【摘要】：目的:1.探索超聲微泡靶向破壞聯(lián)合轉(zhuǎn)染試劑Entranster能否提高aggrecanase-1基因轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞的轉(zhuǎn)染效率;2.制備兔類風(fēng)濕關(guān)節(jié)炎模型,并對其進(jìn)行評估;3.超聲微泡靶向破壞聯(lián)合轉(zhuǎn)染試劑Entranster in vivo介導(dǎo)aggrecanase-1 sh RNA,COX-2 sh RNA,h IGF體內(nèi)轉(zhuǎn)染對類風(fēng)濕性關(guān)節(jié)炎膝關(guān)節(jié)的影響。方法:應(yīng)用全骨髓培養(yǎng)法培養(yǎng)類風(fēng)濕關(guān)節(jié)炎患者骨髓間充質(zhì)干細(xì)胞。將第3代骨髓間充質(zhì)干細(xì)胞接種于24孔板中,共分為4組:1.超聲靶向微泡破壞組(UTMD);2.轉(zhuǎn)染試劑Entranster組(ENTR);3.超聲微泡靶向破壞+轉(zhuǎn)染試劑Entranster組(UTMD+ENTR);4.對照組。轉(zhuǎn)染后第2天、第4天、第6天,在倒置熒光顯微鏡下,觀察aggrecanase sh RNA轉(zhuǎn)染情況;應(yīng)用熒光定量PCR檢測轉(zhuǎn)染后aggrecanase m RNA水平的變化。家兔類風(fēng)濕關(guān)節(jié)炎模型制備:應(yīng)用牛Ⅱ型膠原溶液與完全弗氏佐劑混合成乳劑,取1 ml共分10點(diǎn)注射于家兔肩胛骨區(qū)域。致敏后,膝關(guān)節(jié)注入1ml(5g/L)膠原蛋白溶液,觀察足掌關(guān)節(jié)腫脹程度,對關(guān)節(jié)炎進(jìn)行評分以及病理學(xué)組織檢查。體內(nèi)轉(zhuǎn)染實(shí)驗(yàn):將造模成功的兔類風(fēng)濕關(guān)節(jié)炎模型分為3組:空白對照組:僅在家兔右膝關(guān)節(jié)腔內(nèi)注射等量的PBS;單純質(zhì)粒組:取200ul Aggrecanase-2 sh RNA(10ug)、COX-2 sh RNA(10ug)、h IGF(10ug)質(zhì)粒以及100ul PBS注入家兔右膝關(guān)節(jié)腔內(nèi);質(zhì)粒+Entranster-in vivo+超聲微泡組:各取200ul Aggrecanase-2 sh RNA(10ug)、COX-2 sh RNA(10ug)、IGF(10ug)、Entranster-in vivo轉(zhuǎn)染試劑以及100ul六氟化硫微泡液體注入家兔右膝關(guān)節(jié)腔內(nèi),在4.9MHz頻率下,超聲探頭掃右膝關(guān)節(jié)關(guān)節(jié)5分鐘。大體觀察比較各組間關(guān)節(jié)病變及改善情況;病理組織檢查各組間炎細(xì)胞有無減少;體內(nèi)轉(zhuǎn)染4天后,切取各組右膝關(guān)節(jié)滑膜組織,用冰凍切片機(jī)切片,在熒光顯微鏡下觀察各組間基因轉(zhuǎn)染情況;RT-PCR檢測各組滑膜組織COX-2、aggrecanse表達(dá)水平;ELISA法檢測各組右膝關(guān)節(jié)灌洗液h IGF-I含量。結(jié)果:在倒置熒光顯微鏡下觀察UTMD+ENTR組綠色熒光最強(qiáng);UTMD組和ENTR組次之,對照組最暗。轉(zhuǎn)染后各組間比較aggrecanase m RNA表達(dá)水平;UTMD+ENTR組下降最明顯。UTMD聯(lián)合轉(zhuǎn)染試劑Entranster的轉(zhuǎn)染效率高于單純超聲輻照和Entranster轉(zhuǎn)染(P0.01);牛Ⅱ型膠原與完全弗氏佐劑混合乳劑皮下注射并誘導(dǎo)后,膝關(guān)節(jié)關(guān)節(jié)腫脹,關(guān)節(jié)直徑增加,皮膚溫度升高,1周后,病理切片鏡下滑膜組織內(nèi)大量淋巴細(xì)胞浸潤;體內(nèi)實(shí)驗(yàn):單純質(zhì)粒組,關(guān)節(jié)腔注射后,滑膜組織制成冰凍切片后,熒光顯微鏡下觀察熒光微弱,質(zhì)粒+Entranster-in vivo+超聲微泡組,熒光顯微鏡下可見大量熒光彌漫于滑膜組織。Entranster-in vivo+超聲微泡組的COX-2m RNA、aggrecanase m RNA表達(dá)水平明顯低于空包對照和單純質(zhì)粒組;各組間應(yīng)用ELISA法檢測膝關(guān)節(jié)灌洗液h IGF-I含量比較后Entranster-in vivo+超聲微泡組水平最高。關(guān)節(jié)腔注射后兩周滑膜組織HE染色切片觀察,Entranster-in vivo+超聲微泡組炎細(xì)胞數(shù)量較其他兩組減少,但大體外觀見各組的關(guān)節(jié)修復(fù)沒有明顯差別。結(jié)論:UTMD聯(lián)合轉(zhuǎn)染試劑Entranster應(yīng)用可以作為一種新的轉(zhuǎn)染方法,提高基因轉(zhuǎn)染效率;兩種方法聯(lián)合進(jìn)行體內(nèi)轉(zhuǎn)染后,亦能夠在體內(nèi)表達(dá),從而達(dá)到治療的目的。
[Abstract]:Objective: 1. To explore whether ultrasound microbubble targeted destruction combined with transfection agent Entranster can improve the transfection efficiency of aggrecanase-1 gene transfected bone marrow mesenchymal stem cells; 2. To prepare rabbit rheumatoid arthritis model and evaluate it; 3. ultrasound microbubble targeted destruction combined with transfection agent Entranster vivo mediated aggrecanase-1 sh RNA, COX-2 sh RNA Methods: Bone marrow mesenchymal stem cells from patients with rheumatoid arthritis were cultured by whole bone marrow culture. The 3rd generation bone marrow mesenchymal stem cells were inoculated into 24-well plate and divided into four groups: 1. Ultrasound targeted