The Investigation On The Function And Mechanism Of Myocardin Gene During Adipose Derived Stem Cells Differentiating Into Smooth Muscle Cells In Vitro

BACKGROUND&OBJECTIVEErectile dysfunction (ED) is defined as the persistent inability to achieve or maintain penile erection for successful sexual intercourse, causing decreased quality of life in men. According to a recent study, the prevalence of ED was1%-10%in men younger than40years,2%-9%among men between40and49years, and it increased to20%-40%among men between60-69years, reaching the highest rate in men older than70years (50%-100%). ED has been a common complication of diabetes, while diabetes has been recognized as one of the most important risk factors for ED. The pathogenesis of diabetes-induced erectile dysfunction (DIED) is a complicated process and multifactorial, which has brought great difficulties for the treatment of ED. The treatment of DIED is multimodal. Oral medications (5phosphodiesterasetype5inhibitors, PDE5i) are considered as the first line therapy for management of DIED. If oral agents cannot be used or have insufficient efficacy despite appropriate dosing and education, second-line treatments should be addressed. When there is lack of efficacy or when there is dissatisfaction with other modalities, penile prostheses are often the best alternative for ED and are considered as the third line therapy for DIED. However, a large part of DIED patients are not sensitive to first line drugs, and unable or unwilling to accept the second-and third-line therapy. Therefore, searching a more preferably way for management of DIED is imminent.Corpus cavernosum smooth muscle (CCSM), the structural basis of cavernous space relaxing and penile erection, plays a major role in modulating penile blood flow during erection and detumescence. Therefore, damage of the smooth muscle tissue or cells in the corpora cavernosa is probably the major single factor impairing erectile function in patients with ED. Some scholars have proposed that decreased number significantly, ultrastructure pathological changes and damaged systolic and diastolic function of CCSM cells casued by diabetes mellitus (DM) is an important cause of ED. Our previous study has confirmed that the expression of smooth muscle cell (SMC) marker proteins and contractility-associated genes, such as SMa-actin (SMA), smooth muscle myosin heavy chain (SMMHC), smoothelin and calponin, are significantly reduced in the tissue and cells of CCSM of diabetic rats with ED, and its amount is decresead and function is damaged. Therefore, the incidence of DIED would closely associated with pathological changes and deletions CCSM cells. At present, there is no ideal way to improve these pathological states fundamentally.In recent years, the effects of gene modification on stem cell differentiation has captured people’s attention. Adipose-derived stem cells (ADSCs) reportedly have the potential of self-renewal and multi-directional differentiation. They can be easily obtained and used in auto-transplantation, so ADSCs can be used as seeding cell in cell therapy. Myocardin, a remarkably potent transcriptional coactivator and the founding member of a class of muscle transcription factors expressed exclusively in cardiomyocytes and SMCs, plays an important role in regulating smooth muscle cell differentiation process by binging to serum response factor (SRF). Recent studies have shown that myocardin also play an important role in the ADSCs differentiation, and myocardin gene-modified ADSCs can differentiation into the SMC.Numerous studies have found that gene-modified ADSCs for the treatment of ED are better than gene or stem cells therapy alone. Therefore, we believe that intracavernous injection of myocardin gene-modified ADSCs can restore erectile function in diabetes mellitus patient with ED. This study to explore the effects of myocardin gene transfection on the differentiation of rat ADSCs into smooth muscle cells in vitro and its mechanism, and it can lay the foundation for further study using myocardin gene modification of ADSCs treatment of DIED. METHODS1. Isolation, culture and biological characteristics of adipose tissue-derived stromal cells from SD rat paraepididymal adipose tissues.The adipose tissues were obtained from paraepididymal fat pads of SD rat under sterile condition. Separated rats ADSCs by digested with0.1%I-collagenase. The growth vigor of ADSCs was detected by MTT chromatometry and experiments of freezing and resuscitation were performed. Specific immune antibody labeling combined with flow cytometry and osteogenesis the adipogenic purity of ADSCs are identified.2. Construction of lentivirus vector carrying myocardin gene and its expression in rat ADSCsRat myocardin gene was obtained by full gene synthesis technology and then connected into lentiviral vectors pLVX-IRES-Neo to produce recombinant