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Human β-NGF gene transferred to cat corneal endothelial cells

Wen-Juan Luo¹, Min Liu¹, Gui-Qiu Zhao¹, Chuan-Fu Wang¹, Li-Ting Hu ¹, Xiang-Ping Liu²

¹Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China

²Central Laboratory of the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China

Correspondence to: Wen-Juan Luo. Department of Ophthalmology, the Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China. Luowj76@163.com

Received: 2015-05-18 Accepted: 2016-02-03

Abstract

AIM: To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs.

METHODS: The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco’s modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope.

RESULTS: The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the percentage of G1cells in CEC-AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%.

CONCLUSION: Recombinant AAV-β-NGF promotes proliferation in cat CECs by expressing bioactive β-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.

KEYWORDS: nerve growth factor; corneal endothelial cell; transfect; proliferation

DOI:10.18240/ijo.2016.07.01

Citation: Luo WJ, Liu M, Zhao GQ, Wang CF, Hu LT, Liu XP. Human β-NGF gene transferred to cat corneal endothelial cells. Int J Ophthalmol 2016;9(7):937-942

INTRODUCTION

Cornea is one of the most important refracting media, its transparency and integrity are necessary to maintain visual function. Due to defect of regeneration ability, the healing of trauma in cornea is limited. Genetic therapy is a relatively new method, the high efficiency adeno-associated virus (AAV) vectors greatly facilitated the clinical application of genetic therapy. AAV has great superiorities such as low pathogenicity, extensive host cell, high titre and bigger package capability and so on. Nerve growth factor (NGF) is one of the neurotrophin family, which could promote the proliferation and differentiation of neurocyte and many non-neuron cells^[1]. In this study, we constructed AAV-β-NGF vector, transfered it to in vitro cultured cat corneal endothelial cells (CECs), expressed bioactive β-NGF protein in high efficiency and promoted the proliferation of CECs so as to lay a basis for the treatment of corneal endothelium blind.

MATERIALS AND METHODS

Design and Synthesize of Primers Specifically designed probe primers and fluorescent probes with ABI Primer Express software and analyzed with BLAST. 1) Human β-NGF gene: the forward and reverse primers were as follows 5′-CAC ACT GAG GTG CAT AGC GT -3′, 5′-TGA TGA CCG CTT GCT CCT GT -3′. Fluorescent probes：5′-ATC TGG ACT TCG AGG TCG GTG GTG C -3′, the length of the target gene is 182 bp. 2) Cat glyceraldehyde-3-phosphate dehydrogenase (endo-reference)：the forward and reverse primers were as follows 5′-CTT AGC ACC CCT GGC CAA G -3′, 5′-GAT GTT CTG GAG AGC CCC G -3′. Fluorescent probes: 5′-CAT GCC ATC ACT GCC ACC CAG AAG A -3′, the length of the target gene is 146 bp.

Primary Culture of Cat Corneal Endothelial Cells All animal procedures performed in this study complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of the Affiliated Hospital of Qingdao University. Two months old cats, with no limit to sexuality, healthy without medical history were used in this study. Cats were anesthetized by chloral hydrate. Totally 120 cat eye balls were enucleated and immersed in D-Hanks solution with 100 μg/mL penicillin and 100 μg/mL phytomycin for 30min and then rinsed with sterile water, ruled out abnormality with examination. The corneas were excised under sterile condition and placed in a petri dish containing DMEM. Under a dissecting microscope, Descemet’s membrane attached with endothelium was stripped from the stroma and placed in a 15-mL centrifuge tube containing 0.25% trypsin, incubated for 10min at 37℃. Cells were detached by vigorous disruption with a flame-polished pipette, centrifuged and resuspended in culture medium DMEM with 0.5% fetal bovine serum then were incubated in tissue culture bottles at 37℃ in a 5% CO₂ humidified atmosphere. Medium was changed every other day. Cells reached confluence in 10-14d. Monolayer cultures of cat endothelial cells were harvested using 0.05% trypsin/0.02% EDTA solution.

