Abstract
BACKGROUND: There are still few effective methods to repair injured myocardium after myocardial failure and pathologically rebuild reverral myocardium. As a new therapy, normal myocytes and therapeutic gene to interfere injured myocardium have advantageous effects in improving heart function.
OBJECTIVE: To observe the efficiency and stability of adenovirus-medicated gene transferred into different passages of bone marrow mesenchymal stem cell (MSC) and investigate the effect of MSC-based sarcoplasmic reticulum Ca2+ ATPase gene (SERCA2a) gene therapy for rats with chronic heart failure. To compare the effects of gene therapy, cell transplantation and MSC-based SERCA2a gene therapy for chronic heart failure.
DESIGN: Randomized controlled study.
SETTING: Department of Senile Angiocardiopathy, General Hospital of Chinese PLA; Department of Biochemistry, Beijing Medical University.
MATERIALS: Male Sprague-Dawley (SD) rats with 4 weeks old, clean grade and weighing 45-50 g provided by the Animal Experimental Center, Peking Medical University were used as donators of bone marrow. Other female SD rats of 12 weeks old, clean grade and weighing 200-250 g were used as receptors of cell transplantation and gene therapy. Sry gene of Y chromosome in male rats was used to evaluate whether transplanted cells of donators lived in myocardium of receptor rats. Ad-SERCa2a and Ad-EGFP were constructed by Doctor Lu Xiao-chun; MSC in the 3rd and 8th generations was isolating cultured on its own.
METHODS: The experiment was carried out in the Zhou CY Laboratory (BSL-2), Department of Biochemistry, Beijing Medical University from July 2004 to December 2005. Thirty female SD rats received ligation at the left coronary artery to make models with chronic cardiac failure following acute myocardial infarction. And then, 29 rats were randomly divided into four groups, including gene therapy group (n=7), MSC group (n=7), gene-modified MSC group (n=8) and control group (n=7). Rats in the four groups were given the interventions of SERCA2a gene, MSC transplantation, MSC+Ad/SERCa2a and empty adenoviral vector, respectively. MSCs were separated and cultured, and then Ad-SERCA2a-GFP was used to transfer MSC in the 3rd and 8th generations.
MAIN OUTCOME MEASURES: Ad-SERCA2a-GFP transfection rate of MSC was measured by using flow cytometer. Before and at 14 and 21 days after treatment, cardiac function was evaluated by ultrasonic echocardiogram. Expression of cytokine Ⅷ was tested by immunohistochemical staining. SERCA2a gene and protein expression were evaluated by RT-PCR and Western blot respectively, as well as SERCA2a enzyme activity.
RESULTS: ① Transfection rate: The infection efficiency of adenovirus-medicated gene into different passages of MSC was over 80%, and there was no difference between passage three (P3) MSC and P8 MSC (P > 0.05). ② Heart function: Left ventricle wall was thickened obviously in group MSC and group MSC+Ad/SERCa2a on the 21st day after treatment, while volume was shortened and gradually rounded. Compared to control group, ejection fraction (EF) and shortening fraction (FS) of group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a were elevated significantly on the 14th day after therapy (P < 0.01). While the elevation values of EF and FS began to reduce in group Ad-SERCa2a on 14th day after therapy, it continued to increase in both group MSC and group MSC+Ad/SERCa2a (P < 0.01). Improvement rate of EF at 21 days after therapy (EF D21) increased in group MSC and group MSC+Ad/SERCa2a respectively, but decreased in group Ad-SERCa2a. Compared to group Ad-SERCa2a, peak systolic flow velocity of anterior wall and interventricular septum in group MSC+Ad/SERCa2a increased significantly on the 21st day after therapy, and peak diastolic flow velocity of anterior wall and interventricular septum elevated in group MSC+Ad/SERCa2a, too (P < 0.01). ③ SERCA2a gene, protein expression and enzyme activity in group MSC+Ad/SERCa2a were significantly stronger in group MSC and control group. Parts of MSC transplanted into scar zone expressed Ⅷ.
CONCLUSION: ① MSC is an effective platform for the targeted delivery of therapeutic gene. It suggests that different passages of MSC from P3 MSC to P8 MSC are regarded as high-effectively gene vehicles. MSC-based SERCA2a gene therapy showed much strong and lasting beneficial effect on exhausted myocardium. ② Effect of MSC transplantation on improving heart function may be related to promoting vascular neogenesis.

