pcDNA3.1-Myc-His B（-）/tPA质粒DNA-交联明胶微球复合物的制备及其性质检测和分析
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摘要
实验一交联明胶微球的制备及其物理性质的检测和分析
目的制备交联明胶微球并检测和分析其物理性质。
方法以普通碱性明胶为原料,25%戊二醛水溶液为交联剂,采用改良的双相乳化冷凝交联法制备交联明胶微球。采用扫描电镜观察微球微观形态,测定微球在37℃生理盐水中充分溶胀前后的平均直径和最大溶胀率。微电泳法测定微球的表面电荷。采用紫外分光光度法检测交联明胶微球结合和体外缓释pcDNA3.1-Myc-His B(-)/tPA质粒DNA的性能和剂量-时间规律。
结果交联明胶微球呈分散良好的均匀球体,表面可见孔道散在分布。干燥状态下微球的平均直径为21.13±1.42μm,在37℃生理盐水中充分溶胀后的平均直径为26.72±1.74μm,两者相比具有明显统计学差异(P <0.05)。微电泳法测定微球表面携带正电荷。交联明胶微球在37℃生理盐水中的最大溶胀率为27.32±0.45%。当pcDNA3.1-Myc-His B(-)/tPA质粒DNA与交联明胶微球的质量比为1:10时,微球结合质粒DNA达到饱和状态,此时的包封率为62.75±3.31%。所形成的pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物在体外能够持续缓释质粒DNA超过4周。
结论本实验所制备的交联明胶微球能够有效结合pcDNA3.1-Myc-His B(-)/tPA质粒DNA形成pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物,在体外能够持续缓释pcDNA3.1-Myc-His B(-)/tPA质粒DNA超过4周。
实验二交联明胶微球的细胞毒性和生物相容性研究
目的检测交联明胶微球的细胞毒性和生物相容性,评价其用于体内应用的安全性。
方法采用MTT染色法检测交联明胶微球的细胞毒性程度。通过溶血实验、凝血酶原时间实验和复钙时间实验评价交联明胶微球对红细胞的破坏作用和对凝血功能的影响。通过扫描电镜观察、测定黏附细胞数和细胞迁移实验研究交联明胶微球对体外培养的人脐静脉内皮细胞的黏附和迁移能力的影响。
结果交联明胶微球的细胞毒性程度维持在1级,并且不随微球降解时间延长和浓度增加而增强。交联明胶微球的溶血率为2.27±0.34%,提示微球没有溶血性。测定微球的凝血酶原时间为16.0±0.7 s,与阴性对照组(17.2±0.8 s)相比没有统计学差异(P >0.05);复钙时间为165.4±4.5 s,与普通明胶组(169.2±5.4 s)和阴性对照组(181.2±3.4 s)相比均没有统计学差异(P >0.05),从而提示微球不会促进外源性和内源性凝血途径的功能。通过扫描电镜观察和在1周末和4周末时测定携带交联明胶微球溶液的Dacron补片表面的黏附细胞数量为(1.47±0.30)×106和(65.34±11.25)×106,分别与普通Dacron补片组的黏附细胞数(1.63±0.26)×106和(62.43±10.06)×106相比均没有统计学差异(P >0.05),提示微球不会抑制人脐静脉内皮细胞在Dacron纤维表面的黏附。细胞迁移试验测定交联明胶微球组的细胞迁移距离为29.61±3.54μm,与阴性对照组(32.41±4.31μm)和普通明胶组(30.55±5.08μm)相比均没有统计学差异(P >0.05),提示微球不会抑制细胞的迁移能力。
结论交联明胶微球没有细胞毒性;不会引起溶血和促进外源性和内源性凝血功能,也不会抑制人脐静脉内皮细胞的黏附和迁移能力,具有良好生物相容性。
实验三pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物的体外基因转染实验研究
目的检测pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物体外缓释pcDNA3.1-Myc-His B(-)/tPA质粒DNA转染人脐静脉内皮细胞的瞬时转染效率和转染后的tPA蛋白的表达和分泌情况。
方法以体外培养的人脐静脉内皮细胞HUVECs为转染对象,通过免疫荧光染色技术检测其瞬时转染效率。通过RT-PCR技术检测转染后HUVECs中tPA mRNA转录,通过Western印迹检测技术检测转染后HUVECs中tPA蛋白表达情况。通过ELISA技术检测HUVECs转染后细胞培养上清液中tPA蛋白含量。
结果pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物所缓释的pcDNA3.1-Myc-His B(-)/tPA质粒DNA能够成功转移进入HUVECs,其瞬时转染效率为12.74±2.53%,明显高于单纯pcDNA3.1-Myc-His B(-)/tPA质粒DNA溶液组(4.63±1.75%) (P <0.05),但低于pcDNA3.1-Myc-His B(-)/tPA质粒DNA-阳离子脂质体复合物组(26.49±5.23%) (P <0.05)。RT-PCR提示其转染后转录产生的tPA mRNA电泳条带的积分光密度值与相应内参β-actin的比值为1.65±0.27,明显高于单纯pcDNA3.1-Myc-His B(-)/tPA质粒DNA溶液组(1.22±0.23) (P <0.05),但低于pcDNA3.1-Myc-His B(-)/tPA质粒DNA-阳离子脂质体复合物组(2.07±0.25) (P <0.05),以上3组均显著高于空白pcDNA3.1质粒DNA载体组(0.86±0.21)和正常培养的HUVECs (0.91±0.18) (P <0.05)。Western免疫印迹技术提示其转染后细胞内有tPA蛋白表达,而空白pcDNA3.1质粒DNA载体组和正常培养的HUVECs中则仅有痕量表达。ELISA实验证明HUVECs转染后所表达的tPA蛋白能够被分泌到细胞培养上清液中,其tPA蛋白浓度为497.2±61.9 ng/ml,高于单纯pcDNA3.1-Myc-His B(-)/tPA质粒DNA溶液组(295.7±49.1 ng/ml) (P <0.05),但低于pcDNA3.1-Myc-His B(-)/tPA质粒DNA-阳离子脂质体复合物组(862.1±96.8 ng/ml) (P <0.05)。