microbubble destruction group (UTMD); 2. Entranster group (ENTR); 3. Ultrasound. Microbubble targeted destruction + transfection agent Entranster group (UTMD + ENTR); 4. Control group. On the 2nd, 4th and 6th day after transfection, aggrecanase sh RNA transfection was observed under inverted fluorescence microscope; the level of aggrecanase m RNA was detected by fluorescence quantitative PCR after transfection. After sensitization, the knee joints were injected with 1 ml (5 g / L) of collagen solution to observe the degree of swelling of the foot joints, score arthritis and pathological examination. Blank control group: only the same amount of PBS was injected into the right knee joint cavity of rabbits; simple plasmid group: 200ul Aggrecanase-2 sh RNA (10ug), COX-2 sh RNA (10ug), h IGF (10ug) plasmid and 100ul PBS were injected into the right knee joint cavity of rabbits; plasmid + Entranster-in vivo + ultrasound microbubbles group: 200ul Aggrecanase-2 sh RNA (10ug), COX-2 sh RNA (10ug), COX-2 sh RNA (10ug), respectively. IGF (10ug), Entranster-in vivo transfection reagent and 100ul sulfur hexafluoride microbubbles were injected into the right knee joint of rabbits. The right knee joint was scanned by ultrasonic probe at 4.9MHz for 5 minutes. The expression of COX-2 and aggrecanse in synovial tissue was detected by RT-PCR, and the content of h-IGF-I in right knee lavage fluid was detected by ELISA. After transfection, the expression level of aggrecanase m RNA was compared between the groups of UTMD + ENTR. The transfection efficiency of UTMD combined transfection reagent Entranster was higher than that of ultrasound irradiation and Entranster transfection alone (P 0.01). The knee joint was induced by subcutaneous injection of bovine type II collagen and complete Freund's adjuvant emulsion. Joint swelling, joint diameter increased, skin temperature increased, 1 week later, a large number of lymphocyte infiltration in synovial tissue under pathological section; in vivo experiment: simple plasmid group, synovial tissue after intra-articular injection into the frozen section, fluorescence microscopy observation of weak fluorescence, plasmid + Entranster-in vivo + ultrasound microbubble group, fluorescence microscopy can be used. The expression of COX-2m RNA and aggrecanase-m RNA in the Entranster-in vivo+ultrasound microbubbles group was significantly lower than that in the blank control group and the pure plasmid group, and the highest level was found in the Entranster-in vivo+ultrasound microbubbles group two weeks after intra-articular injection. HE staining of synovial tissue showed that the number of inflammatory cells in Entranster-in vivo + ultrasound microbubbles group was less than that in the other two groups, but there was no significant difference in joint repair between the two groups. After transfection, it can also be expressed in vivo, so as to achieve the goal of treatment.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R593.22

【共引文獻(xiàn)】

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本文編號：2216519