INTRODUCTION

Chronic heart failure (CHF) is a serious public health problem all over the world. CHF has a high prevalence and mortality[1- 2]. The alarming reality is our current lack of an effective therapy to repair or otherwise reverse severe forms of cardiac dysfunction and pathological remodeling associated with heart failure. So there is a well-recognized urgent need for development of novel alternative therapeutic approaches. As a new strategy, cell-based therapeutic maneuver, which aims to introduce healthy myogenic cells and therapeutic gene into the myocardium of the disease heart, is showing bright future in this field. Until now, a number of cell types, including skeletal myoblasts, fetal myocardial cell，smooth muscles, embryonic stem cells and marrow stromal cell (MSC),have been employed for transplantation, and have shown benefits on heart functions in various animal models of myocardium injury.

But MSC is considered as easy culture and good proliferation, no moral controversy and no immunoreaction in auto-grafting, and it has been taken as the most potential candidate cell for clinical therapy[3-6].
Besides, sarcoplasmic reticulum calcium ATPase (SERCA2a) gene has already been taken as the most valuable target gene for heart failure, especially end-stage heart failure. Data from animal models have emerged in recent years suggest that the therapeutic benefits of SERCA2a targeted enhancement in cardiac contractility and relaxation as a way to prevent or reverse heart failure[7-9].
The current studies investigate that the feasibility of SERCA2a gene transferring into MSC by using the adenovirus vector, explores the effect of MSC-based SERCA2a gene therapy of heart failure and provides the experimental data for cell-based gene therapy of cardiovascular disease.

MATERIALS AND METHODS

Materials
Animals
The experiment was carried out in the Zhou CY Laboratory (BSL-2), Department of Biochemistry, Beijing Medical University from July 2004 to December 2005. Male Sprague-Dawley (SD) rats of 4 weeks old, clean grade and weighing 45-50 g provided by the Animal Experimental Center, Peking Medical University [SCXK (jing) 2006-0008] were used as donators of bone marrow. Other female SD rats of 12 weeks old, clean grade and weighing 200-250 g were used as receptors of cell transplantation and gene therapy. All animal experiments were approved by the Animal Care and Use Committee of Academy of health science center of Peking University and were in compliance with the European Convention on Animal Care.

Main equipments and reagents
Small animal breathing machine (Medical Tool Factory of Zhejiang Medical University); freezing microtome (Leica Company, Germany); cardiac ultrasonic echocardiogram (HDI5000 model device, Philips); Ad-SERCa2a, Ad-EGFP (constructed by Doctor Kelu Xiaofu); P3MSC, P8MSC self-isolated culture; Iscove's modified Dulbecco's medium (Gibco, Grand Island, N.Y. USA); 20% fetal bovine serum (Gibco, Grand Island, N.Y. USA); Hoechst-33342 at a concentration of 10 μg/mL at 37℃ for 30 minutes (Sigma-Aldrich, St. Louis, Missouri, USA); AMV reverse transcriptase (Promega); Trizol reagent (Promega); Rabbit polyclonal antibodies against either rat Ⅷ (ab6994, Abcam) or α-actinin (A7811, Sigma).
FACS Vantage flow cytometer (Becton Dickinson, San Jose, USA); FACS flow (Becton Dickinson); Calibrite beads (Becton Dickinson); a pressure transducer (model ML110, AD Instruments, Australia); a transducer amplifier (SP8520, Powerlab/8SP, AD Instruments Pty Ltd, Castle Hill, Australia); digital data acquisition system (Chart V4.2, Powerlab, AD Instruments ).

