Ultrasound-targeted microbubble destruction (UTMD) has been proposed as a new technique for organ-specific gene transfer and drug delivery. This study was performed to investigate the effect of UTMD on marrow mesenchymal stem cells (MSCs) transfected with pcDNA3.1−-hVEGF165.pcDNA3.1−-hVEGF165 were transfected into the third passage of MSCs, with or without UTMD under different ultrasound conditions. Protein expression was quantified by hVEGF165-ELISA kit after transfection for 24, 48, and 72 hours. UTMD-mediated transfection of MSCs yielded a significant protein expression. UTMD of mechanic index (MI) 0.6 for 90 seconds led to the highest level of protein expression.
Heart disease currently remains the leading cause of death worldwide. With the development of tissue engineering, stem cell technology has been widely used and highlights the latest advances in these exciting fields [
Our experiment was performed in the Clinical Research Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, China. Five male Sprague-Dawley rats, weighing 80–100 g, were provided by the animal center of Zhejiang University. All experiments have adhered to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications no. 8023, revised 1978). Approval from the Institutional Animal Care and Use Committee at Zhejiang University Health Science Centre was also obtained to perform the described experiments. MSCs were harvested from the bone marrow of femurs of these rats. Briefly, bone marrow cells were flushed out with 30 mL complete Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA), 5 mg/mL glutamine (Gibco, USA), 100 U/mL penicillin (Gibco, USA), and 100 U/mL streptomycin (Gibco, USA). The cells were grown in a humidified atmosphere containing 5% CO2 and 95% O2. The medium was replaced 24 hours later and refreshed every 2 days. Cells were subcultured according to 1 : 2 ratio when they reached approximately 80% confluence by trypsinization (0.25% trypsin, Gibco, USA). The third passage of MSCs was adopted for transfection.
The third passage of MSCs were planted into three 6-well plate (Becton Dickinson, USA) at 1.0 × 105 cells/per well and cultured for 24 hours in 37°C, 5% CO2 conditions. Before transfection, 5 mL normal saline were added to the microbubble contrast agent SonoVue (Bracco, Italy) powder (25 mg) and thoroughly mixed for 20 seconds. 4
In this study, all the cells were divided intothe following five groups: the blank control group: MSCs with culture fluid, the control group A: 4 the control group B: 4 the control group C: 4 the UTMD group: 4
The UTMD group was also divided into three groups according to different MI and ET.
Ultrasound-targeted microbubble was ruptured as following: Acuson Sequoia 512 ultrasound’s 3V2C transducer (Siemens, German) was placed on the bottom of each well plate according to the preset ultrasonic exposure condition. The ultrasound parameters were set as follows: the frequency was 4 MHz, the depth was 4 cm, MI was 0.6, 1.0, and 1.4 respectively, and ET was 30 s, 60 s, and 90 s, respectively.
MSCs cultural supernatant was collected after transfection for 24, 48, and 72 hours, respectively. Five samples were applied in each group.
hVEGF165-ELISA kit (Jingmei, China) was used to determine VEGF165 protein expression after transfection for 24, 48, and 72 hours according to the instructions.This was repeatedfive times in this experiment.
All the parameters were expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA), followed by a LSD (least significant difference) test was used to compare VEGF165 protein expression among different groups. All analyses were performed using SPSS statistical software, version 13.0 (SPSS, Inc., USA). A two-sided
The results showed that the VEGF165 protein expression increased at 24 hours and reached the maximum level at 48 hours, then decreased at 72 hours (Table
Protein expression of VEGF165 in mesenchymal stem cells supernatant after transfection (
Groups | Protein expression of VEGF165 | ||
24 h | 48 h | 72 h | |
(1) The blank control group | |||
(2) The control group A | |||
(3) The control group B | |||
(4) The control group C | |||
(5) The UTMD group |
*
(1) The blank control group: MSCs with culture fluid.
(2) The control group A: 4
(3) The control group B: 4
(4) The control group C: 4
(5) The UTMD group: 4
Table
Protein expression of VEGF165 in mesenchymal stem cells supernatant after transfection under different ultrasound conditions (
Various ultrasound conditions | Protein expression of VEGF165 | |||
ET | MI | 24 h | 48 h | 72 h |
0.6 | ||||
30 s | 1.0 | |||
1.4 | ||||
0.6 | ||||
60 s | 1.0 | |||
1.4 | ||||
0.6 | ||||
90 s | 1.0 | |||
1.4 |
*
The lack of suitable autologous grafts has produced a need for artificial grafts, but the patency of such grafts is limited compared to natural materials. Tissue engineering, whereby living tissue replacements can be constructed, has emerged as a solution to some of these difficulties [
VEGF, a class of molecular weight of 34~45 KD glycoprotein, could induce vascular endothelial cell proliferation and angiogenesis. VEGF165 protein-induced differentiation of MSCs directional vascular endothelial cells plays a vital role in neovascularization of ischemic tissues [
UTMD has evolved as a promising tool for organ-specific gene and drug delivery [
As the target cell of gene transfer, MSCs could promote expression of VEGF protein and vascularization of tissue engineering bone by transfected VEGF165. VEGF165 was a kind of secretary protein, whether the transfected gene could express effectively was the critical point of the present experiment.
Table
Table
The first limitation of this present study is that the number of samples is small. However, even with this small number of samples, we were able to reach our primary goal of investigating the protein expression of UTMD on MSCs transfected with pcDNA3.1−-hVEGF165. Secondly, the cell proliferation and angiogenesis of transfected MSCs by UTMD will not be traced, which is very important for tissue engineering. Thirdly, this study is limited in vitro. So further investigation, especially in larger animal models, is needed.
UTMD-mediated transfection of MSCs yielded a significant protein expression. UTMD of mechanic index (MI) 0.6 for 90 seconds led to the highest level of protein expression.
This study was supported by the Health Bureau of Zhejiang Provincial (2010KYB056).