Surgical and chemical castration induce differential histological response in prostate lobes of Mongolian gerbil

The present study describes the short-term alterations in the prostate ventral and dorsal lobe of the adult Mongolian gerbil, in response to two different androgen suppression approaches. Groups (n=6) of 16-week-old gerbils were maintained intact or subjected, either to the bilateral surgical castration 1 week previously or to daily subcutaneous injections of Flutamide (10mg/kg body weight) for 7 days. The main microscopic features of both prostate lobes in these groups were compared using conventional paraffin tissue sections, measurements of acinar epithelial height and stereological data of main gland components (acini, collagen fibers and fibromuscular stroma). Marked alterations were observed in the basement membrane of the ventral lobe after both surgical and chemical castration, such as an increase in thickness and collagen staining. A low degree of epithelial atrophy was detected in the dorsal lobe following both androgen suppression approaches in comparison with that found in the ventral lobe, indicating that this lobe is not so responsive to testosterone ablation induced by castration or Flutamide treatment, at least insofar as secretory activity is concerned. However, the dorsal lobe exhibited marked stromal modification, such as an increase in collagen fibers following castration and an increase in fibromuscular stroma following Flutamide-treatment. Thus, the histological and quantitative data indicates a differential short-term response of the prostate dorsal lobe to surgical castration and Flutamide therapy, suggesting the existence of lobe-specific mechanisms for stromal remodeling.     

A quantitative study of sodium tungstate protective effect on pancreatic beta cells in streptozotocin-induced diabetic rats

Diabetes is a major public health problem. Development of new therapies that are able to improve glycemia management, cure diabetes, and can even protect from it, are of great interest. This study investigated the protective effect of sodium tungstate against STZ-induced beta-cell damages by means of stereological methods. Sixty rats were divided into six groups: control (C), tungstate-treated control (TC), STZ-induced diabetic (D), STZ-induced diabetic rats were treated by sodium tungstate from 1 week before STZ injection (TDB), food-restricted diabetic (FRD), and diabetic rats treated with sodium tungstate 1 week after STZ administration (TDA). Stereological estimation of pancreas volume, islets volume density, volume-weighted mean islets volume and mass of beta cells, islets, and pancreas and total number of islets were done. Islets volume density, volume-weighted mean islets volume, and mass of beta cells, islets, and pancreas of TDB group was significantly higher than D, FRD and TDA groups (P < 0.001) and was comparable to controls (C and TC groups). Total number of islets, pancreas wet weight and volume did not show any significant changes between these groups (P > 0.05).Results suggested that sodium tungstate preserves pancreatic beta cells from STZ-induced damages and diabetes induction in rats.     

Hormonal therapy in the senescence: Prostatic microenvironment structure and adhesion molecules

Hormonal replacement has been utilized to minimize the harmful effects of hormonal imbalance in elderly men. The development and progression of prostatic diseases and their relation to hormone therapy is still unclear. Thus, the aim herewith was to characterize the structure and dystroglycan molecule (DGs) reactivities in the ventral prostatic lobe from elderly rats submitted to steroid hormone replacement. Male rats (Sprague-Dawley) were divided into one Young group and six senile groups. The Young group (YNG) (4 months old) received peanut oil (5mL/kg, s.c.). The senile rats (10 months old) were submitted to the following treatments: Senile group (SEN) (5mL/kg peanut oil, s.c.); Testosterone group (TEST) (5mg/kg testosterone cipionate, s.c.); Estrogen group (EST) (25μg/kg 17β-estradiol, s.c.); Castrated group (CAS) (surgical castration); Castrated-Testosterone (CT) (surgical castration and treatment similar to TEST group); and Castrated-Estrogen (CE) (surgical castration and treatment similar to EST group). After 30 days treatment, blood samples were collected for hormonal analysis and ventral prostate samples were processed for light and transmission electron microscopies, morphometrical analysis, immunohistochemistry and Western Blotting. The results showed decreased serum testosterone levels in the senescence and increased testosterone and estrogen plasmatic levels after hormone administration in the TEST and EST groups, respectively, highlighting the therapy efficiency. Hypertrophied stroma and inflammatory cells were verified in the SEN group. After hormone replacement in the senescence or following castration, atrophic epithelium, epithelial cells with clear cytoplasmic halo around the nucleus, microacini and maintenance of hypertrophied stroma were seen. Decreased DG levels were verified in the senescence. After hormonal therapy, increased protein levels of these molecules were observed, especially in those groups which received estradiol. Thus, the occurrence of inflammatory cells, stromal hypertrophy and the presence of cells with clear halo around the nucleus after hormonal therapy probably indicated prostatic paracrine signaling imbalance, suggesting a stromal reactive microenvironment favorable to the development of glandular lesions. However, the increase of DG levels characterized positive effect of steroid hormone replacement on the prostate in the senescence. Thus, it could be concluded that despite having positive effects on important molecules involved in the maintenance of epithelial-stromal interaction and glandular cytoarchitecture, such as DGs, hormonal therapy enhanced structural changes associated with senescence, probably due to increased hormonal imbalance between androgens and estrogens in the prostatic tissue.     