Methods
Cell culture
Bone marrow was obtained from the femur of 4-week old male donor SD rats. Nucleated cells were isolated by Ficoll-Hypaque gradient centrifugation and cultured at 37℃ in humidified air with 5% CO2 in Iscove's modified Dulbecco's medium (Gibco, Grand Island, N.Y. USA) containing 20% fetal bovine serum (Gibco, Grand Island, N.Y. USA), penicillin (100 μg/mL) and streptomycin (100 μg/mL). The medium was changed to remove non-adherent cells 24 hours after seeding and every 3 days thereafter. At 80% confluence, cells were in passage at a ratio of one to three and cultured for 8 passages. Cells were stained with Hoechst-33342 at a concentration of 10 μg/mL at 37 ℃ for 30 minutes (Sigma-Aldrich, St. Louis, Missouri, USA) before transplantation.

Transferred with Ad-SERCA2a-GFP into MSC
The viral titers of Ad-SERCA2a-GFP and Ad-GFP were 1×1012 pu/L. Adenovirus-mediated gene transfer was performed as previously described[10]. Briefly, the cells were seeded at a density of 2×106 cells per 15-cm plate. MSC were exposed to the infectious viral particles in 7.5 mL IMDM at 37 ℃ medium for 60 minutes; cells were infected with Ad-SERCA2a-GFP at a multiplicity of infection (MOI) of 300. The medium was then removed, and the cells were washed once with IMDM and then re-cultured with normal medium for 24 hours, after which transplantation was performed.

Infection efficiency by flow cytometry
Cell suspension was centrifuged at 1 000 r/min for 3 minutes. The pellet was re-suspended in 0.5 mL of PBS and filtered through a 41 μm nylon mesh and subjected to flow cytometry. Flow cytometric analysis was performed using a FACS Vantage flow cytometer (Becton Dickinson, San Jose, USA). Cells were analyzed at a rate of 500 to 1 000 cells/s using FACS flow (Becton Dickinson) as sheath fluid and 10 000 events for each sample were recorded for analysis. The flow cytometer was standardized for each analysis session by using Calibrite beads (Becton Dickinson). Data were analyzed with WinMDI version 2.8.

Myocardial infarction model and MSC transplantation, gene therapy and MSC-based SERCA2a gene therapy
A previously described rat myocardial infarction model[11] was used in this study. Female recipient SD rats were anesthetized with 5% isoflurane. The proximal left coronary artery was circumferentially ligated with a 6-0 prolene suture. After myocardial ischemia was confirmed by regional myocardial color changes, the incision was closed in layers with 3-0 silk continuous sutures. 0.4 mL penlong XL (penicillin G benzathine, 150 000 U/mL, and penicillin G procaine, 150 000 U/mL) was injected intramuscularly and 0.3 mg morphine hydrochloride was injected subcutaneously as analgesics.
Four weeks after coronary artery ligation, rats with ejection fraction < 45% were divided into 4 groups: Group Ad-SERCa2a (n=7) received SERCA2a gene therapy, group MSC (n=7) received MSC transplantation, group MSC+Ad/SERCa2a (n=8) received MSC-based SERCA2a gene transplantation, and control group (n=7) received empty adenoviral vector. Every rat had been treated for 21 days. Cyclosporine A (10 mg/kg) was injected subcutaneously on a daily basis.

Assessment of infarct size and left ventricular morphological analysis
At 21 days after therapy, all animals were killed. The heart was transected parallel to the atrioventricular groove at the center of the infarct. Three slices of the left ventricle were used for hematoxylin-eosin (HE) staining and Masson's staining, for immunohistochemical staining, for western blotting and PCR of Sry gene. The distal portion of the heart was fixed with 10% formalin, embedded in molten paraffin, and 5 μm sections were prepared for subsequent HE staining, Masson's staining and immunohistochemical staining for Ⅷ. A Masson's stained cross-section from each heart was used to assess infarct size in each animal. The H&&E stained cross-section was used to analyze left ventricular morphology. The rest of the proximal portion of the ventricle was frozen for further analysis.

Left ventricular function
Rats underwent 2-dimensional transthoracic echocardiography with a HDI 5000 model device (Philips) with sectorial transducers S12 (5-12 MHz) and threshold of 15L6 (7- 15 mHz), which allowed an analysis of up to 160 Hz before 1 day, after 4weeks, 6 weeks and 7 weeks of coronary artery ligation.
The maximal and minimal values of the first derivative of LV pressure (dp/dtmax and dp/dtmin, respectivel) were measured using a pressure transducer (model ML110, AD Instruments, Australia) amplified with a transducer amplifier (SP8520, Powerlab/8SP, AD Instruments Pty Ltd, Castle Hill, Australia), and data were recorded using a digital data acquisition system (Chart V4.2, Powerlab, AD Instruments).
All measurements were taken 3 times by the same echocardiographer and the mean for each parameter was analyzed by an examiner who was unaware of the phase of the study and the groups' identities.