结论由pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物在体外缓释的pcDNA3.1-Myc-His B(-)/tPA质粒DNA能够成功转移进入HUVECs中并进行转录和表达,转染后细胞所产生的tPA蛋白能够被分泌到细胞外。
Experiment I Preparation of Cross Linking Gelatin Microspheres, Detection and Analysis of Physical Properties
Objective To prepare cross linking gelatin microspheres (CLG microspheres), analyze and detect physical properties.
Method Modified diphasic emulsified cold-condensation method was adapted to prepare CLG microspheres, with basic gelatin as material and 25% glutaral solution as cross linking agent. SEM was used to observe morphology and detect the average diameter of CLG microspheres before and after fully swelling in physiologic saline at 37?C. The maximum swelling radio of CLG microspheres was also evaluated. Microelectrophoresis was used to detect the surface charge of CLG microspheres. The ability of binding and delayed releasing dose-time profile of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA by CLG microspheres was also detected by ultraviolet spectrophotometry.
Results CLG microspheres were uniform spheroids with small pores on surface and sporadically dispersed without conglutination. The average diameter of CLG microspheres was 21.13±1.42μm and 26.72±1.74μm before and after fully swelling in physiologic saline at 37?C, and there was statistical difference between the average diameter of microspheres before and after fully swelling. The maximum swelling radio was 27.32±0.45% in physiologic saline at 37?C. The surface charge was positive. When the mass proportion of plasmid DNA vs. CLG microspheres reached 1:10, the absorption for plasmid DNA by CLG microspheres reached saturation, with entrapment radio of 62.75±3.31%. pcDNA3.1-Myc-His B(-)/tPA plasmid DNA could be continuously delayed released by CLG microspheres over 4 weeks in vitro.
Conclusion CLG microspheres possessed good ability of binding pcDNA3.1-Myc-His B(-)/tPA plasmid DNA to form pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, and pcDNA3.1-Myc-His B(-)/tPA plasmid DNA could be delayed released continuously from the complex over 4 weeks in vitro.
