刊期:双月刊
主管单位:四川省科学技术协会
主办单位:四川省动物学会/成都大熊猫繁育研究基金会/四川省野生动植物保护协会/四川大学
地址:四川省成都市武侯区望江路29号四川大学生命科学学院内
邮编:610065
电话:028-85410485; 15881112385
传真:028-85410485
E-Mail:scdwzz@vip.163.com & scdwzz001@163.com
刊号:ISSN 1000-7083
        CN 51-1193/Q
国内发行代号:
国际发行代号:
发行范围:国内外公开发布
定价:50元/册
定价:300元/年

您所在位置:首页->过刊浏览->2019年第38卷第6期

利用小鼠骨骼肌细胞持续表达单链胰岛素类似物进行1型糖尿病模型鼠的长效基因治疗
Continuous Expression of Single-Strand Insulin Analogue in Skeletal Muscles for the Long-Term Efficient Gene Therapy of Type 1 Diabetic Mice
杨平, 苏若珂, 邓璐, 杨月瑶, 王刚*
点击:103次 下载:3次
DOI:10.11984/j.issn.1000-7083.20190173
作者单位:四川大学生物材料工程研究中心, 成都 610064
中文关键字:胰岛素类似物;Pluronic L64-电脉冲体系;1型糖尿病小鼠;骨骼肌基因治疗
英文关键字:insulin analogue; Pluronic L64-electropulse system; type 1 diabetic mice; skeletal muscle gene therapy
中文摘要:目的 探讨Pluronic L64-电脉冲(L/E)体系介导的单链胰岛素类似物基因在骨骼肌组织的传递表达对1型糖尿病小鼠的治疗效果和影响。方法 将胰岛素基因中的C肽区域换成(GGGGS)3,克隆到pcDNA3.1(+)质粒中,构建载体pcDNA-INS。使用链脲佐菌素腹腔注射雄性BALB/c小鼠建立1型糖尿病小鼠模型,并分为L/E联合pcDNA3.1(+)空质粒治疗的L/E-pcDNA3.1(+)组、仅使用pcDNA-INS裸质粒治疗的pcDNA-INS组和L/E联合pcDNA-INS裸质粒治疗的L/E-pcDNA-INS组。模型小鼠在胫骨前肌注射相关质粒进行治疗,且根据治疗方案在注射1 h后进行Pluronic L64-电脉冲处理,并以生理盐水处理的正常小鼠(NC)组作为对照。记录各组小鼠的血糖、体质量、血清胰岛素含量、生存率、免疫组化、外形特征及组织病理等指标。结果 (1)pcDNA-INS质粒通过酶切和测序鉴定无误;(2)与NC组相比,模型小鼠血糖显著上升,体质量显著下降(P<0.05),且胰岛遭到严重破坏;(3)L/E-pcDNA-INS组的血糖水平和血清胰岛素含量显著缓解,与NC组较为接近,pcDNA-INS组的血糖缓解和胰岛素补偿远不及L/E-pcDNA-INS组(P<0.05),且与NC组之间的差异一直有统计学意义(P<0.05)。第4周免疫组化结果显示,L/E-pcDNA-INS组的胰腺胰岛素表达较少,但肌肉胰岛素表达量较高,且该组生存率较高,与NC组均为100%(12/12),L/E-pcDNA-INS组的病理结果和外形特征与NC组小鼠最为接近,L/E-pcDNA3.1(+)组与pcDNA-INS组在不同部位均有不同程度的病变发生,且鼠毛疏少粗糙,体形较小。结论 Pluronic L64-电脉冲体系与单链胰岛素类似物基因联合治疗1型糖尿病的效果较好,且无明显不良影响,可为相关研究提供实验依据。
英文摘要:Objective To investigate the treatment effects of naked plasmid containing single-strand insulin gene via Pluronic L64-electropulse system (L/E) on type 1 diabetes. Methods C-peptide sequences of original insulin gene was replaced by linker (GGGGS)3, and pcDNA-INS plasmid was constructed based on pcDNA3.1(+). BALB/c diabetic mice were established by streptozotocin (STZ) via intraperitoneal injection, and divided into 3 groups:Group pcDNA-INS was treated by pcDNA-INS naked plasmid, Group L/E-pcDNA3.1(+) was treated by L/E system combined with pcDNA3.1(+) naked plasmid treatments, and Group L/E-pcDNA-INS was treated by L/E system combined with pcDNA-INS naked plasmid treatments. All these groups were injected with corresponding plasmids and treated by L/E system after 1 hour according to therapy protocols, and the mice of control group (Group NC) were injected with the same volume of saline in tibialis anterior muscles on both sides. Blood glucose, serum insulin and other indices were detected. Results The pcDNA-INS plasmid was constructed and identified. The STZ-diabetic mice models with higher blood glucose and lower body mass than those of Group NC mice were also successfully established (P<0.05). Group L/E-pcDNA-INS showed lower blood glucose level and higher serum insulin content than both Group pcDNA-INS and Group L/E-pcDNA3.1(+)(P<0.05), and all the other indices such as survival rates, HE staining and immunohistochemical results, and appearances were much closer to the Group NC mice. Moreover, the immunohistochemical results at the end of therapy reflected that the pancreas insulin content of all diabetic mice was still much lower than normal mice, while the muscle insulin content of the L/E-pcDNA-INS-treated mice was much adequate, and the survival rates of L/E-pcDNA-INS-treated mice were 100%(12/12)(vs. Group NC, P > 0.05). Conclusion Insulin analogue gene combined with Pluronic L64-electropulse system apparently improve the remission of type 1 diabetic mice, and thus provide some experimental cues for the future related gene therapy.
2019,38(6): 607-615 收稿日期:2019-05-17
分类号:R318.08;Q95-33
基金项目:国家自然科学基金项目(31370972)
作者简介:杨平(1994-),女,硕士研究生,研究方向:生物医学工程,E-mail:pyang082@outlook.com
*通信作者:王刚,研究员,研究方向:靶向基因传递、基因治疗,E-mail:wgang@scu.edu.cn
参考文献:
Abai AM, Hobart PM, Barnhart KM. 1999. Insulin delivery with plasmid DNA[J]. Human Gene Therapy, 10(16):2637-2649.
Alakhov V, Lemieux P, Klinski E, et al. 2001. Block copolymeric biotransport carriers as versatile vehicles for drug delivery[J]. Expert Opinion on Biological Therapy, 1(4):583-602.
