基因沉默与诱导双向治疗类风湿性关节炎
发布时间：2018-09-01 07:28
【摘要】：目的:1.探索超声微泡靶向破坏联合转染试剂Entranster能否提高aggrecanase-1基因转染骨髓间充质干细胞的转染效率;2.制备兔类风湿关节炎模型,并对其进行评估;3.超声微泡靶向破坏联合转染试剂Entranster in vivo介导aggrecanase-1 sh RNA,COX-2 sh RNA,h IGF体内转染对类风湿性关节炎膝关节的影响。方法:应用全骨髓培养法培养类风湿关节炎患者骨髓间充质干细胞。将第3代骨髓间充质干细胞接种于24孔板中,共分为4组:1.超声靶向微泡破坏组(UTMD);2.转染试剂Entranster组(ENTR);3.超声微泡靶向破坏+转染试剂Entranster组(UTMD+ENTR);4.对照组。转染后第2天、第4天、第6天,在倒置荧光显微镜下,观察aggrecanase sh RNA转染情况;应用荧光定量PCR检测转染后aggrecanase m RNA水平的变化。家兔类风湿关节炎模型制备:应用牛Ⅱ型胶原溶液与完全弗氏佐剂混合成乳剂,取1 ml共分10点注射于家兔肩胛骨区域。致敏后,膝关节注入1ml(5g/L)胶原蛋白溶液,观察足掌关节肿胀程度,对关节炎进行评分以及病理学组织检查。体内转染实验:将造模成功的兔类风湿关节炎模型分为3组:空白对照组:仅在家兔右膝关节腔内注射等量的PBS;单纯质粒组:取200ul Aggrecanase-2 sh RNA(10ug)、COX-2 sh RNA(10ug)、h IGF(10ug)质粒以及100ul PBS注入家兔右膝关节腔内;质粒+Entranster-in vivo+超声微泡组:各取200ul Aggrecanase-2 sh RNA(10ug)、COX-2 sh RNA(10ug)、IGF(10ug)、Entranster-in vivo转染试剂以及100ul六氟化硫微泡液体注入家兔右膝关节腔内,在4.9MHz频率下,超声探头扫右膝关节关节5分钟。大体观察比较各组间关节病变及改善情况;病理组织检查各组间炎细胞有无减少;体内转染4天后,切取各组右膝关节滑膜组织,用冰冻切片机切片,在荧光显微镜下观察各组间基因转染情况;RT-PCR检测各组滑膜组织COX-2、aggrecanse表达水平;ELISA法检测各组右膝关节灌洗液h IGF-I含量。结果:在倒置荧光显微镜下观察UTMD+ENTR组绿色荧光最强;UTMD组和ENTR组次之,对照组最暗。转染后各组间比较aggrecanase m RNA表达水平;UTMD+ENTR组下降最明显。UTMD联合转染试剂Entranster的转染效率高于单纯超声辐照和Entranster转染(P0.01);牛Ⅱ型胶原与完全弗氏佐剂混合乳剂皮下注射并诱导后,膝关节关节肿胀,关节直径增加,皮肤温度升高,1周后,病理切片镜下滑膜组织内大量淋巴细胞浸润;体内实验:单纯质粒组,关节腔注射后,滑膜组织制成冰冻切片后,荧光显微镜下观察荧光微弱,质粒+Entranster-in vivo+超声微泡组,荧光显微镜下可见大量荧光弥漫于滑膜组织。Entranster-in vivo+超声微泡组的COX-2m RNA、aggrecanase m RNA表达水平明显低于空包对照和单纯质粒组;各组间应用ELISA法检测膝关节灌洗液h IGF-I含量比较后Entranster-in vivo+超声微泡组水平最高。关节腔注射后两周滑膜组织HE染色切片观察,Entranster-in vivo+超声微泡组炎细胞数量较其他两组减少,但大体外观见各组的关节修复没有明显差别。结论:UTMD联合转染试剂Entranster应用可以作为一种新的转染方法,提高基因转染效率;两种方法联合进行体内转染后,亦能够在体内表达,从而达到治疗的目的。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22
本文编号：2216519
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22
【共引文献】
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