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Gene Transfection to Retinal Ganglion Cells Mediated by Ultrasound Microbubbles in vitro

Posted Aug 31, 2009 Updated Apr 7, 2023
By Wei Li MS , Su Liu , Jianli Ren MD , Haibo Xiong MS , Xingyun Yan MS , Zhigang Wang
9 min read

Rationale and Objectives

To investigate the expression levels of green fluorescence protein (GFP) into retinal ganglion cells (RGCs) in vitro by ultrasound-mediated microbubble destruction (UMMD) and assess the effect of bcl-xl gene on N-methyl-D-aspartate (NMDA)-induced apoptosis in the cultured RGCs by UMMD.

Materials and Methods

pEGFP-N1 was transfected to RGCs in vitro by UMMD and liposome was used as the control. The transfection effect was detected using microscope and flow cytometry qualitatively and quantitatively. Monotetrazolium was adopted to measure the cell vitality. NMDA was used to induce apoptosis in the cultured RGCs, and the bcl-xl gene was transfected into RGCs by UMMD before NMDA-induced apoptosis. The expression of bcl-xl protein in RGCs was assessed by immunohistochemistry assay. The amorphous character of RGCs was revealed by acridine orange and ethidium bromide staining. DNA fragment was detected by agarose gel electrophoresis.

Results

Ultrasound combined with microbubbles enhanced gene transfection to the cultured cells in some condition. The average transfection rate of pEGFP-N1 with UMMD was 25%. Both ultrasound and microbubble had no effect on cell viability. The expression of bcl-xl protein in transfected and non-transfected RGCs was significantly different. Less apoptotic bodies and no representative DNA fragment were detected in the treatment group.

Conclusions

Microbubble destruction can enhance the reporter gene transfection and expression and have a good target. Transfection of bcl-xl gene has an anti-apoptosis effect on the cultured RGCs induced by NMDA with UMMD.

In recent years, numerous studies have indicated that ultrasonic irradiation itself not only promotes gene transfection and expression in vitro and in vivo and ultrasound-mediated microbubble destruction (UMMD) can further enhance gene transfection efficiency . It has become the research hotspot because it is safe, reliable, and low in cost . This study explored the gene transfection efficiency mediated by UMMD in retinal ganglion cells (RGCs) and the related condition in vitro. With the in-depth research in pathogenesis of glaucoma, it is recognized that to reduce intraocular pressure alone is not enough. Other ways to further protect the optic nerve and prevent or delay damage to RGCs need to be considered . Thus the new concept of optic nerve protection treatment has become the glaucoma research direction and focus in 21st century . Because RGC apoptosis is the key link to glaucoma, we established an RGC excitotoxicity injury model and used ultrasound microbubble–mediated anti-apoptotic gene bcl-xl and determined the optimum parameters for transfecting RGCs with the bcl-xl gene and subsequently investigated its effects on RGC apoptosis. We hope we can protect the optic nerve with new ideas.

Materials and methods

Preparation of Plasmid Enhanced Green Fluorescence Protein N1

Bacillus coli DH5 (Invitrogen, Carlsbad, CA) was resuscitated by the conventional method and plasmid enhanced green fluorescence protein N1 (pEGFP-N 1 ) was inverted into bacteria by classical calcium chloride method. We extracted plasmid according to Kit (EndoFree Plasmid Maxi Kit; Qiagen, Germany) requirements. A small amount of plasmid was electrophoresis, and plasmid concentration was adjusted to 1 μg/μL by spectrophotometry.

Culture and Identification of Retinal Ganglion Cells

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Experimental Groups and Transfection of EGFP Gene

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Cell Transfection Analysis

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Flow Cytometric Analysis of Transfected Quantitative Gene Expression

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Methyl Thiazolyl Tetrazolium Assay

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Anti-apoptosis Effect of bcl-xl on RGCs by UMMD

Preparation and identification of the plasmid SFFV.neo-bcl-xl

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Cultivated RGCs were identified by immunocytochemical method

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Ultrasound-mediated Microbubble Transfection Cell Model

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Immunohistochemical Detection

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The Morphology Detection of Apoptosis

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Agarose Electrophoresis Detection

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Statistical Methods

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Results

UMMD Parameter Selection

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Figure 1, Retinal ganglion cells (RGCs) cultured for 3 days under a contrast phase microscope (×400). RGCs body cell grew larger and further increased. Axons grew longer and connected with each other after culturing for 72 hours. Neurite grew further and a few cells had typical cell processes and axons connect.