DNA and RNA preparation and conventional RT-PCR
Genomic DNA of tissue from the injection site was isolated using the phenol-chloroform method. PCR amplification of the Sry gene (forward primer: 5'AGTGTTCAGCCCACAGCCTT
GAGGAC3'; reverse primer: 5'GTGTGTAGGTTGTTGTCC
CATTGCAGC3', annealing temperature 74 ℃, 1 minute, 40 cycles) was used to identify male donor-derived cells in female recipient tissues[12].
Total RNA was isolated using Trizol reagent (Promega) according to the protocol and converted to cDNA by AMV reverse transcriptase (Promega) according to the protocol. RT-PCR amplification of the SERCA2a gene (forward primer: 5' TGATCATTTTGTATTCTGGA3', reverse primer: 5'TTAACAAGAACGGTCAGCAG3', annealing temperature 54 ℃, 45 seconds, 40 cycles) was used to examine target gene expression.

Immunohistochemical staining of Ⅶ
The immunofluorescence staining for Ⅶ and α-actinin used frozen sections that were 8 μm thick. After the sections were fixed with cool acetone, nonspecific binding was blocked with normal goat serum. Rabbit polyclonal antibodies against either rat Ⅶ (ab6994, Abcam) or α-actinin (A7811, Sigma) were used as the primary antibodies, which were diluted 1:100 and incubated with the tissue sections for 60 minutes at 37 ℃. The second antibody, which was the second antibody with FITC-conjugated affinipure goat anti-rabbit IgG, was incubated with the tissue sections for 30 minutes at 37 ℃. Between each step, the sections were washed with 10 mmol/L sodium PBS (pH 7.2).

SERCA2a enzyme activity
Prepared for 1% myocardium homogenate and tested protein concentration by BioRad model 550 micro-plate reader. According to the protocol (A070, NJBI, China), SERCA2a enzyme activity was evaluated on the 21st day after therapy.

Western blot analysis
Total protein was extracted from cultured MSC or the left ventricle. The cells were washed with ice-cold PBS, scraped off the dish and transferred into centrifuge tubes. And approximately 60 mg of heart tissue was placed in 300 μL lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate: 0.1% sodium dodecyl sulfate SDS in 1 PBS), and homogenized at 4°C for 20 s, incubated on ice for 30 minutes, centrifuged at 15 000 r/min for 10 minutes, and the supernatant removed.
Protein concentration was measured using a BioRad model 550 micro-plate reader. Total protein (30 μg) was mixed with loading buffer, boiled for 5 minutes, and loaded onto a 10% Tris-glycine gel. Gels were run with a full range molecular weight ladder (Rainbow MW marker, Amersham Pharmacia, Biotech UK Ltd, Bucks, UK). Proteins were transferred to an Immobilon-P membrane (IPVH 00010, Millipore, MA) by semidry blotting. Membranes were blocked with 5% milk in TBST (20 mmol/L Tris HCl pH 7.6, 137 mmol/L NaCl, 0.05% Tween-20) for 2 hours at 37 ℃. Membranes were subsequently exposed to rabbit polyclonal anti-rat SERCA2a (ab3625, Abcam) at 1:500 dilutions in PBS-Tween for 4 hours. Bound antibody was detected by horseradish peroxidase conjugated anti-rabbit IgG. Finally, 3, 3'-diaminobenzidine tetra hydrochloride was employed to visualize the peroxidase reaction products (Amersham). SERCA2a was detected as a 110 ku band.

Statistically analysis
Two statisticians carried out statistical analyses. Statistical analyses were performed with ANOVA or Chi-square test followed by SPSS 9.0 software (SPSS Science, Chicago, Illinois) analysis. Data (Mean±SD) were considered statistically significant at a value of P < 0.05.

