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

作　　者：杨平 苏若珂 邓璐 杨月瑶 王刚[1] YANG Ping;SU Ruoke;DENG Lu;YANG Yueyao;WANG Gang(Engineering Research Center in Biomaterials,Sichuan University,Chengdu 610064,China)

机构地区：[1]四川大学生物材料工程研究中心

出　　处：《四川动物》2019年第6期607-615,共9页Sichuan Journal of Zoology

基　　金：国家自然科学基金项目(31370972)

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。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.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.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).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.

关 键 词：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 

分 类 号：R318.08[医药卫生—生物医学工程] Q95-33[医药卫生—基础医学]