Experiment II Research of Cytotoxicity and Biocompatibility of Cross Linking Gelatin Microspheres
Objective To detect cytotoxicity and biocompatibility of CLG microspheres in order to evaluate safety of CLG microspheres used in vivo.
Method MTT assay was used to detect cytotoxicity of CLG microspheres. Haemolysis test, prothrombin time and recalcification time experiment were used to evaluate influence on erythrocyte and blood coagulation function by CLG microspheres. The influence on ability of adhesion and migration on Dacron fibers surface of human umbilical vein endothelium cells (HUVECs) by CLG microspheres were also evaluated through SEM, adhesion cells numbers counting and cell migration test.
Results Cytotoxicity of CLG microspheres retained as grade 1, and would not increase along with degradation time and concentration of CLG microspheres. The Haemolysis degree was 2.27±0.34%, which indicated that CLG microspheres would not destroy erythrocytes. Prothrombin time was 16.0±0.7 s, and there was no statistical difference with that of negative control group (17.2±0.8 s) (P >0.05). Recalcification time was 165.4±4.5 s, and there was no significantly statistical difference with that of common gelatin (169.2±5.4 s) or negative group (181.2±3.4 s) (P >0.05). Those results indicated that CLG microspheres would not facilitate exogenous and intrinsic coagulation. The amount of HUVECs on the surface of Dacron fibers coated with CLG microspheres solution was (1.47±0.30)×106 and (65.34±11.25)×106 at the end of week 1 and 4 respectively, and there were no statistical difference between that of untreated Dacron fibers, (1.63±0.26)×106 and (62.43±10.06)×106 respectively (P >0.05). The migration distance in CLG microspheres group was 29.61±3.54μm, and there was no statistical difference between that of negative control group (32.41±4.31μm) or common gelatin group (30.55±5.08μm) (P >0.05). Those results indicated that CLG microspheres would not inhibit the ability of adhesion and migration of HUVECs.
Conclusion CLG microspheres had little cytotoxicity and would not facilitate coagulation, or inhibit the ability of adhesion and migration of HUVECs, which indicated that CLG microspheres possessed good biocompatibility.
Experiment III Experimental Research of Gene Transfection of pcDNA3.1-Myc-His B(-)/tPA Plasmid DNA-Cross Linking Gelatin Microspheres Complex in vitro
Objective To detect transient gene transfection efficiency of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA being transferred into HUVECs, which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex in vitro, and gene expression after transfection.
Method Immunofluorescence staining was used to detect the transient gene transfection efficiency of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex. RT-PCR was used to detect tPA mRNA and Western blotting was used to detect tPA protein synthesized in HUVECs after transfection. ELISA was used to evaluate tPA content in culture medium supernatant, which was secreted by HUVECs after transfection.
Results pcDNA3.1-Myc-His B(-)/tPA plasmid DNA, which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, could be successfully transferred into HUVECs in vitro, with transient transfection efficiency of 12.74±2.53%, and it was significantly higher than that of simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution (4.63±1.75%) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex (26.49±5.23%) (P <0.05). The integral optical density ratio value of tPA mRNA electrophoresis strip in pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex group was 1.65±0.27, which was higher than simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution group (1.22±0.23) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex group (2.07±0.25) (P <0.05). The integral optical density ratio values of those 3 groups were all significantly higher than that of blank pcDNA3.1 plasmid DNA vector group (0.86±0.21) and HUVECs in control culture (0.91±0.18) (P <0.05). tPA expression could be detected in HUVECs after transfection by pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, however it was expressed in trace level in blank pcDNA3.1 plasmid DNA vector group and HUVECs in control culture. tPA could be secreted into culture medium supernatant by HUVECs after transfection, and tPA content in pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex grorp was 497.2±61.9 ng/ml, which was higher than simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution group (295.7±49.1 ng/ml) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex group (862.1±96.8 ng/ml) (P <0.05).
Conclusion pcDNA3.1-Myc-His B(-)/tPA plasmid DNA which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex could be successfully transferred into HUVECs and tPA synthesized in HUVECs could be secreted extracellularly after transfection.
