ABSTRACT
Background: Ultrasound-targeted microbubble destruction has been utilized to deliver a drug/gene into cells in vitro and in vivo. Objective: To access the effects of ultrasound or/and microbubbles on recombinant adeno-associated virus (rAAV)-mediated transgene expression in rat retinal pigmented epithelium-J (RPE-J) cells in vitro and Wistar rat retina in vivo and to compare the difference between them. Methods: Different doses of serotype-2 rAAV (rAAV2) encoding an enhanced green fluorescent protein gene and microbubbles were administered to RPE-J cells in vitro and Wistar rat retina in vivo under different ultrasound conditions. Transfection efficiency was assessed by fluorescence microscopy, fluorescence stereoscope and flow cytometry analysis. Cell viability and tissue damage were assessed by trypan blue staining and hematoxylineosin staining. Results: rAAV2-mediated transgene expression in vivo was significantly enhanced by ultrasound and microbubbles, while rAAV2-mediated gene transfection in vitro was significantly enhanced by ultrasound or microbubbles alone. Conclusion: Ultrasound or/and microbubbles-mediated rAAV delivery strategies must be chosen for retinal gene therapy based on their effects in vitro and in vivo.
ABSTRACT
Background: Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment surgery or ocular trauma. Growth factors are believed to play an important role in promoting the events that contribute to PVR. It is important to study the pathogeneic of PVR and possible therapies. Objective: To determine whether heterogenic differentiated retinal pigmented epithelial (RPE) cells and/or syngeneic platelet-rich plasma (PRP) injected into the vitreous cavity of Wistar rats could create a model for PVR, and to quantitatively detect the expression levels of transforming growth factor-\β2 (TGF-\β2), platelet-derived growth factor-AA and BB (PDGF-AA,PDGF-BB) in this rat model. Methods: RPE-J cells and autogeneic PRP were injected separately (or in combination) into the vitreous cavity of adult Wistar rats (4.5x10⁵ RPE-J cells /eye and 3.7x10⁹ platelets/eye). Seven, 14, 21, and 28 days after cell injections, PVR was quantified using surgical microscopy and histopathologic examination. Three, seven, 14, 21, and 28 days after cell injections, the expression levels of TGF-\β2, PDGF-AA and PDGF-BB were tested using enzyme-linked immunosorbent assay (ELISA). Results: The intravitreal injection of RPE-J cells alone could not initiate a proliferative process in the eyes. However, PRP alone or in combination with RPE-J cells, induced a PVR-like process characterized by inflammatory cell infiltration, extracellular collagen production, and formation of epiretinal membranes. The strongest proliferative process was induced by the co-injection of RPE-J cells and PRP into the eyes of Wistar rats. In this process, the expression levels of TGF-\β2, PDGF-BB were significantly up-regulated. Conclusion: Intravitreal co-injection of RPE-J cells and PRP in Wistar rat eyes could effectively induce a model of PVR. PRP clearly promoted the proliferative process of PVR. TGF-\β2 and PDGF-BB took part in the pathogenesis of this model.
ABSTRACT
Objective To investigate the transfection efficiency of combination of ultrasound (US) microbubbles (MBs) mediated liposome small interference RNA (siRNA) to human and rat retinal pigment epithelium (RPE) cells. Methods Human and rat RPE cells and siRNA were incubated in 24-well plates (2×10~5/well and 1×10~5/well, respectively). The cells were devided into 5 groups: siRNA+US, siRNA+MBs+US, siRNA+L (liposome), siRNA+L+US, siRNA+L+US+MBs. After 12 h, transfection efficiency was observed with fluorescence microscopy and flow cytometry. Results US or ultrasound targeted microbubbles destruction without liposome-mediated could not promote siRNA transfection efficiency to human and rat RPE cells. The transfection efficiency of human and rat RPE cells significantly decreased in the siRNA+L+US+MBs group, but increased in siRNA+L+US group. Conclusion Ultrasonic irradiation can promote lipid-mediated siRNA transfected human RPE cells.
ABSTRACT
Objective To investigate the practical efficacy and safety of ultrasound with microbubbles mediated rAAV2-EGFP to retina of rat after intravitreal and subretinal injection. Methods Gene transfer was examined by rAAV2-EGFP intravitreal and subretinal injection into the Wistar rats with or without microbubbles. The eyes were exposed to US (1 MHz,2 W/cm2, duration 5 minutes,duty cycle 50%,pulse recurrent frequency 100 Hz). The onset of EGFP gene expression, lightness of fluorescence, area of fluorescence and its distribution in the fundus in vivo via fluorescence stereosocope were investigated on the 4th,7th, 35th,49th and 120th day respectively. The value of gene transfer was quantified through the EGFP fluorescence quantitative methods by Axiovision 3. 1 software. HE staining was used to observe tissue damage. Results There was no fluorescence observed by fluorescence stereosocope after intravitreal injection after two-month study. After subretinal injection, ultrasound-targeted microbubbles destruction (UTMD) strongly increased gene transfer efficiency. UTMD used in the experiment did no harm to the rat retina structure. Conclusions UTMD could not enhance rAAV2-EGFP transfecion efficiency to rat retina after intravitreal injection but the transduction could be enhanced significantly after subretinal injection.