RESULTS

Quantitative analysis of the experimental animals
Fifty SD rats were established chronic heart failure models. After 4 weeks, heart failure models of 29 rats were established successfully with ejection fraction < 45%. All 29 rats were randomly divided into 4 groups.

Morphology and growth properties of MSC and the efficiency of adenovirus-medicated gene transferred into different passages of MSC
After discarding the non-adherent cells by the first medium change and by washing with PBS three times at 24 hours of primary culture, MSC were seen to attach to culture dishes sparsely and the majority of the cells displayed a spindle-like shape. These cells began to proliferate at about day 4, and gradually grew to form small colonies. By day 7, the number of cellular colonies with different size had obviously increased (8-12 colonies / 60-mm dish). In large colonies cells were more densely distributed and showed a spindle or triangle shape. As growth of cells continued, colonies gradually expanded in size with the adjacent ones internally connected with each other. Passage MSC behaved similarly to those in primary cultures (Figure 1a).
Two days after infection with Ad-GFP, green fluorescence could be observed. Green fluorescence strengthened at 7 days and lasted 2 weeks. It was still observed at 21 days (Figure 1b). The infection efficiency of P3 MSC and P8 MSC with Ad-GFP by flow cytometry was not different at 2 days, 4 days and 7 days [(52.42±0.17)%, (63.29±0.8)% and (83.89±0.29)%; (58.89±0.53)%, (61.13±1.27)% and (82.58±0.46)%, P=0.06].

Serca2a gene and protein expression in MSC after transferred with Ad-SERCA2a
RT-PCR products showed 518 bp gene band in MSC transferred with Ad-SERCA2a (Figure 2a) and special protein band occurred in Mr 110 000 with Western blotting (Figure 2b).

Survival of MSC and MSC modified with SERCA2a gene
A 400 bp specific Sry gene fragment was amplified by PCR in the MSC group. Ad/SERCa2a samples 21 days after transplantation, indicating that male donor cells survived in female recipients. MSC labeled with Hoechst-33342 could be observed in frozen sections after transplantation under fluorescence microscopy (Figure 3a). And green fluorescence was observed in the MSC group. Ad/SERCa2a could be observed in the similar zone too (Figure 3b). From continuous frozen sections on the same site in cardiac of MSC + Ad/SERCa2a group, green fluorescence expressed much stronger than blue fluorescence.

Left ventricular morphologic analysis and cardiac function
At 21 days after treatment, left ventricle wall thickened obviously, while compared with control group (Figure 4a), volume shortened and geometry tended to round in group Ad-SERCa2a (Figure 4b), MSC group (Figure 4c), and MSC modified with SERCA2a gene group (Figure 4d).

Compared with control group, EF and FS of group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a were elevated significantly at 14 days after therapy (P < 0.01). Both EF and FS of group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a at 21 days after therapy were greater than those in control group (P < 0.01). While the elevation values of EF and FS began to reduce in group Ad-SERCa2a at 14 days after therapy, it continued to increase in both group MSC and group MSC+Ad/SERCa2a. Improvement rate of EF at 21 days after therapy (EF D21) increased in group MSC, group MSC+Ad/SERCa2a and control group-SERCa2a (P =0.01) (Tables 1, 2).

The groups were pointed the same as Table; Group Ⅰ: Received SERCA2a gene therapy; Group Ⅱ: Received MSC transplantation; Group Ⅲ: Received MSC-based SERCA2a gene transplantation; Group Ⅳ: Received empty adenoviral vector.
Absolute value of DP/dtmin at 21 days after treatment was increased in group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a compared to control group (P < 0.01), as well as DP/dtmax at 21 days after treatment increased in group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a compared to control group (P < 0.01) (Figure 5).

Compared with group Ad-SERCa2a, peak systolic flow velocity of anterior wall and interventricular septum in group MSC+Ad/SERCa2a increased obviously at 21 days after therapy (P < 0.01), and peak diastolic flow velocity of anterior wall and interventricular septum elevated in group MSC+Ad/SERCa2a too (P < 0.01) (Figure 6).