Immunocytochemistry Stainning of Cat Corneal Endothelium Cells Neuron specific enolase (NSE) is the specific mark protein of cat corneal endothelial cell, which could effectively distinguish endothelial cell from keratocyte. CEC could keep expressing NSE even after 20 passages while keratocyte never express NSE. Immunocytochemistry stainning was performed to identify the corneal endothelium cells with anti-NSE antibodies. Briefly, 1´10⁴ cells growing in chamber slides (Nalge Nunc International, Rochester, NY, USA) were fixed with 4% paraformaldehyde, rinsed with phosphate buffered saline(PBS) and permeabilized with ice-cold acetone. Non-specific binding was blocked by incubating cells in 1% bovine serum albumin (BSA) for 30min at room temperature. Added with anti-NSE antibodies (1:250 in PBS, Invitrogen Molecular Probes), cells were incubated overnight at 4℃, then rinsed with PBS. The second antibody was then applied for 1h at room temperature. After rinsed with PBS, cells were applied with ABC elite and DAB. Rinsed with ddH₂O for 3 times, cells were then coverslipped with Geltol (Thermo Electron Corp., Waltham, MA, USA) as a mounting media and viewed under inverted phase contrast microscope.

Transfer β-NGF Gene to in Vitro Cultured Corneal Endothelial Cells There are three groups in the study: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. The adeno-associated virus was diluted with DMEM to 1×10⁹ in tite, discard the supernatant liquid 4h later, then was added to DMEM with 10% blood serum. Two repeated wells were designed for each group. All the cells were harvested at 12h, 1, 3, 5 and 7d after transfection.

β-NGF Gene Expression Assay Total CEC RNA in three groups was isolated by Trizol reagent. 1) The purified RNA was analyzed by agrose gel electrophoresis and quantified spectraphotometrically. The β-NGF cDNA was synthesized in 10 μL real-time polymerase chain reaction (RT-PCR) mixture: 5×ExScript^TM Buffer 2 μL, 10 mmol/L dNTP Mixture 0.5 μL, 100 μmol/L Random 6mers 0.5 μL，ExScript^TM Rtase (200 U/μL) 0.25 μL，RNase Inhibitor(40 U/μL) 0.25 μL，total RNA 0.5 μg. Reverse transcription at 42℃ for 12min, then reverse transcriptase was deactivation at 95℃ for 2min. 2) Synthesize with specific primers: specifically designed probe primers of humanβ-NGF gene and cat glyceraldehyde-3-phosphate dehydrogenase were applied to PCR reaction. The reaction mixture was firstly pre-denatured at 95℃ for 10s, denatured at 95℃ for 5s, and 45s extension at 60℃, amplified in 40 cycles. 3) fluorescent quantitation PCR reaction: the reaction mixture was 20 μL includeμL: 2 ×Premix ExTaqTM buffer 10 μL, primers concentration 0.2 μmol/L, probes concentration 0.05 μmol/L, 50×ROX Reference Dye Ⅱ 0.4 μL, cDNA 2 μL. The reaction mixture was firstly pre-denatured at 95℃ for 10s, denatured at 95℃ for 5s, and 45 s extension at 60℃, amplified in 40 cycles. The mean CT value of the two values in each group was analyzed by 2^－△△CT method to get the relative express quantity. △△CT=(CTtarget gnen-CTendo-reference gene)transfected-(CTtarget gnen-CTendo-reference gene) control. Here the CT value means the cycles for the reaction mixture to reach the fluorescent threshold in PCR reaction.

Cell Proliferation Assay Cell proliferation was tested by modified MTT method. Forty-eight hours after being transfected, CECs in three groups were subcultured in 5.0×10⁴/mL to 96-well tissue culture plate for another 24h. For MTT assay, cells were switched to MTT solutions (5 mg/mL) 20 μL, 37℃, 5% CO₂ for 4h, then solution was discarded and 150 μL dimethyl sulfoxide (DMSO) was added to each well, traced blender shock for 10min, then detected the OD value in 490 nm.

Detect percentage of G1 cells by flow cytometry method Twenty-four hours after transfect passaged the CEC by 1:2 and subcultured for 48h. Digested cells with 0.05% trypsin, centrifuged and resuspended cells in ice cold PBS to remove cell debris in medium. Filetered the cell suspension with 500 mesh nylon network to get cell in 1×10⁶/mL. Stained cells according to the study plan and detected the cell cycle with FCM.

Morphology observation Keep observing the cells growth condition under inverted phase contrast microscope after transfection. Pick up 10 fields randomly, count the cell numbers and get the mean values. At 24, 48h and 5d after transfection, evaluated the differences of CECs proliferation between recombinant CEC-AAV-β-NGF group and two control groups .