目的制备交联明胶微球并检测和分析其物理性质。
方法以普通碱性明胶为原料,25%戊二醛水溶液为交联剂,采用改良的双相乳化冷凝交联法制备交联明胶微球。采用扫描电镜观察微球微观形态,测定微球在37℃生理盐水中充分溶胀前后的平均直径和最大溶胀率。微电泳法测定微球的表面电荷。采用紫外分光光度法检测交联明胶微球结合和体外缓释pcDNA3.1-Myc-His B(-)/tPA质粒DNA的性能和剂量-时间规律。
结果交联明胶微球呈分散良好的均匀球体,表面可见孔道散在分布。干燥状态下微球的平均直径为21.13±1.42μm,在37℃生理盐水中充分溶胀后的平均直径为26.72±1.74μm,两者相比具有明显统计学差异(P <0.05)。微电泳法测定微球表面携带正电荷。交联明胶微球在37℃生理盐水中的最大溶胀率为27.32±0.45%。当pcDNA3.1-Myc-His B(-)/tPA质粒DNA与交联明胶微球的质量比为1:10时,微球结合质粒DNA达到饱和状态,此时的包封率为62.75±3.31%。所形成的pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物在体外能够持续缓释质粒DNA超过4周。
结论本实验所制备的交联明胶微球能够有效结合pcDNA3.1-Myc-His B(-)/tPA质粒DNA形成pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物,在体外能够持续缓释pcDNA3.1-Myc-His B(-)/tPA质粒DNA超过4周。
实验二交联明胶微球的细胞毒性和生物相容性研究
目的检测交联明胶微球的细胞毒性和生物相容性,评价其用于体内应用的安全性。
方法采用MTT染色法检测交联明胶微球的细胞毒性程度。通过溶血实验、凝血酶原时间实验和复钙时间实验评价交联明胶微球对红细胞的破坏作用和对凝血功能的影响。通过扫描电镜观察、测定黏附细胞数和细胞迁移实验研究交联明胶微球对体外培养的人脐静脉内皮细胞的黏附和迁移能力的影响。
结果交联明胶微球的细胞毒性程度维持在1级,并且不随微球降解时间延长和浓度增加而增强。交联明胶微球的溶血率为2.27±0.34%,提示微球没有溶血性。测定微球的凝血酶原时间为16.0±0.7 s,与阴性对照组(17.2±0.8 s)相比没有统计学差异(P >0.05);复钙时间为165.4±4.5 s,与普通明胶组(169.2±5.4 s)和阴性对照组(181.2±3.4 s)相比均没有统计学差异(P >0.05),从而提示微球不会促进外源性和内源性凝血途径的功能。通过扫描电镜观察和在1周末和4周末时测定携带交联明胶微球溶液的Dacron补片表面的黏附细胞数量为(1.47±0.30)×106和(65.34±11.25)×106,分别与普通Dacron补片组的黏附细胞数(1.63±0.26)×106和(62.43±10.06)×106相比均没有统计学差异(P >0.05),提示微球不会抑制人脐静脉内皮细胞在Dacron纤维表面的黏附。细胞迁移试验测定交联明胶微球组的细胞迁移距离为29.61±3.54μm,与阴性对照组(32.41±4.31μm)和普通明胶组(30.55±5.08μm)相比均没有统计学差异(P >0.05),提示微球不会抑制细胞的迁移能力。
结论交联明胶微球没有细胞毒性;不会引起溶血和促进外源性和内源性凝血功能,也不会抑制人脐静脉内皮细胞的黏附和迁移能力,具有良好生物相容性。
实验三pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物的体外基因转染实验研究
目的检测pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物体外缓释pcDNA3.1-Myc-His B(-)/tPA质粒DNA转染人脐静脉内皮细胞的瞬时转染效率和转染后的tPA蛋白的表达和分泌情况。
方法以体外培养的人脐静脉内皮细胞HUVECs为转染对象,通过免疫荧光染色技术检测其瞬时转染效率。通过RT-PCR技术检测转染后HUVECs中tPA mRNA转录,通过Western印迹检测技术检测转染后HUVECs中tPA蛋白表达情况。通过ELISA技术检测HUVECs转染后细胞培养上清液中tPA蛋白含量。
结果pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物所缓释的pcDNA3.1-Myc-His B(-)/tPA质粒DNA能够成功转移进入HUVECs,其瞬时转染效率为12.74±2.53%,明显高于单纯pcDNA3.1-Myc-His B(-)/tPA质粒DNA溶液组(4.63±1.75%) (P <0.05),但低于pcDNA3.1-Myc-His B(-)/tPA质粒DNA-阳离子脂质体复合物组(26.49±5.23%) (P <0.05)。RT-PCR提示其转染后转录产生的tPA mRNA电泳条带的积分光密度值与相应内参β-actin的比值为1.65±0.27,明显高于单纯pcDNA3.1-Myc-His B(-)/tPA质粒DNA溶液组(1.22±0.23) (P <0.05),但低于pcDNA3.1-Myc-His B(-)/tPA质粒DNA-阳离子脂质体复合物组(2.07±0.25) (P <0.05),以上3组均显著高于空白pcDNA3.1质粒DNA载体组(0.86±0.21)和正常培养的HUVECs (0.91±0.18) (P <0.05)。Western免疫印迹技术提示其转染后细胞内有tPA蛋白表达,而空白pcDNA3.1质粒DNA载体组和正常培养的HUVECs中则仅有痕量表达。ELISA实验证明HUVECs转染后所表达的tPA蛋白能够被分泌到细胞培养上清液中,其tPA蛋白浓度为497.2±61.9 ng/ml,高于单纯pcDNA3.1-Myc-His B(-)/tPA质粒DNA溶液组(295.7±49.1 ng/ml) (P <0.05),但低于pcDNA3.1-Myc-His B(-)/tPA质粒DNA-阳离子脂质体复合物组(862.1±96.8 ng/ml) (P <0.05)。