SERCA2a gene, protein and activity in infracted zone
SERCA2a gene and protein expressions were significantly stronger in group Ad-SERCa2a and group MSC+Ad/SERCa2a than group MSC and control group (Figure 7). Enzyme activity in group Ad-SERCa2a and group MSC+Ad/SERCa2a was higher than that in control group at 21 days after therapy [(116.19±8.34) μkat/g, (65.35±3.50) μkat/g versus (4.67±0.67) μkat/g, P < 0.01].

Immunohistochemical Staining of Ⅷ
It was observed that parts of MSC expressed Ⅷ (Figure 8).

DISCUSSION

Heart failure is a final stage of the development of many chronic heart diseases. Current therapeutic modalities for the treatment of heart failure, especially end-stage cardiac failure is limited. Gene therapy and cell transplantation have been developed the most potential strategy for heart disease in recent years. Though great improvements of gene therapy has already obtained, there are still some problems to need to be resolved further, fox examples, how to find the key target gene, build the safe and high effective carrying platform of target gene and protect out of control of gene expression. On the other hand, cell transplantation has its limits, for instance, how to increase survival rate of transplanted cells and boost up the function of cells. Production of functional protein of trans-gene expression might improve cell survival, while gene manipulation in cell level in vitro could control gene expression to avoid maladjustment and enhance the safety of gene therapy, and it speculated that MSC-based gene therapy could be the safe and effective approach for heart failure.
Our in vitro experiments demonstrated that MSC could be efficiently transferred by Ad-GFP with more than 80% of the infection efficiency by FACS analysis. Subsequent Ad-SERCA2a infection analysis suggested that SERCA2a trans-gene expression existed in MSC. This indicated that infection with Ad-SERCA2a could reconstitute SERCA2a at a cell level. Moreover, there was no cytotoxic reaction in the process of AV-SERCA2a transferring into MSC in vitro, and then there were no rats which die at 21 days after MSC-based SERCA2a gene therapy in vivo, so it indicated that MSC-based SERCA2a gene therapy for heart failure was safe in earlier period after treatment.
Furthermore, in vivo experiments confirmed that all SERCA2a gene therapy, MSC transplantation and MSC-based SERCA2a gene therapy could enhance cardiac function. MSC-based SERCA2a gene therapy showed many strong and long-term effects on exhausted cardiac. EF and FS were higher at 21 days after therapy in MSC-based SERCA2a gene therapy than in SERCA2a gene therapy alone, as well as PSFV and PDFV. Morphology analysis still found that left ventricle wall thickened obviously, volume shortened and geometry tended to round in MSC-based SERCA2a gene therapy. It identified that the feasibility of MSC as therapeutic gene platform for heart failure and MSC-based SERCA2a gene therapy was the effective approach towards the improvement of cardiac performance in the diseased heart.
Lots of animal models and clinical experiments show that SERCA2a gene and protein deregulate in myocardium with heart failure[13]. It does not only aggravate myocardium contractility descent, but also spurs cardiac myocyte apoptosis and LV remodeling, then causes progressive deterioration of the left ventricular function and severity heart failure. Further studies demonstrated that the ventricle chamber enlarged in trans-gene rats which knocked off SERCA2a gene, heart function dropped and was apt to suffer heart failure[14]. On the other hand, high expression of SERCA2a protein in those trans-gene rats improved heart function and did not decrease survival rate in the future[15-16]. Based on them, the researches are explored to use SERCA2a gene