Statistical Analysis Statistical analyses of quantity-PCR and MTT results were performed with SigmaStat 11.5 (SPSS) software. Differences between groups were assessed with a t-test and followed by a Tukey (Student-Neumann-Keuls) multiple comparisons of means tests. Data are expressed as means SD. A value of P<0.05 was considered to be statistically significant.

RESULTS

Primary Cultured Cell Morphology Observation CECs adhered in 2-3d primary culture, two weeks later expanded to massive single layer cells in shapes of similar circular and polygon. Cells were passaged and inoculated in 96-well board, 24h later most of them adhered.

Immunocytochemistry Stainning of Cat Corneal Endothelial Cells NSE staining show buffy macrobead could be found in the cytoplasm of corneal endothelium cells with over 98% positive rate (Figure 1A), while NSE was negative in keratocyte control group (Figure 1B).

Figure 1 NSE staining of cat endothelial cells and keratocyte A: NSE staining was positive in cat endothelial cells (×400); B: Keratocyte had no specific antigen staining (×400).

Synthesize with Specific Primers The total RNA of all groups is 1.8-2.0 in A₂₆₀/A₂₈₀. The electrophoresis result of PCR product shows a line in 182 bp and 146 bp, which is consist with human β-NGF gene and cat glyceraldehyde-3-phosphate dehydrogenase gene (Figure 2). The sequencing result shows the 182 bp line is human β-NGF gene which is totally consist with genebank.

Figure 2 Agarose gel electrophoresis of polymerase chain reaction products of target genes M: 100-600 bp marker; 1,2: cat glyceraldehyde-3-phosphate dehydrogenase gene (146 bp); 3,4: human β-NGF gene (182 bp).

Fluorescent Quantitation Polymerase Chain Reaction The results show that fluorescent quantitation PCR in every group got classic S shape curve. The Ct value of glyceraldehyde-3-phosphate dehydrogenase group diversified trifle which means the initiate quantity of templates in each group is almost the same. At 12h, 1, 3, 5 and 7d after transfection, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed: there were no significant differences between normal CEC control group and CEC-AAV control group (P>0.05); there were significant differences between the two control groups and recombinant CEC-AAV-β-NGF group in all the five time point (P<0.05); there were significant difference of β-NGF gene mRNA express in recombinant CEC-AAV-β-NGF group of different time points (Table 1, Figure 3).

Table 1 Relative quantitative analysis of human β-NGF Mrna (2^-ΔΔCt)

Group	Time	2^-ΔΔCt
A1	12h	1.0044±0.1812
A2	1d	0.9612±0.2031
A3	3d	1.1329±0.3062
A4	5d	1.1265±0.2843
A5	7d	0.9857±0.1328
B1	12h	1.0142±0.2039
B2	1d	1.1032±0.2121
B3	3d	1.1858±0.2962
B4	5d	1.2533±0.3127
B5	7d	0.9766±0.2051
C1	12h	2.8481±0.3985
C2	1d	4.9018±0.5290
C3	3d	9.5574±0.9528
C4	5d	16.9411±2.1384
C5	7d	13.0653±1.8124

A: Normal CEC control group; B: CEC-AAV control group; C: Recombinant CEC-AAV-β-NGF group.

Figure 3 Relative quantitative analysis of human β-NGF m RNA.

Biological Activities of the Expressed β-NGF In this study, the bioactivity of expressed β-NGF was detected based on the fact that it could promote CEC cells survival and proliferation. The MTT results showed 3d after transfection, the expressed β-NGF could obviously promote survival and proliferation of cat endothelial cells in CEC-AAV-β-NGF group (P<0.01 vs the two control groups). Two control groups showed no significant differences (P>0.05) in between. Blank AAV did not change the proliferation activity of CEC and there was no toxic effect of AAV to CEC (Table 2).

Table 2 MTT result of 3d after transfection

Blank medium	Normal CEC control	CEC-AAV control	CEC-AAV- β-NGF
0.255	1.625	1.502	2.638
0.242	1.641	1.531	2.753
0.273	1.614	1.624	2.687
0.234	1.586	1.543	2.861
0.263	1.609	1.590	2.569
0.257	1.592	1.649	2.826

Figure 4 FCM result of percentage of G1 cells 3d after transfection.

