Using Gene Transfection Of Gene Silencing And Induction To Treat The Rheumatoid Arthritis

Objection:1. To investigate the efficiency of aggrecanase-1 sh RNA transfection on MSCs mediated by ultrasound-targeted microbubble and Entranster;2. Establishment of the rheumatoid arthritis model of the rabbit and evaluation; 3. To explore the therapeutic effect of aggrecanase-1 sh RNA,COX-2 sh RNA, h IGF transfection mediated by ultrasound-targeted microbubble and Entranster in the rheumatoid arthritis model of rabbits.Method: According to the standard bone marrow puncture procedure, the marrow was aspirated to culture the human bone marrow mesenchymal stem cells(h BMSCs). h BMSCs were cultured and passaged to third generation. Then the third generation cells were cultured in 24-well plates for 24 hours, and divided into four groups:(a) plasmid + ultrasound-targeted microbubble destruction group(UTMD),(b) plasmid + transfection reagent Entranster group(ENTR),(c) plasmid + ultrasound-targeted microbubble destruction group + transfection reagent Entranster group(UTMD+ENTR),(d) plasmid(CONTR). To observe the fluorescence of every group on day 2, 4, 6 by the inverted fluorescence microscope; To detect the m RNA expression of aggrecanase-1 by RT-PCR. The establishment of the rheumatoid arthritis model of the rabbit: Mix the bovine type II collagen solution with the complete Freund’s adjuvant into emulsion. Then continuous subcutaneous injection of 1ml emulsion at 10 points of scapular area. To inject 1ml collagen solution into knee joint to sensitize RA group rabbits. And then After operative, we evaluated the model through the degree of joint swelling, the grade of the arthritis and pathohistological examitation. In vivo transfection experiments: The successful rheumatoid arthritis model of rabbits were divided into 3 groups: The control group: Only given the intra-articular injection the same amount of PBS into the right knee of rabbit. The plasmid group: Given the intra-articular injection plasmid of 200 ul Aggrecanase-2 sh RNA(10ug)、COX-2 sh RNA(10ug)、h IGF(10ug)and 100 ul PBS into the right knee of rabbit. Plasmid+ Entranster-in vivo + Ultrasound group:Given the intra-articular injection plasmid of 200 ul Aggrecanase-2 sh RNA(10ug),COX-2 sh RNA(10ug),h IGF(10ug),100 ul PBS, Entranster-in vivo and sulfur hexafluoride microbubbles into the right knee of rabbit. In 4.9MHz frequency, ultrasonic probe right knee joints for 5 minutes. Observation the difference of articular cavity; To compared every group about the amount of inflammatory cells by the pathological tissue examine; After transfection in vivo 4 days, to cut synovial of the right knee,Preparation of frozen sections by freezing microtome, and observed under fluorescence microscopy. To detect the m RNA expression of COX-2、aggrecanse by RT-PCR.To detect the right knee joint lavage fluid content of h IGF-I by ELISA method.Result: The sequence of the green fluorescence intensity from high to low was UTMD+ENTR, UTMD、ENTR,CONTR. The m RNA expression of aggrecanase-1 was suppressed significantly in UTMD+ENTR group. The effect of aggrecanase suppression of UTMD+ENTR group was the best than other groups. After sensitized by collagen protein, the knee joint swelled, articular diameter increased, the skin temperature increased too. After 1 week, the biopsie saw many lymphocytes infilled in the synovial tissue. In vivo transfection experiments: After transfection in vivo 4 days in the plasmid group, the frozen section was observed the weak fluorescence under fluorescence microscopy. Plasmid+ Entranster-in vivo + Ultrasound group: We saw the strong fluorescence diffused in the synovial tissue. The m RNA expression of COX-2 and aggrecanse was suppressed significantly in Plasmid+ Entranster-in vivo + Ultrasound group. The amount of h IGF-I of the right knee joint lavage fluid content in Plasmid+ Entranster-in vivo + Ultrasound group was the highest compared with other groups. The HE staining of synovial tissue showed that Plasmid+ Entranster-in vivo + Ultrasound group the number if inflammatory cells were reduced than the other two groups. But in gross appearance there were no significant differences for each group.Conclusion: The combined application of UTMD and transfection reagent Etranster could be as a new method of gene transfection to enhance the transfection efficiency. Using Gene Transfection of Aggrecanase-2 sh RNA, COX-2 sh RNA, h IGF mediated by UTMD and transfection reagent Etranster could able to treat the Rheumatoid Arthritis.