EXPERIMENTAL STUDY ON LENTIVIRUS-MEDIATED MULTI-GENES CO-TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF KNEE OSTEOARTHRITIS IN CYNOMOLGUS MONKEY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGZhao ¹ , LIXiaofei ² ^Δ , WANGYanyan ³ , CUIZhaowei ² , CHENZhuke ² ,  ZHANGHaining ²

1. Department of Joint Surgery, Qingdao Hiserve Medical Center, Qingdao Shandong, 266033, P. R. China;
2. Department of Joint Surgery, the Affiliated Hospital of Qingdao University;
3. Department of Oncology, Central Hospital of Qingdao;

Corresponding author：

ZHANGHaining, Email: zhhaining@hotmail.com

Keywords：

Cyclooxygenase 2; Aggrecanase-1; Insulin-like growth factor 1; Bone marrow mesenchymal stem cells; Lentivirus; Cynomolgus monkey

DOI：

10.7507/1002-1892.20160235

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Objective To observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA). Methods BMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×10⁷ cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR. Results No significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P < 0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P < 0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P < 0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P < 0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P < 0.05). Conclusion Lentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.

Citation： ZHANGZhao, LIXiaofei, WANGYanyan, CUIZhaowei, CHENZhuke, ZHANGHaining. EXPERIMENTAL STUDY ON LENTIVIRUS-MEDIATED MULTI-GENES CO-TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF KNEE OSTEOARTHRITIS IN CYNOMOLGUS MONKEY. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(9): 1153-1159. doi: 10.7507/1002-1892.20160235 Copy

1.	Prieto-Alhambra D, Judge A, Javaid MK, et al. Incidence and risk factors for clinically diagnosed knee, hip and hand osteoarthritis:influences of age, gender and osteoarthritis affecting other joints. Ann Rheum Dis, 2014, 73(9):1659-1664.
2.	陈福灵, 张健.膝关节骨性关节炎治疗进展.检验医学与临床, 2013, 10(12):1596-1598.
3.	Jung HS, Kwon BS, Lee SW. Tumor-specific gene delivery using RNA-targeting Tetrahymena group I intron. Biotechnol Lett, 2005, 27(8):567-574.
4.	Alvarez-Soria MA, Largo R, Santillana J, et al. Long term NSAID treatment inhibits COX-2 synthesis in the knee synovial membrane of patients with osteoarthritis:differential proinflammatory cytokine profile between celecoxib and aceclofenac. Ann Rheum Dis, 2006, 65(8):998-1005.
5.	Naito S, Shiomi T, Okada A, et al. Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage. Pathol Int, 2007, 57(11):703-711.
6.	黄宗强, 刘尚礼, 郑召民, 等.腺病毒介导的hIGF-1基因转染对软骨细胞增殖的影响.中国临床解剖学杂志, 2005, 23(1):9-13.
7.	Hulth A, Lindberg L, Talhag H. Experimental osteoarthritis in rabbits. Preliminary report. Acta Orthop Scand, 1970, 41(5):522-530.
8.	Pineda S, Pollack A, Stevenson S, et al. A semiquantitative scale for histologic grading of articular cartilage repair. Acta Anat (Basel), 1992, 143(4):335-340.
9.	Mankin HJ, Dorfman H, Lippiello L, et al. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. Ⅱ. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg (Am), 1971, 53(3):523-537.
10.	王嫱, 梅焕平.骨性关节炎软骨修复与基因治疗.中华临床免疫和变态反应杂志, 2008, 2(1):41-44.
11.	Hagmann S, Moradi B, Frank S, et al. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells. BMC Musculoskelet Disord, 2013, 14:223.
12.	Seo S, Na K. Mesenchymal stem cell-based tissue engineering for chondrogenesis. J Biomed Biotechnol, 2011, 2011:806891.
13.	Ulivi V, Lenti M, Gentili C, et al. Anti-inflammatory activity of monogalactosyldiacylglycerol in human articular cartilage in vitro:activation of an anti-inflammatory, cyclooxygenase-2 (COX2) pathway. Arthritis Res Ther, 2011, 13(3):R92.
14.	Wang ZH, Yang ZQ, He XJ, et al. Lentivirus-mediated knockdown of aggrecanase-1 and-2 promotes chondmcyte-engineered cartilage formation in vitro. Biotechnol Bioeng, 2010, 107(4):730-736.
15.	张绍昆, 刘一, 吴宏, 等.外源性人胰岛素样生长因子-1基因在关节软骨细胞中的表达.中华实验外科杂志, 2005, 22(3):278-280.
16.	Weimer A, Madry H, Venkatesan JK, et al. Benefits of recombinant adeno-associated virus (rAAV)-mediated insulinlike growth factor I (IGF-I) overexpression for the long-term reconstruction of human osteoarthritic cartilage by modulation of the IGF-I axis. Mol Med, 2012, 18:346-358.
17.	Yuan YQ, Zhang HN, Kong X, et al. Lentivirus-mediated cyclooxygenase 2 and aggrecanase 1 silencing and insulin-like growth factor 1 overexpression in human bone marrow mesenchymal stem cells.中国组织工程研究, 2015, 19(10):1488-1494.
18.	Evans CH, Gouze E, Gouze JN, et al. Gene therapeutic approaches-transfer in vivo. Adv Drug Deliv Rev, 2006, 58(2):243-258.
19.	Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 2003, 9(4):493-501.
20.	Martin SE, Caplen NJ. Applications of RNA interference in mammalian systems. Annu Rev Genomics Hum Genet, 2007, 8:81-108.