结论由pcDNA3.1-Myc-His B(-)/tPA质粒DNA-交联明胶微球复合物在体外缓释的pcDNA3.1-Myc-His B(-)/tPA质粒DNA能够成功转移进入HUVECs中并进行转录和表达,转染后细胞所产生的tPA蛋白能够被分泌到细胞外。
Experiment I Preparation of Cross Linking Gelatin Microspheres, Detection and Analysis of Physical Properties
Objective To prepare cross linking gelatin microspheres (CLG microspheres), analyze and detect physical properties.
Method Modified diphasic emulsified cold-condensation method was adapted to prepare CLG microspheres, with basic gelatin as material and 25% glutaral solution as cross linking agent. SEM was used to observe morphology and detect the average diameter of CLG microspheres before and after fully swelling in physiologic saline at 37?C. The maximum swelling radio of CLG microspheres was also evaluated. Microelectrophoresis was used to detect the surface charge of CLG microspheres. The ability of binding and delayed releasing dose-time profile of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA by CLG microspheres was also detected by ultraviolet spectrophotometry.
Results CLG microspheres were uniform spheroids with small pores on surface and sporadically dispersed without conglutination. The average diameter of CLG microspheres was 21.13±1.42μm and 26.72±1.74μm before and after fully swelling in physiologic saline at 37?C, and there was statistical difference between the average diameter of microspheres before and after fully swelling. The maximum swelling radio was 27.32±0.45% in physiologic saline at 37?C. The surface charge was positive. When the mass proportion of plasmid DNA vs. CLG microspheres reached 1:10, the absorption for plasmid DNA by CLG microspheres reached saturation, with entrapment radio of 62.75±3.31%. pcDNA3.1-Myc-His B(-)/tPA plasmid DNA could be continuously delayed released by CLG microspheres over 4 weeks in vitro.
Conclusion CLG microspheres possessed good ability of binding pcDNA3.1-Myc-His B(-)/tPA plasmid DNA to form pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, and pcDNA3.1-Myc-His B(-)/tPA plasmid DNA could be delayed released continuously from the complex over 4 weeks in vitro.