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The Best Transfection Efficiency Parameters of UMMD

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Table 1

The Role of Different Ultrasound Intensity, Exposure Time and Plasmid Concentration to the Transfection Efficiency in the RGC and the Activity of the RGC

Groups Examples n Transfected cells rate AbsorbanceUltrasound Intensity Control group 8 0.62±0.04 0.245±0.032 0.25 8 17.3±4.21 0.246±0.029 0.50 8 19.6±3.22 0.237±0.041 0.75 8 22.8±5.63 0.221±0.034 1.00 8 23.1±4.32 0.185±0.017▴ 1.25 8 20.2±4.25 0.172±0.009Exposure time Control group 8 0.59±0.02 0.241±0.025 30s 8 16.4±3.16 0.238±0.019 60s 8 22.5±4.23 0.226±0.031 90s 8 24.1±3.12 0.195±0.023▴ 120s 8 21.5±2.58 0.181±0.012Plasmid concentration Control group 8 0 0.252±0.034 0.8μg/100μl 8 14.5±2.82 0.243±0.015 2μg/100μl 8 21.6±3.26 0.241±0.021 4μg/100μl 8 25.8±3.83 0.237±0.023 6μg/100μl 8 19.3±3.10 0.185±0.017▴

The role of different machinery index, exposure time and plasmid concentration to the transfection efficiency in the RGC and the activity of the RGC (X±S) note compared with group ahead P<0.05. ▴P<0.05.

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Cytoactive Determination

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Comparative Analysis of the Cells Transfection Positive Rate and Expression Level between UMMD and Liposome

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Figure 2, Fluorescent microscopic images of green fluorescence protein N1 (pEGFP-N 1 ) expression in retinal ganglion cells (RGCs) (×200). Cells were transfected with all treated factors. Cells were cultured for 48 hours and the enhanced green fluorescent protein (EGFP) gene expression was observed using fluorescent microscopy. (a) RGCs transfected by EGFP gene with ultrasound-mediated microbubble destruction. (b) RGCs transfected by EGFP gene with liposome. The average positive rate of EGFP gene by ultrasound-mediated microbubble destruction is 26.2 ± 1.3%, and the liposomes method is 18.1 ± 1.5% ( P < .05).

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Identify SFFV.neo-bcl-xl Plasmid by Enzyme Digestion

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Figure 3, Identify SFFV.neo-bcl-xl plasmid by enzyme digestion. M: λ-EcoT14I digest DNA maker. I: SFFV.neo-bcl-xl plasmid by enzyme digestion.

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RGCs Morphology Observation and Identification

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Figure 4, Retinal ganglion cells (RGCs) transfected enhanced green fluorescent protein (EGFP) gene for 2 days from a phase contrast microscope (×200).

Figure 5, After being cultured 2 days, retinal ganglion cells (RGCs) labeled by Thy1.1 monoclonal antibodies were yellowish green (FITC ×200).

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Immunohistochemical Detection

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Figure 6, bcl-xl protein expresses in retinal ganglion cells (RGCs) that are not transfected. Many cells are light brown DAB coloration ×200). Most bcl-xl protein in RGCs without extra transfected bcl-xl are rendered light brown.

Figure 7, bcl-xl protein express in retinal ganglion cells (RGCs), which are transfected. Many cells are deep brown (DAB coloration ×200). Most bcl-xl protein in RGCs with extra transfected bcl-xl are rendered dark brown.

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The Morphology Detection of Apoptosis

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Figure 8, Retinal ganglion cells (RGCs) that are not transfected are mainly jacinth. A typical apoptotic body has nucleus dense dyed and karyopyknosis (AO-EB stain, laser confocal microscopy ×400). Most RGCs without extra transfected bcl-xl stained by acridine orange and ethidium bromide were orange.

Figure 9, Retinal ganglion cells (RGCs) that are transfected are mainly green. Minority cells have nucleus dense dyed and karyopyknosis (acridine orange and ethidium bromide stain, laser confocal microscopy ×400). Most RGCs with extra transfected bcl-xl stained by acridine orange and ethidium bromide stain were green.

Figure 10, DNA agarose gel electrophoresis. M: DNA Marker; Group A: normal contrast group, ladder strap; Group C: little ladder strap. (a) control, (b) DNA “ladder,” (c) DNA without typical “ladder.”

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Discussion

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Academic Radiology, Volume 16, Issue 9
Journals General Radiology
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