1. Prieto-Alhambra D, Judge A, Javaid MK, et al. Incidence and risk factors for clinically diagnosed knee, hip and hand osteoarthritis:influences of age, gender and osteoarthritis affecting other joints. Ann Rheum Dis, 2014, 73(9):1659-1664.
2. 陈福灵, 张健.膝关节骨性关节炎治疗进展.检验医学与临床, 2013, 10(12):1596-1598.
3. Jung HS, Kwon BS, Lee SW. Tumor-specific gene delivery using RNA-targeting Tetrahymena group I intron. Biotechnol Lett, 2005, 27(8):567-574.
4. Alvarez-Soria MA, Largo R, Santillana J, et al. Long term NSAID treatment inhibits COX-2 synthesis in the knee synovial membrane of patients with osteoarthritis:differential proinflammatory cytokine profile between celecoxib and aceclofenac. Ann Rheum Dis, 2006, 65(8):998-1005.
5. Naito S, Shiomi T, Okada A, et al. Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage. Pathol Int, 2007, 57(11):703-711.
6. 黄宗强, 刘尚礼, 郑召民, 等.腺病毒介导的hIGF-1基因转染对软骨细胞增殖的影响.中国临床解剖学杂志, 2005, 23(1):9-13.
7. Hulth A, Lindberg L, Talhag H. Experimental osteoarthritis in rabbits. Preliminary report. Acta Orthop Scand, 1970, 41(5):522-530.
8. Pineda S, Pollack A, Stevenson S, et al. A semiquantitative scale for histologic grading of articular cartilage repair. Acta Anat (Basel), 1992, 143(4):335-340.
9. Mankin HJ, Dorfman H, Lippiello L, et al. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. Ⅱ. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg (Am), 1971, 53(3):523-537.
10. 王嫱, 梅焕平.骨性关节炎软骨修复与基因治疗.中华临床免疫和变态反应杂志, 2008, 2(1):41-44.
11. Hagmann S, Moradi B, Frank S, et al. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells. BMC Musculoskelet Disord, 2013, 14:223.
12. Seo S, Na K. Mesenchymal stem cell-based tissue engineering for chondrogenesis. J Biomed Biotechnol, 2011, 2011:806891.
13. Ulivi V, Lenti M, Gentili C, et al. Anti-inflammatory activity of monogalactosyldiacylglycerol in human articular cartilage in vitro:activation of an anti-inflammatory, cyclooxygenase-2 (COX2) pathway. Arthritis Res Ther, 2011, 13(3):R92.
14. Wang ZH, Yang ZQ, He XJ, et al. Lentivirus-mediated knockdown of aggrecanase-1 and-2 promotes chondmcyte-engineered cartilage formation in vitro. Biotechnol Bioeng, 2010, 107(4):730-736.
15. 张绍昆, 刘一, 吴宏, 等.外源性人胰岛素样生长因子-1基因在关节软骨细胞中的表达.中华实验外科杂志, 2005, 22(3):278-280.
16. Weimer A, Madry H, Venkatesan JK, et al. Benefits of recombinant adeno-associated virus (rAAV)-mediated insulinlike growth factor I (IGF-I) overexpression for the long-term reconstruction of human osteoarthritic cartilage by modulation of the IGF-I axis. Mol Med, 2012, 18:346-358.
17. Yuan YQ, Zhang HN, Kong X, et al. Lentivirus-mediated cyclooxygenase 2 and aggrecanase 1 silencing and insulin-like growth factor 1 overexpression in human bone marrow mesenchymal stem cells.中国组织工程研究, 2015, 19(10):1488-1494.
18. Evans CH, Gouze E, Gouze JN, et al. Gene therapeutic approaches-transfer in vivo. Adv Drug Deliv Rev, 2006, 58(2):243-258.
19. Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 2003, 9(4):493-501.
20. Martin SE, Caplen NJ. Applications of RNA interference in mammalian systems. Annu Rev Genomics Hum Genet, 2007, 8:81-108.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON LENTIVIRUS-MEDIATED MULTI-GENES CO-TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF KNEE OSTEOARTHRITIS IN CYNOMOLGUS MONKEY

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