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Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction.

Xu Y, Xie Z, Zhou Y, Zhou X, Li P, Wang Z, Zhang Q - Mol. Vis. (2015)

Bottom Line: Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes.Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01).These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ultrasound Imaging and Department of Ultrasound, Second Affiliated Hospital of Chongqing Medical University; Chongqing Key Laboratory of Ultrasound Molecular Imaging, Chongqing, P.R. China.

ABSTRACT

Purpose: The purpose of this study was to investigate whether ultrasound-targeted cationic microbubble destruction could effectively deliver endostatin-green fluorescent protein (ES-GFP) plasmids to human retinal vascular endothelial cells (HRECs).

Methods: Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes. First, the two types of microbubbles were characterized in terms of size and zeta potential. The cell viability of the HRECs was measured using the 3-(4,5-dimthylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. The transcription and expression of endostatin, VEGF, Bcl-2, and Bcl-xl were measured via quantitative real-time PCR (qPCR) and western blotting, respectively.

Results: CMBs differed significantly from NMBs in terms of the zeta potential, but no differences in size were detected. Following ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were highest in the CMB group (p<0.05), while the transcription and expression of VEGF, Bcl-2, and Bcl-xl were lowest compared with the other groups. Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01).

Conclusions: This study demonstrated that ultrasound-mediated cationic microbubbles could enhance the transfection efficiency of ES-GFP, which had obvious impacts on the inhibition of the growth process of HRECs in vitro. These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization.

No MeSH data available.


Related in: MedlinePlus

qPCR analysis of the transcription of the ES-GFP plasmid and the effect on VEGF, Bcl-2, and Bcl-xl. Endostatin (A), VEGF (B) Bcl-2(C), and Bcl-xl (D) mRNA levels were measured via quantitative real-time PCR (qPCR) 48 h after ultrasound-targeted microbubble destruction (UTMD)-mediated embryonic stem (ES) gene delivery. The levels of endostatin mRNA were significantly higher following cationic microbubble (CMB) delivery of the ES gene compared with the neutral microbubble (NMB), liposome, and control groups. VEGF, Bcl-2, and Bcl-xl mRNA levels were significantly lower following CMB delivery of the ES gene compared with the NMB, liposome, and control groups (* p<0.05 relative to the control and CMB groups, #p<0.01 relative to the control; n=6 per group).
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f4: qPCR analysis of the transcription of the ES-GFP plasmid and the effect on VEGF, Bcl-2, and Bcl-xl. Endostatin (A), VEGF (B) Bcl-2(C), and Bcl-xl (D) mRNA levels were measured via quantitative real-time PCR (qPCR) 48 h after ultrasound-targeted microbubble destruction (UTMD)-mediated embryonic stem (ES) gene delivery. The levels of endostatin mRNA were significantly higher following cationic microbubble (CMB) delivery of the ES gene compared with the neutral microbubble (NMB), liposome, and control groups. VEGF, Bcl-2, and Bcl-xl mRNA levels were significantly lower following CMB delivery of the ES gene compared with the NMB, liposome, and control groups (* p<0.05 relative to the control and CMB groups, #p<0.01 relative to the control; n=6 per group).

Mentions: To evaluate the transcription of the ES-GFP gene after transfection into HRECs in vitro, the relative ratio of ES RNA in the treated and control HRECs was determined via qPCR at 48 h post-transfection (Figure 4A). To evaluate the effect of endostatin on VEGF, Bcl-2, and Bcl-xl expression, the relative ratios of VEGF, Bcl-2, and Bcl-xl RNA were also determined (Figure 4B–D). Following transfer, the transcription levels of the ES gene were highest in the CMB group (p<0.05 relative to the NMB and liposome groups, p<0.01 relative to the control group), while there was almost no difference between the NMB and liposome groups (p>0.05). With the increasing levels of ES RNA, the level of VEGF, Bcl-2, and Bcl-xl mRNA decreased (Figure 4). Thus, these effects may be due to the therapeutic effect induced by endostatin.


Experimental endostatin-GFP gene transfection into human retinal vascular endothelial cells using ultrasound-targeted cationic microbubble destruction.

