本文選題：慢病毒　切入點(diǎn)：基因轉(zhuǎn)染　出處：《福建醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】 1.構(gòu)建骨形態(tài)發(fā)生蛋白-2（BMP-2）重組慢病毒表達(dá)載體； 2.TGF-β1、BMP-2重組慢病毒轉(zhuǎn)染BMSCs后向成骨、軟骨細(xì)胞的分化,為探索體外組織工程化骨軟骨的構(gòu)建提供新的思路。 【方法】 1.構(gòu)建攜帶BMP-2基因的重組慢病毒表達(dá)載體：將提取的BMP-2cDNA包裝至慢病毒表達(dá)載體，在脂質(zhì)體Lipofectamine2000作用下轉(zhuǎn)染293T細(xì)胞，通過(guò)酶切電泳分析和及DNA測(cè)序的方法對(duì)重組DNA進(jìn)行鑒定，RT-PCR測(cè)定病毒滴度；Westernblot法檢測(cè)所構(gòu)建質(zhì)粒能在293T細(xì)胞中表達(dá)活性。 2.以BMP-2重組慢病毒載體轉(zhuǎn)染BMSCs，通過(guò)熒光表達(dá)法判定感染復(fù)數(shù)（Multiplicitiesofinfection,MOI），免疫組化、Real-timePCR、Westernblot和酶聯(lián)免疫吸附試驗(yàn)(Enzymelinkedimmunosorbentassay,ELISA)判定感染后的BMSCs中BMP-2的表達(dá)情況；MTT法檢測(cè)其轉(zhuǎn)染后的增殖活性；ALP活性檢測(cè)、堿性磷酸酶染色和茜素紅染色觀察礦化結(jié)節(jié)形成能力對(duì)其成骨分化能力進(jìn)行分析；以TGF-β1重組慢病毒載體轉(zhuǎn)染BMSCs，經(jīng)一段時(shí)間的培養(yǎng)后，II型膠原免疫組織化學(xué)染色及RT-PCR法檢測(cè)經(jīng)TGF-β1轉(zhuǎn)染后BMSCs的成軟骨分化能力。 【結(jié)果】 1.成功構(gòu)建BMP-2重組慢病毒表達(dá)載體，經(jīng)酶切及測(cè)序結(jié)果完全正確，轉(zhuǎn)染293T細(xì)胞后獲得滴度為4.07×108/ml慢病毒濃縮液。 2.BMP-2重組慢病毒可高效轉(zhuǎn)染BMSCs，最佳MOI值100，轉(zhuǎn)染后BMSCs可高效穩(wěn)定表達(dá)BMP-2RAN及蛋白；MTT法檢測(cè)顯示轉(zhuǎn)染后BMSCs的生長(zhǎng)增殖明顯增強(qiáng)；堿性磷酸酶染色、堿性磷酸酶活性檢測(cè)及鈣結(jié)節(jié)染色發(fā)現(xiàn)轉(zhuǎn)染兩周后堿性磷酸酶和鈣結(jié)節(jié)表達(dá)陽(yáng)性；免疫組織化學(xué)染色及RT-PCR法發(fā)現(xiàn)TGF-β1轉(zhuǎn)染后的BMSCs可表達(dá)II型膠原蛋白及II型膠原RNA。 【結(jié)論】 成功構(gòu)建BMP-2重組慢病毒表達(dá)載體，轉(zhuǎn)染BMSCs后可持續(xù)高效表達(dá)BMP-2RAN及蛋白；BMSCs經(jīng)BMP-2、TGF-β1慢病毒轉(zhuǎn)染后，，經(jīng)一定時(shí)間的培養(yǎng)后可誘導(dǎo)其向成骨、軟骨細(xì)胞分化。
[Abstract]:[purpose]. 1.Recombinant lentivirus expression vector of bone morphogenetic protein-2BMP-2 was constructed. 2. The recombinant lentivirus TGF- 尾 1 and BMP-2 were transfected into BMSCs and differentiated into osteoblasts and chondrocytes, which provided a new idea for the construction of tissue engineered bone cartilage in vitro. [methods]. 1. Construction of recombinant lentivirus expression vector carrying BMP-2 gene: the extracted BMP-2cDNA was packaged into the lentivirus expression vector and transfected into 293T cells under the action of liposome Lipofectamine2000. The recombinant DNA was identified by restriction endonuclease electrophoresis and DNA sequencing. RT-PCR was used to detect the expression activity of the constructed plasmid in 293T cells by Western blot. 2. The BMP-2 recombinant lentivirus vector was used to transfect BMSCs. The expression of BMP-2 in infected BMSCs was determined by fluorescence expression method. The expression of BMP-2 in infected BMSCs was determined by Western blot and enzyme-linked immunosorbent assay (Elisa). Alkaline phosphatase staining and alizarin red staining were used to observe the ability of mineralized nodule formation to analyze its osteogenic differentiation ability. BMSCs were transfected with TGF- 尾 1 recombinant lentivirus vector. After a period of culture, the ability of cartilage differentiation of BMSCs transfected with TGF- 尾 1 was detected by immunohistochemical staining of type II collagen and RT-PCR method. [results]. 1.Recombinant lentivirus expression vector of BMP-2 was successfully constructed. The results of restriction endonuclease digestion and sequencing were correct. After transfection into 293T cells, the titer of lentivirus concentrate was 4.07 脳 10 8 / ml. 2. BMP-2 recombinant lentivirus could efficiently transfect BMSCs, the best MOI value was 100. After transfection, BMSCs could express BMP-2RAN efficiently and stably. Alkaline phosphatase activity and calcium nodule staining showed that the expression of alkaline phosphatase and calcium nodule was positive after two weeks of transfection, and the BMSCs transfected with TGF- 尾 1 could express type II collagen and type II collagen RNAA by immunohistochemical staining and RT-PCR method. [conclusion]. The recombinant lentivirus expression vector of BMP-2 was successfully constructed. After transfection of BMSCs, BMP-2RAN and BMSCs could be induced to differentiate into osteoblasts and chondrocytes after transfection with BMP-2TGF- 尾 1 lentivirus.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329


本文編號(hào)：1627081