuǎn)基因細(xì)胞片中糖胺聚糖（Glycosaminoglycan,GAG）、CDMP1及Π型膠原的表達(dá)。將上述3組組織工程細(xì)胞片移植入兔甲狀軟骨缺損處，，常規(guī)喂養(yǎng)，于術(shù)后4周和8周無(wú)痛處死，行大體和組織切片觀察。 結(jié)果 體外培養(yǎng)的BMSCs增殖活躍，腺病毒介導(dǎo)的hCDMP1轉(zhuǎn)基因并未對(duì)其增殖活性產(chǎn)生影響，采用溫度敏感性培養(yǎng)皿體外成功收獲了完整的細(xì)胞片結(jié)構(gòu)，且通過(guò)CST成功構(gòu)建了多層三維立體組織工程細(xì)胞片，有一定韌性和可操作性。Real time PCR和Western blot可檢測(cè)到CDMP1和Π型膠原的表達(dá)。HE染色顯示A組細(xì)胞片基質(zhì)豐富，阿利新藍(lán)染色呈均一藍(lán)色異染，Π型膠原表達(dá)陽(yáng)性，B、C組細(xì)胞片未見(jiàn)明顯特異性藍(lán)染顆粒，Π型膠原表達(dá)陰性，A組與B、C組比較有統(tǒng)計(jì)學(xué)差異（P0.05）。體外實(shí)驗(yàn)結(jié)果顯示，A組軟骨缺損修復(fù)區(qū)有類軟骨樣細(xì)胞生長(zhǎng)，質(zhì)韌，表面光滑， GAG和Π型膠原表達(dá)陽(yáng)性，而B組和C組基本為纖維結(jié)締組織和肌肉組織填充，表面凹陷粗糙，GAG和Π型膠原表達(dá)陰性。 結(jié)論 1、體外成功構(gòu)建CDMP1轉(zhuǎn)基因細(xì)胞片。 2、細(xì)胞片對(duì)修復(fù)兔甲狀軟骨的缺損有促進(jìn)作用。
[Abstract]:Purpose The chondrogenic morphogenetic protein-1 derived cartilage derived CDMP1 was constructed and its biological activity was identified. In order to provide a new idea and method for cartilage reconstruction and regenerative medicine, the method of cell sheet technology combined with tissue engineering technique was used to study the repair effect of new scaffolding tissue engineering cell piece in rabbit thyroid cartilage defect. Method BMSCs were isolated from New Zealand white rabbits at the age of about 1 month under aseptic condition. BMSCs were isolated and purified by optimization method and cultured in vitro. The fourth generation of rabbit BMSCs was divided into three groups: Ad-CMV-hCDMP1-IRES-eGFP group was transfected with the following three treatments: Ad-CMV-hCDMP1-IRES-eGFP group (BMSCs) were transfected into Ad-CMV-eGFP group. The proliferative activity of Ad-CMV-eGFP group was measured by MTT. After 14 days of continuous culture, the cells were cultured in a temperature-sensitive dish to prepare the cells. The expression of glycosaminoglycan (Gago) CDMP1 and collagen 蟺 in transgenic cells was detected by Real time PCR Western blot and histological method. The above three groups of tissue engineering cells were transplanted into the defect of thyroid cartilage in rabbits. The rabbits were given routine feeding and were killed painlessly at 4 and 8 weeks after operation. The gross and histological sections were observed. Result The proliferation of BMSCs in vitro was active, and the hCDMP1 gene mediated by adenovirus had no effect on its proliferative activity. The whole cell piece structure was successfully harvested in vitro by using temperature-sensitive culture dish. The multilayer three-dimensional tissue engineering cell pieces were successfully constructed by CST. The results showed that the cells in group A were rich in matrix. Real time PCR and Western blot could detect the expression of CDMP1 and 鈪

本文編號(hào)：1943486