therapy for heart failure. Our study demonstrated that SERCA2a trans-gene expression produced functional protein in exhausted cardiac, and then improved cardiac contractility and relaxation and LV remolding. It conformed to Barry London and Hajjar RJ groups' research. They have found that SERCA2a gene therapy benefits for cardiac function. Besides, recent studies have shown that the beneficial effect of SERCA2a on heart is different from classical β-adrenergic activation path. SERCA2a is the most relevant biological effectors of β-adrenergic receptors, so manipulation of SERCA2a could only impart a more unitary effect on myocardial contractility without impacting on other signaling pathways through β-adrenergic receptors[17]. The latter may cause occurrence of arrhythmia and mortality[18]. Beside that, it is observed that volume is shortened and gradually rounded after SERCA2a gene therapy too. This indicated that SERCA2a gene therapy could alleviate left ventricle remodeling. It may be for improved force-frequency relationship of myocardium by over-expression of SERCA2a[19]. So over-expression of SERCA2a protein seems to be more promising to enhance myocardial contractility without intrinsically harmfulness to the heart and SERCA2a gene therapy is promising for gene therapy with heart failure, especially end-stage heart failure.
However, whether MSC-based SERCA2a gene therapy can play a synergistic role in treatment of heart failure remains unclear. In this study, MSC-based SERCA2a gene therapy displayed much lasting and steady-going effects on cardiac performance. There is following possible explanations: SERCA2a may protect MSC from apoptosis. From continuous frozen sections on the same site in cardiac of MSC transplantation alone and MSC-based SERCA2a gene therapy, it indicated that cell density of MSC modified with SERCA2a gene therapy was higher than that of MSC alone. It concluded to that there are many improvement cell survivals of the former. So it suggested that beneficial effect of MSC-based SERCA2a gene therapy partly owed to trans-SERCA2a gene expression inhibiting the transplanted MSC apoptosis or necrosis, which may reduce calcium overload. When the homeostatic mechanisms responsible for regulating cellular Ca2+ are compromised, cells will die. This can be either in a disordered manner by necrosis, or by the more deliberate apoptotic mechanism. And it is reported that apoptosis induced by prolonged endoplasmic reticulum stress under calcium overload exerted the critical roles in post-infarction ventricular remodeling and heart failure[20]. But over-expression of SERCA2a was helpful for cellular calcium homeostatic recovery so as to protect MSC modified with SERCA2a from apoptosis induced by calcium overload[21].
Moreover, this study confirmed that MSC transplantation could enhance myocardium contractility and ejection fraction, reduce ventricular chamber dilatation, reverse ventricular wall thinning and regulate fluid homeostasis. It could be observed that MSC expressed Ⅷ. The benefits of restoration of cardiac function for heart failure would cause revascularization or vascular regeneration. One of the greatest attributes of MSC has been proved to be their potential to supply growth factors and cytokines to repairing tissue[22-23]. So it is possibly to produce revascularization of the injured region, accordingly improve tissue perfusion, save hibernating myocardium and ameliorate left remodeling.
Our current study suggests taking MSC as vehicles for gene therapies is the valuable strategy and MSC-based SERCA2a gene therapy may be the safe and effective approach for heart failure. Furthermore, it should be observed on long-term safety and explore deeply the effect and mechanism MSC-based SERCA2a gene therapy for heart failure.