健康和糖尿病大鼠红细胞荧光光谱非线性程度差异

糖尿病是严重威胁人类健康的慢性疾病之一,对其进行早期诊断尤为重要。由于糖尿病病程的发展会对血液中红细胞的结构和功能产生影响,因此利用红细胞荧光光谱在特征峰位和峰高等方面的差异性,可以对糖尿病状态进行诊断。在选取光谱信号的差异性作为特征向量方面,可以采用荧光光谱信号的非线性程度作为特征向量。为更好地描述荧光光谱信号的非线性,采用延迟向量方差(DVV)法描述信号的非线性程度。采用迭代幅度匹配傅里叶变换(IAAFT)法产生原始数据的替代数据,通过比较原始数据与替代数据的延迟向量方差确定原始数据的非线性特征。以原始数据目标方差为横坐标,以其替代数据目标方差为纵坐标,画出延迟向量方差散布图。健康大鼠红细胞荧光光谱的延迟向量方差散布图与其对角线几乎重合,说明健康大鼠红细胞荧光光谱非线性程度较低;而糖尿病大鼠红细胞荧光光谱的延迟向量方差散布图偏离其对角线,说明糖尿病大鼠红细胞荧光光谱非线性程度较高,并且氨基酸对应的光谱段非线性程度更深。据此,提出健康和糖尿病荧光光谱之间的非线性差异程度可以作为糖尿病早期诊断的特征之一。     

Calponin is expressed by subpopulations of connective tissue cells but not olfactory ensheathing cells in the neonatal olfactory mucosa

Background  

Debate has been ongoing on the relative merits of olfactory ensheathing cells (OECs) and Schwann cells as candidates for transplant-mediate repair of CNS lesions. Both glial cells exhibit similar molecular and cellular properties and to date there has been no antigenic marker identified that can clearly distinguish the two cell types. This inability to distinguish between the two cells types prevents confirmation of a controversial statement that cultures of OECs are contaminated with Schwann cells. Recently, proteomic analysis of foetal OECs and adult Schwann cells identified an actin-binding protein, calponin, as a specific marker for OECs. However, at the same time a recent report suggested that adult OECs do not express calponin. It was not clear if this discrepancy was due to methodology, as cells had to be treated with proteinase K to maximize calponin staining or developmental differences with only foetal/neonatal OECs expressing calponin. For this reason we have examined calponin expression in the peripheral olfactory system of embryonic and neonatal rats in vivo and from cells in vitro to assess if calponin is expressed in a developmental manner.     

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.     

Androgen receptor in the Mongolian gerbil ventral prostate: evaluation during different phases of postnatal development and following androgen blockage