Detect Percentage of G1 Cells by Flow Cytometry Method FCM showed 3d after transfection, percentage of G1 cells in normal CEC control group is 46.6%, in CEC-AAV control group is 49.8% while in recombinant CEC-AAV-β-NGF group is 76.8%. The result means that transfection of AAV-β-NGF could put more cells to G1 stage while blank AAV hadn’t show this function (Figure 4).

Morphologic Changes of Corneal Endothelial Cells After Being Transfected CEC being transfected with recombinant CEC-AAV-β-NGF group proliferated obviously faster compared to the two control groups. After the first 24h, cells in all three groups proliferated in shapes of roundness and polygon in small scales. However, 48h later, cells transfected with recombinant CEC-AAV-β-NGF proliferated into bigger scales in shapes of regular triangle to hexagon with distinct boundary, while the number of cells was significantly less in the two control groups (Figure 5).

A B

Figure 5 Cells cultured for 48h after AAV-β-NGF transfection (×200) A: Blank control group; B: AAV-β-NGF group.

DISCUSSION

NGF is the first discovered and best-characterized member of the neurotrophin family ^[1]. It is produced by and acts upon cells of the visual system, both in vitro and in vivo and it is able to promote the functional recovery of retinal ganglion cells (RGCs) in an animal model of ocular ischemia and following optic nerve section, to reduce retinal cell damage induced by intraocular hypertension and to delay retinal cell degeneration in rodents with retinitis pigmentosa^[2-7]. These effects are mediated by two NGF-receptors, the high-affinity receptor tyrosine kinase (TrkA), and the low-affinity receptor p75 neurotrophin receptor (p75), both located on the surface of NGF-responsive cells. Altered expression of these receptors and/or their ligands can lead to NGF-target cell degeneration^[8]. NGF is present in the aqueous humor, increases following ocular injuries, and binds to its specific receptors expressed by the corneal endothelium. It has also been demonstrated that topical NGF eye drops administration promotes corneal healing and exerts anti-inflammatory and immunomodulatory actions on corneal endothelial cells^[9-11].

The NGF in the anterior segment played an important role in tissue maintenance and wound healing^[12]. High-affinity receptors of NGF are readily expressed on corneal tissues and are able to bind NGF^[10,13]. Topical NGF treatment was found to have a profound effect on corneal wound healing while restoring corneal epithelium and improving stromal and endothelial cell function^[9,14]. In conclusion, NGF has not only effects of nerve growth and regeneration but also pleiotropic effects on wound healing and tissue reconstruction^[15-20].

AAV vectors can efficiently initiate sustained transgene expression in vivo and appear to be safe. With the identification of different serotypes and recent progress in the improvement of AAV vectors, such as dual vectors to overcome the limited packaging capacity, self-complementary vectors to increase the level and onset of transgene expression, and capsid modifications to mediate cell specific transduction, it will be possible in the future to design more specific and efficient therapies for use in the gene treatment area ^[21-23].

In this study, we clone β-NGF gene into AAV vector to construct AAV-β-NGF vector and transfer it to in vitro cultured second passage cat corneal endothelial cells. There are three groups: normal CEC group, CEC-AAV control group and CEC-AAV-β-NGF group. Forty-eight hours after transfection, we detected the expression of β-NGF in CEC-AAV-β-NGF group was much higher than the other two control groups. The result of MTT test also showed the proliferation ability was much higher in CEC-AAV-β-NGF group than the other two control groups. Twenty-four hours after transfection, CECs in all three groups were passaged by 1:2 and cultured for another 48h followed by FCM test. The result showed percentage of G1 cells in CEC-AAV-β-NGF group is 76.8%, while that is 46.6% in CEC group and 49.8% in CEC-AAV group. All the results above showed that transfection of CEC-AAV-β-NGF could prominently promote the proliferation of in vitro cultured cat CECs and AAV has no toxicity in the transfection.

Herein, we report the potential of β-NGF gene therapy as a promising approach to promote corneal endothelial cells survival and proliferation thus provide further insight into the mechanisms of corneal endothelium blind treatment.

ACKNOWLEDGEMENTS

Foundations: Supported by National Natural Science Foundation (No.30572011); Natural Science Foundation of Shandong Province (No.ZR2010HQ041).

Conflicts of Interest: Luo WJ, None; Liu M, None; Zhao GQ, None; Wang CF, None; Hu LT, None; Liu XP, None.

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