ACKNOWLEDGEMENTS

The authors appreciate the technical assistance of Mrs. Yang Yang-ling in the Department of Pathology of the Third Hospital of Peking University. We also thank Mr. Wang Jie in the Experimental Animal Center in General Air Force Hospital for his support in animal breeding.

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钙离子ATP酶2a基因修饰骨髓间充质干细胞移植改善慢性心力衰竭大鼠的心功能***☆

郭豫涛1，李小鹰1，鲁小春1，吴 迪2，姚克群2，陈 平3，马康 涛3，周春燕3
1解放军总医院老年心血管病一科，北京市 100853；2解放军空军总医院超声科，北京市 100036；3北京大学医学部生物化学系，北京市 100083
郭豫涛☆，女，1973年生，四川省内江市人，汉族，1995年泸州医学院大学毕业，博士，主治医师，主要从事心力衰竭的细胞和基因治疗的研究。
国家重点基础研究发展“九七三”计划（G2000056906）*；北京市科技计划重大项目基金资助项目（H020220010490）*；教育部211工程和北京大学985基金资助项目*
摘要
背景：目前仍缺乏有效手段修复心力衰竭后受损心肌，逆转心肌病理性重塑。作为一种新的治疗策略，用正常肌细胞和治疗性基因干预受损心肌正逐渐显示出其在改善心功能方面的优势。
目的：观察腺病毒转染不同代骨髓间充质干细胞的有效性和稳定性。并以携带肌浆网钙离子ATP酶（sarcoplasmic reticulum Ca(2+) ATPase，SERCA2a) 基因的腺病毒转染骨髓间充质干细胞，治疗心力衰竭大鼠，比较SERCA2a基因治疗，骨髓间充质干细胞移植以及骨髓干细胞基础的SERCA2a基因治疗慢性心力衰竭大鼠的效果。
设计：随机对照实验。
单位：解放军总医院老年心血管病科和北京医科大学生物化学系。
材料：选取购于北京医科大学动物实验中心的4周龄雄性SD大鼠作为骨髓供体。选取体质量200~250 g雌性成年SD大鼠作为细胞移植和基因治疗受体。以雄性大鼠Y染色体sry基因鉴定供体移植细胞是否在雌性大鼠受体心肌内存活。实验所用Ad-SERCa2a，Ad-EGFP的构建由我科鲁小春博士完成；第3和8代骨髓间充质干细胞自行分离培养。
方法：实验于2004-07/2005-12在北京医科大学生物化学系周春燕实验室完成。对30只雌性SD大鼠进行左冠状动脉结扎，制作急性心肌梗死后慢性心力衰竭大鼠模型。将造模成功的29只大鼠随机分为4组：基因治疗组7只，干细胞移植治疗组7只，基因修饰的干细胞移植组8只，腺病毒空载体对照组7只，分别予以单纯SERCA2a基因、MSC移植、SERCA2a基因修饰的骨髓间充质干细胞移植及腺病毒空载体干预。分离培养大鼠骨髓间充质干细胞，用携带SERCA2a及绿色荧光蛋白的腺病毒（Ad-SERCA2a-GFP）转染第3和8代骨髓间充质干细胞。
主要观察指标：采用流式细胞仪检测不同代骨髓间充质干细胞的Ad-SERCA2a-GFP转染率。分别在治疗前及治疗后14，21 d采用超声心动图检测大鼠心功能。采用免疫组化法检测大鼠心脏Ⅷ因子表达；采用RT-PCR 和Western杂交检测SERCA2a的基因和蛋白水平表达，并按照说明书检测宿主SERCA2a功能活性。
结果：①转染率：腺病毒转染不同代骨髓间充质干细胞的转染率超过80%，第3代骨髓间充质干细胞与第8代的转染率比较，差异无显著性意义（P > 0.05）。②大鼠心功能：治疗后14 d，与腺病毒空载体对照组相比，其余3组左室射血分数均明显升高（P < 0.01）。治疗后 21 d，与腺病毒空载体对照组相比，干细胞移植治疗组和基因修饰的干细胞移植组大鼠室壁增厚；干细胞移植治疗组和基因修饰的干细胞移植组左室射血分数和左室短轴缩短率改善率持续升高（P < 0.01），基因治疗组两指标改善率较治疗14 d时下降。与腺病毒空载体对照组相比，基因修饰的干细胞移植组左室前壁和室间隔收缩期纵向峰值速度显著升高（P < 0.01），左室前壁和室间隔舒张期纵向峰值速度亦呈现相同改善(P < 0.01)。③心肌SERCA2a的基因、蛋白水平表达和功能活性，以及Ⅷ因子表达：携带SERCA2a基因的骨髓干细胞在宿主心肌能有效地合成和表达有功能的SERCA2a蛋白。与腺病毒空载体对照组相比，基因修饰的干细胞移植组SERCA2a基因、蛋白表达和酶活性均显著增强。干细胞移植组心力衰竭大鼠心脏瘢痕区可观察到Ⅷ因子表达。
结论：①第3和8代骨髓间充质干细胞均具有高腺病毒转染率，骨髓间充质干细胞是基因治疗的良好细胞载体，其介导的SERCA2a基因治疗对衰竭心肌具有持续稳定的心功能改善作用。②骨髓间充质干细胞移植改善心功能的作用可能与促进血管新生有关。
关键词:细胞移植；干细胞；基因治疗；心力衰竭，充血性；肌浆网Ca2+ATP酶2a
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01550-08
郭豫涛，李小鹰，鲁小春，吴迪，姚克群，陈平，马康涛，周春燕.钙离子ATP酶2a基因修饰骨髓间充质干细胞移植改善慢性心力衰竭大鼠的心功能[J].中国组织工程研究与临床康复，2008，12(8):1550-1557
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1550(ps).pdf]
(Edited by Thiele/Ji H/Wang L)