Batrakova EV, Kabanov AV. 2008. Pluronic block copolymers:evolution of drug delivery concept from inert nanocarriers to biological response modifiers[J]. Journal of Controlled Release, 130(2):98-106.
Blaveri K, Heslop L, Yu DS, et al. 1999. Patterns of repair of dystrophic mouse muscle:studies on isolated fibers[J]. Developmental Dynamics, 216(3):244-256.
Bureau MF, Gehl J, Deleuze V, et al. 2000. Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer[J]. Biochimica et Biophysica Acta-General Subjects, 1474(3):353-359.
Chen J, Luo J, Zhao Y, et al. 2015. Increase in transgene expression by pluronic L64-mediated endosomal/lysosomal escape through its membrane-disturbing action[J]. ACS Applied Materials & Interfaces, 7(13):7282-7293.
Coster HGL. 1965. A quantitative analysis of the voltage-current relationships of fixed charge membranes and the associated property of ‘punch-through’[J]. Biophysical Journal, 5(5):669-686.
Fukazawa T, Matsuoka J, Naomoto Y, et al. 2006. Development of a novel β-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures[J]. Experimental Cell Research, 312(17):3404-3412.
Han J, Mclane B, Kim EH, et al. 2011. Remission of diabetes by insulin gene therapy using a hepatocyte-specific and glucose-responsive synthetic promoter[J]. Molecular Therapy, 19(3):470-478.
Handorf AM, Sollinger HW, Alam T. 2015. Insulin gene therapy for type 1 diabetes mellitus[J]. Experimental & Clinical Transplantation Official Journal of the Middle East Society for Organ Transplantation, 13(Suppl 1):37-45.
Hartikka J, Sukhu L, Buchner C, et al. 2001. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle:plasmid dependence of muscle damage and effect of poloxamer 188[J]. Molecular Therapy, 4(5):407-415.
He YT, Liu YL, Sun Z, et al. 2019. The proper strategy to compress and protect plasmid DNA in the Pluronic L64-electropulse system for enhanced intramuscular gene delivery[J]. Regenerative Biomaterials. DOI:10.1093/rb/rby028.
Hua QX, Nakagawa SH, Jia W, et al. 2008. Design of an active ultrastable single-chain insulin analog:synthesis, structure, and therapeutic implications[J]. Journal of Biological Chemistry, 283(21):14703-14716.
Kabanov AV. 2002. Pluronic block copolymers:novel functional molecules for gene therapy[J]. Advanced Drug Delivery Reviews, 54(2):223-233.
Kessler PD, Podsakoff GM, Chen X, et al. 1996. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein[J]. Proceedings of the National Academy of Sciences of the United States of America, 93(24):14082-14087.
Kon OL, Sivakumar S, Teoh KL, et al. 1999. Naked plasmid-mediated gene transfer to skeletal muscle ameliorates diabetes mellitus[J]. The Journal of Gene Medicine, 1(3):186-194.
Kufer P, Mack M, Gruber R, et al. 1997. Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer[J]. Cancer Immunology Immunotherapy, 45(3-4):193-197.
Liu S, Ma L, Tan R, et al. 2014. Safe and efficient local gene delivery into skeletal muscle via a combination of Pluronic L64 and modified electrotransfer[J]. Gene Therapy, 21(6):558-565.
Navarro-Gonzalez JF, Mora-Fernandez C. 2008. The role of inflammatory cytokines in diabetic nephropathy[J]. Journal of the American Society of Nephrology, 19(3):433-442.
Olfert IM, Breen EC, Mathieucostello O, et al. 2001. Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia[J]. Journal of Applied Physiology, 91(3):1176-1184.
Pu L, Geng Y, Liu S, et al. 2014. Electroneutralized amphiphilic triblock copolymer with a peptide dendron for efficient muscular gene delivery[J]. ACS Applied Materials & Interfaces, 6(17):15344-15351.
Whiting DR, Guariguata L, Weil C, et al. 2011. IDF diabetes atlas:global estimates of the prevalence of diabetes for 2011 and 2030[J]. Diabetes Research and Clinical Practice, 94(3):311-321.
Yoon JW, Jun HS. 2002. Recent advances in insulin gene therapy for type 1 diabetes[J]. Trends in Molecular Medicine, 8(2):62-68.
读者评论

      读者ID: 密码:   
我要评论:
国内统一连续出版物号:51-1193/Q |国际标准出版物号:1000-7083
主管单位:四川省科学技术协会  主办单位:四川省动物学会/成都大熊猫繁育研究基金会/四川省野生动植物保护协会/四川大学
开户银行:中国工商银行四川分行营业部东大支行(工行成都东大支行营业室)  帐户名:四川省动物学会  帐号:4402 2980 0900 0012 596
版权所有©2019四川动物》编辑部 蜀ICP备08107403号-3
您是本站第7866505名访问者

川公网安备 51010702000173号