Xu Y, Xie Z, Zhou Y, Zhou X, Li P, Wang Z, Zhang Q - Mol. Vis. (2015)

qPCR analysis of the transcription of the ES-GFP plasmid and the effect on VEGF, Bcl-2, and Bcl-xl. Endostatin (A), VEGF (B) Bcl-2(C), and Bcl-xl (D) mRNA levels were measured via quantitative real-time PCR (qPCR) 48 h after ultrasound-targeted microbubble destruction (UTMD)-mediated embryonic stem (ES) gene delivery. The levels of endostatin mRNA were significantly higher following cationic microbubble (CMB) delivery of the ES gene compared with the neutral microbubble (NMB), liposome, and control groups. VEGF, Bcl-2, and Bcl-xl mRNA levels were significantly lower following CMB delivery of the ES gene compared with the NMB, liposome, and control groups (* p<0.05 relative to the control and CMB groups, #p<0.01 relative to the control; n=6 per group).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548789&req=5

f4: qPCR analysis of the transcription of the ES-GFP plasmid and the effect on VEGF, Bcl-2, and Bcl-xl. Endostatin (A), VEGF (B) Bcl-2(C), and Bcl-xl (D) mRNA levels were measured via quantitative real-time PCR (qPCR) 48 h after ultrasound-targeted microbubble destruction (UTMD)-mediated embryonic stem (ES) gene delivery. The levels of endostatin mRNA were significantly higher following cationic microbubble (CMB) delivery of the ES gene compared with the neutral microbubble (NMB), liposome, and control groups. VEGF, Bcl-2, and Bcl-xl mRNA levels were significantly lower following CMB delivery of the ES gene compared with the NMB, liposome, and control groups (* p<0.05 relative to the control and CMB groups, #p<0.01 relative to the control; n=6 per group).
Mentions: To evaluate the transcription of the ES-GFP gene after transfection into HRECs in vitro, the relative ratio of ES RNA in the treated and control HRECs was determined via qPCR at 48 h post-transfection (Figure 4A). To evaluate the effect of endostatin on VEGF, Bcl-2, and Bcl-xl expression, the relative ratios of VEGF, Bcl-2, and Bcl-xl RNA were also determined (Figure 4B–D). Following transfer, the transcription levels of the ES gene were highest in the CMB group (p<0.05 relative to the NMB and liposome groups, p<0.01 relative to the control group), while there was almost no difference between the NMB and liposome groups (p>0.05). With the increasing levels of ES RNA, the level of VEGF, Bcl-2, and Bcl-xl mRNA decreased (Figure 4). Thus, these effects may be due to the therapeutic effect induced by endostatin.

Bottom Line: Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes.Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01).These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ultrasound Imaging and Department of Ultrasound, Second Affiliated Hospital of Chongqing Medical University; Chongqing Key Laboratory of Ultrasound Molecular Imaging, Chongqing, P.R. China.

ABSTRACT

Purpose: The purpose of this study was to investigate whether ultrasound-targeted cationic microbubble destruction could effectively deliver endostatin-green fluorescent protein (ES-GFP) plasmids to human retinal vascular endothelial cells (HRECs).

Methods: Cationic microbubbles (CMBs) were prepared and then compared with neutral microbubbles (NMBs) and liposomes. First, the two types of microbubbles were characterized in terms of size and zeta potential. The cell viability of the HRECs was measured using the 3-(4,5-dimthylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. The transcription and expression of endostatin, VEGF, Bcl-2, and Bcl-xl were measured via quantitative real-time PCR (qPCR) and western blotting, respectively.

Results: CMBs differed significantly from NMBs in terms of the zeta potential, but no differences in size were detected. Following ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were highest in the CMB group (p<0.05), while the transcription and expression of VEGF, Bcl-2, and Bcl-xl were lowest compared with the other groups. Moreover, the inhibition of HREC growth was enhanced following treatment with CMBs compared with NMBs or liposomes in vitro (p<0.01).

Conclusions: This study demonstrated that ultrasound-mediated cationic microbubbles could enhance the transfection efficiency of ES-GFP, which had obvious impacts on the inhibition of the growth process of HRECs in vitro. These results suggest that the combination of UTMD and ES-GFP compounds might be a useful tool for gene therapy targeting retinal neovascularization.

No MeSH data available.


Related in: MedlinePlus