Experiment II Research of Cytotoxicity and Biocompatibility of Cross Linking Gelatin Microspheres
Objective To detect cytotoxicity and biocompatibility of CLG microspheres in order to evaluate safety of CLG microspheres used in vivo.
Method MTT assay was used to detect cytotoxicity of CLG microspheres. Haemolysis test, prothrombin time and recalcification time experiment were used to evaluate influence on erythrocyte and blood coagulation function by CLG microspheres. The influence on ability of adhesion and migration on Dacron fibers surface of human umbilical vein endothelium cells (HUVECs) by CLG microspheres were also evaluated through SEM, adhesion cells numbers counting and cell migration test.
Results Cytotoxicity of CLG microspheres retained as grade 1, and would not increase along with degradation time and concentration of CLG microspheres. The Haemolysis degree was 2.27±0.34%, which indicated that CLG microspheres would not destroy erythrocytes. Prothrombin time was 16.0±0.7 s, and there was no statistical difference with that of negative control group (17.2±0.8 s) (P >0.05). Recalcification time was 165.4±4.5 s, and there was no significantly statistical difference with that of common gelatin (169.2±5.4 s) or negative group (181.2±3.4 s) (P >0.05). Those results indicated that CLG microspheres would not facilitate exogenous and intrinsic coagulation. The amount of HUVECs on the surface of Dacron fibers coated with CLG microspheres solution was (1.47±0.30)×106 and (65.34±11.25)×106 at the end of week 1 and 4 respectively, and there were no statistical difference between that of untreated Dacron fibers, (1.63±0.26)×106 and (62.43±10.06)×106 respectively (P >0.05). The migration distance in CLG microspheres group was 29.61±3.54μm, and there was no statistical difference between that of negative control group (32.41±4.31μm) or common gelatin group (30.55±5.08μm) (P >0.05). Those results indicated that CLG microspheres would not inhibit the ability of adhesion and migration of HUVECs.
Conclusion CLG microspheres had little cytotoxicity and would not facilitate coagulation, or inhibit the ability of adhesion and migration of HUVECs, which indicated that CLG microspheres possessed good biocompatibility.
Experiment III Experimental Research of Gene Transfection of pcDNA3.1-Myc-His B(-)/tPA Plasmid DNA-Cross Linking Gelatin Microspheres Complex in vitro
Objective To detect transient gene transfection efficiency of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA being transferred into HUVECs, which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex in vitro, and gene expression after transfection.
Method Immunofluorescence staining was used to detect the transient gene transfection efficiency of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex. RT-PCR was used to detect tPA mRNA and Western blotting was used to detect tPA protein synthesized in HUVECs after transfection. ELISA was used to evaluate tPA content in culture medium supernatant, which was secreted by HUVECs after transfection.
Results pcDNA3.1-Myc-His B(-)/tPA plasmid DNA, which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, could be successfully transferred into HUVECs in vitro, with transient transfection efficiency of 12.74±2.53%, and it was significantly higher than that of simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution (4.63±1.75%) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex (26.49±5.23%) (P <0.05). The integral optical density ratio value of tPA mRNA electrophoresis strip in pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex group was 1.65±0.27, which was higher than simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution group (1.22±0.23) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex group (2.07±0.25) (P <0.05). The integral optical density ratio values of those 3 groups were all significantly higher than that of blank pcDNA3.1 plasmid DNA vector group (0.86±0.21) and HUVECs in control culture (0.91±0.18) (P <0.05). tPA expression could be detected in HUVECs after transfection by pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, however it was expressed in trace level in blank pcDNA3.1 plasmid DNA vector group and HUVECs in control culture. tPA could be secreted into culture medium supernatant by HUVECs after transfection, and tPA content in pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex grorp was 497.2±61.9 ng/ml, which was higher than simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution group (295.7±49.1 ng/ml) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex group (862.1±96.8 ng/ml) (P <0.05).
Conclusion pcDNA3.1-Myc-His B(-)/tPA plasmid DNA which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex could be successfully transferred into HUVECs and tPA synthesized in HUVECs could be secreted extracellularly after transfection.
引文
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