【摘要】：第一部分骨髓間充質(zhì)干細(xì)胞中環(huán)氧化酶2及聚蛋白多糖酶1沉默與胰島素樣生長(zhǎng)因子1表達(dá)【目的】觀察攜帶基因環(huán)氧化酶2、聚蛋白多糖酶1的sh RNA干擾載體和攜帶胰島素樣生長(zhǎng)因子1的過(guò)表達(dá)慢病毒載體在骨髓間充質(zhì)干細(xì)胞中的表達(dá)情況�！痉椒ā繎�(yīng)用重組慢病毒技術(shù)構(gòu)建攜帶沉默基因環(huán)氧化酶2、聚蛋白多糖酶1、過(guò)表達(dá)基因胰島素樣生長(zhǎng)因子1和綠色熒光蛋白基因的重組慢病毒表達(dá)載體,并用其轉(zhuǎn)染體外培養(yǎng)的第三代人骨髓間充質(zhì)干細(xì)胞(實(shí)驗(yàn)組),并以無(wú)目的基因的慢病毒載體轉(zhuǎn)染人骨髓間充質(zhì)干細(xì)胞和未做處理的人骨髓間充質(zhì)干細(xì)胞分別作為陰性對(duì)照組和空白組進(jìn)行對(duì)比�！窘Y(jié)果】慢病毒轉(zhuǎn)染3d后,熒光顯微鏡下可見(jiàn)人骨髓間充質(zhì)干細(xì)胞發(fā)出綠色熒光,陰性對(duì)照組、實(shí)驗(yàn)組轉(zhuǎn)染效率均達(dá)90%以上。RT-PCR、Western blot和ELISA檢測(cè)結(jié)果顯示,環(huán)氧化酶2和聚蛋白多糖酶1在人骨髓間充質(zhì)干細(xì)胞的基因和蛋白水平有受到了明顯抑制,胰島素樣生長(zhǎng)因子1基因和蛋白水平的表達(dá)均高于陰性對(duì)照組及空白組。【結(jié)論】應(yīng)用慢病毒可在人骨髓間充質(zhì)干細(xì)胞成功沉默環(huán)氧化酶2和聚蛋白多糖酶1基因,同時(shí)將胰島素樣生長(zhǎng)因子1高表達(dá),為系統(tǒng)性治療類風(fēng)濕性關(guān)節(jié)炎帶來(lái)新的希望。第二部分超聲空化效應(yīng)促人骨髓間充質(zhì)干細(xì)胞體內(nèi)歸巢的實(shí)驗(yàn)研究【目的】研究觀察超聲空化效應(yīng)對(duì)慢病毒介導(dǎo)h COX-2 sh RNA、h Aggrecanse-1sh RNA、h IGF-1基因轉(zhuǎn)染人骨髓間充質(zhì)干細(xì)胞移植的可行性及干細(xì)胞在兔關(guān)節(jié)炎模型體內(nèi)歸巢的影響。【方法】關(guān)節(jié)炎模型造模成功的新西蘭兔分為3組:轉(zhuǎn)染基因人骨髓間充質(zhì)干細(xì)胞+微泡+超聲組(實(shí)驗(yàn)組)、轉(zhuǎn)染基因人骨髓間充質(zhì)干細(xì)胞+超聲組(對(duì)照組)、轉(zhuǎn)染基因人骨髓間充質(zhì)干細(xì)胞+無(wú)處理組(空白組),移植細(xì)胞后檢測(cè)兔后肢膝關(guān)節(jié)滑膜組織學(xué)觀察及RT-PCR基因檢測(cè)。【結(jié)果】超聲空化效應(yīng)可以增加慢病毒介導(dǎo)h COX-2 sh RNA、h Aggrecanse-1sh RNA、h IGF-1基因轉(zhuǎn)染人骨髓間充質(zhì)干細(xì)胞在兔關(guān)節(jié)炎模型進(jìn)入靶器官的比例�！窘Y(jié)論】為臨床應(yīng)用骨髓間充質(zhì)干細(xì)胞治療遺傳免疫性疾病提供了有效途徑及實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Part I silencing and expression of insulin-like growth factor 1 in bone marrow mesenchymal stem cells by cyclooxygenase 2 and polyproteoglycan enzyme 1 [objective] to observe the expression of cyclooxygenase 2, polyproteoglycan 1 and sh RNA interference vector. Overexpression of lentivirus vector carrying insulin-like growth factor 1 in bone marrow mesenchymal stem cells. [methods] Recombinant lentivirus technique was used to construct cyclooxygenase 2, polyproteoglycan 1, and excess genes carrying silencing genes. Recombinant lentivirus expression vector expressing insulin-like growth factor 1 and green fluorescent protein gene. The third generation of human bone marrow mesenchymal stem cells (experimental group) were transfected with it and transfected into human bone marrow mesenchymal stem cells and untreated human bone marrow mesenchymal stem cells respectively. The negative control group and the blank group were compared. [results] after 3 days of lentivirus transfection, Fluorescence microscope showed that human bone marrow mesenchymal stem cells emit green fluorescence, but negative control group. The transfection efficiency of the experimental group was above 90%. The results of Western blot and ELISA detection showed that the transfection efficiency of the experimental group was above 90%. The gene and protein levels of human bone marrow mesenchymal stem cells were significantly inhibited by cyclooxygenase 2 and polyproteoglycan 1. The expression of insulin-like growth factor 1 gene and protein were higher than those of negative control group and blank group. [conclusion] Lentivirus can successfully silence cyclooxygenase 2 and polyproteoglycan 1 genes in human bone marrow mesenchymal stem cells. At the same time, the high expression of insulin-like growth factor 1 brings new hope for systemic treatment of rheumatoid arthritis. Part 2: experimental study on the effect of ultrasound cavitation on homing of human bone marrow mesenchymal stem cells in vivo [objective] to study the effect of ultrasonic cavitation on lentivirus mediated transfer of human bone marrow mesenchymal stem cells into h COX-2 sh RNA-hRNA-h IGF-1 gene The feasibility of transplantation and the effect of stem cells on homing in rabbits with arthritis. [methods] New Zealand rabbits with successful arthritis model were divided into three groups: microbubble ultrasound group of transfected human bone marrow mesenchymal stem cells (experiment) The ultrasonic group was transfected with human bone marrow mesenchymal stem cells (control group), and the control group was transfected with human bone marrow mesenchymal stem cells (blank group). The synovial tissue of rabbit hind limb and knee joint and RT-PCR base were detected after transplantation. [results] Ultrasonic cavitation effect could increase the proportion of human bone marrow mesenchymal stem cells transfected with lentivirus-mediated h COX-2 sh RNA-hRNA-h IGF-1 gene into target organs in rabbit arthritis model. [conclusion] it is a clinical application of bone marrow. Mesenchymal stem cells provide an effective approach and experimental basis for the treatment of hereditary immune diseases.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 趙勇,覃榮周,李海;細(xì)胞凋亡與骨關(guān)節(jié)炎[J];阿壩師范高等專科學(xué)校學(xué)報(bào);2004年04期

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本文編號(hào)：2196801