lentivirus vector called as pLVX-IRES-Neo-myocardin, then packaged by293T cells. The virus supernant was harvested, concentrated and titrated. The rat ADSCs were transfected with recombinant lentivirus pLVX-IRES-Neo at the appropriate MOI. The mRNA and protein expression of myocardin were detected by RT-QPCR and Western blot, and statistical analysis were conducted by SPSS13.0.3. Myocardin gene to promote the effect and mechanism of rat ADSCs to SMC differentiationMyocardin gene modification of rat ADSCs were screened with G418. Two weeks later, the expression of SMCs-specific marker proteins, such as SMA, SMMHC, smoothelin, calponin, myocardin and SRF, were detected by immunofluorescence, and the expression of SMA, SMMHC, smoothelin and calponin mRNA were detected by RT-QPCR, and the expression of SMA, SMMHC and myocardin protein were detected by Western blot. A modified collagen type I lattice contractility assay was used to determine the cell contractility in vitro. The interaction between myocardin and SRF was detected by co-immunoprecipitation. Statistical analysis were conducted by SPSS13.0. RESULTS1. Isolation, culture and biological characteristics of adipose tissue-derived stromal cells from SD rat paraepididymal adipose tissues.ADSCs were easily amplified and could propagated rapidly in vitro. The primary cultured ADSCs grew into polygonal and clostridial cells, and mainly spindle-shaped, whirlpool or fasciculation-shaped arranged and homogenous fibroblast-like morphology growth after passage. Following many passages, ADSCs could maintain strong propagative capacity. The growth curve was "S" shape. After cultured by adipogenic-inducing liquid, they displayed adipogenic characteristics, and lipid droplet was detected in cytoplasm by oil red O staining. After cultured by osteogenesis-inducing liquid, they showed osteogenic propertiy, and calcium nodes were observed in the ADSCs by alizarin red staining. The ADSCs of CD29and CD44-positive were87.2%and93.5%, respectively. The ADSCs maintained better viability and displayed the higher survival rate after cryopreserved in liquid nitrogen for three months.2. Construction of lentivirus vector carrying myocardin gene and its expression in rat ADSCsThe result of sequencing showed that the obtained myocardin gene was consistent with the sequence reported in GeneBank. The lentiviral expression vector pLVX-IRES-Neo-myocardin had been successfully constructed, confirmed to be correct clone by enzyme digestion and DNA sequencing, producted to lentivirus.One titer of recombinant lentivirus was2.3X109cfu/ml, the other was4.6X109cfu/ml. After the constructed lentiviral vectors infected the rat ADSCs, the results showed that there were the expression of myocardin mRNA and protein in the three groups by the RT-QPCR and Western blot. Compared with’pLVX-IRES-Neo’group and CK group, the expressions of myocardin mRNA and protein were significantly increased in ’pLVX-IRES-Neo-myocardin’ group (p<0.05). There was no significant difference between the ’pLVX-IRES-Neo’ group and CK group (P>0.05).3. Myocardin gene to promote the effect and mechanism of ADSCs to SMC differentiation After the constructed lentiviral vectors infected the rat ADSCs, the results showed that myocardin gene modified ADSCs are strong expression of SMCs-specific marker protein, such as SMa-actin、SMMHC、smoothelin and calponin, myocardin and SRF, and control with empty virus infection was relatively weak. Compared with’pLVX-IRES-Neo’group, the expressions of SMa-actin、SMMHC、 smoothelin and calponin mRNA were significantly increased in’pLVX-IRES-Neo-myocardin’ group (p=0.000、0.001、0.000、0.001, respectively). The results of Western Blot showed that, Compared with the control group, the expressions of SMa-actin、SMMHC and myocardin protein were significantly increased in the experimental group (all P=0.000). These results indicate that myocardin gene can promote rat ADSCs to SMCs differentiation. According to the results of the lattice contractility assay, we found that the contractility of ADSCs is significantly enhanced after modified by myocardin gene. The interaction between myocardin and SRF were detected by co-immunoprecipitation both the experimental group and control group, but the former is stronger.CONCLUSION1. In this study, successfully separated to high purity ADSCs.2. The recombinant lentiviral vector pLVX-IRES-Neo-myocardin was successfully constructed, the carrier can be a virus packaging, and access to a titer of2.3×109cfu/ml infectious virus.3. Successful implementation of exogenous myocardin gene expression in SD rats ADSCs.4. Myocardin gene can promote ADSCs to SMCs differentiation in vitro.5. The contractility of ADSCs is significantly enhanced after modified by myocardin gene.6. That the interaction between myocardin and SRF plays an important role during rats ADSCs differentiating into SMCs in vitro.