The normal growth, differentiation and maintenance of the morphofunctional integrity of the prostate gland are dependent on the interaction of constant levels of androgens with their receptors. The need to study the responses to hormones under several conditions and the effect of their blockage is due to the fact that the human prostate is the site of a great number of age-related diseases, and the ones with a major medical importance are prostate cancer (CaP) and benign prostatic hyperplasia (BPH), which can both be treated with androgen suppression. Seventy-five male gerbils were divided, randomly, into 3 groups of 25 animals each, where each group corresponded to one phase of postnatal development. In each phase, it was possible to morphologically and stereologically analyze the compartments of prostatic ventral lobe, as well as to immunohistochemically analyze the degree of expression of androgen receptors (ARs) after the androgen blockage therapies. In addition, it was possible to establish the hormonal dosage of serum testosterone levels given the comparative approach of the expression of androgen receptors. There is a pattern of AR distribution in the prostatic ventral lobe throughout postnatal development, in which the younger the animal is the higher, the interaction of circulating androgens that stimulate the AR expression in both the epithelial and stromal compartments. The androgen blockage therapies decreased AR expression in the prostatic compartments, but the androgen reposition after these blockages was not sufficient to recover the glandular structure or stimulate the AR expression up to normal physiological conditions. Both the regulation and distribution of androgen receptors along the gerbil prostatic tissues are complex mechanisms that are likely to be genetically regulated by androgens prenatally or by other factors that are still unknown. This rodent species seems to be a valuable model in the attempt to improve the understanding of the morphophysiological and pathological behavior of this important gland in humans throughout aging and to stimulate new therapeutic ideas to fight prostate cancer.     

Ⅱ型糖尿病人与大鼠红细胞拉曼光谱学比较

使用激光共聚焦拉曼光谱仪测量正常大鼠红细胞、正常人红细胞、糖尿病STZ造模大鼠红细胞、糖尿病四氧嘧啶造模大鼠红细胞和人Ⅱ型糖尿病红细胞的拉曼光谱,应用主成分分析(principal component analysis,PCA)结合支持向量机(support vector machines,SVM)分类器对数据进行判别分析,然后采用类间距离判断两种造模方法与人Ⅱ型糖尿病的接近程度。结果发现糖尿病红细胞与正常红细胞的拉曼光谱存在明显差异,糖尿病在酰胺 ⅥCO变形振动谱带处峰高显著,并在酰胺ⅤN—H变形振动谱带处谱线出现偏移,属于磷脂的脂酰基C—C骨架1 130 cm-1谱线增强,1 088 cm-1谱线强度减弱,说明糖尿病红细胞膜的通透性增强。PCA结合SVM可以很好地区分以上5类红细胞的拉曼光谱,分类器测试结果表明分类准确度达100%。通过分别计算两种造模方法与人Ⅱ型糖尿病的类间距离,发现STZ造模法更接近人Ⅱ型糖尿病。由此得出结论：拉曼光谱法可以用于糖尿病诊断,大鼠糖尿病STZ造模法更接近人类Ⅱ型糖尿病。     

Intracardiac lipid accumulation, lipoatrophy of muscle cells and expansion of myocardial infarction in type 2 diabetic patients

The overall mortality of diabetic patients after myocardial infarction is 3-4 times higher than non-diabetics. The cellular mechanisms underlying such a poor clinical prognosis remain incompletely understood. Recent reports suggest that lipotoxicity associated with impaired liporegulation is among the leading factors in the pathogenesis of type 2 diabetes. The goal of this study was to investigate whether excess lipid accumulation specifically in heart muscle cells contributes to the expansion of myocardial infarction in type 2 diabetic patients. Comparative structural analysis of cardiac tissue was performed on autopsy samples from the infracted hearts of diabetic and non-diabetic individuals with special reference to the expansion of the infarction, degenerative changes, lipoatrophy, cell death, and replacement fibrosis. We found that progressive accumulation of lipids in cardiac myocytes was accompanied by considerable loss of myofibrils and was frequently observed in the heart tissue of type 2 diabetic patients. This indicates that disassembly of the contractile apparatus in the cells infiltrated with lipids weakens their capability for functional activity. Analysis of degenerative changes in the diabetic tissue has shown that lipid-laden cardiac myocytes were more susceptible to necrotic and apoptotic cells death leading to expansion of the infarction and the development of progressive focal replacement fibrosis both in the perinecrotic zone and in the areas located far from the site of injury. Our data show that lipoatrophy and loss of muscle cells during the post-infarction period aggravate the functional impairment in the diabetic heart and limits its adaptive capacity for compensatory remodeling. This suggests that lipotoxic myocardial injury associated with defects of lipid metabolism in type 2 diabetes predisposes its evolution toward congestive heart failure and is an important factor contributing to a high mortality following infarction.     

Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue

A spectroscopy system is developed for studying the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) of epithelial tissue in backscattering geometry. Our findings show that TPEF signals from epithelial and underlying stromal layers exhibit different spectral characteristics, providing information on the biomorphology and biochemistry of tissue. The SHG signal serves as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The polarization dependence of the SHG signal reveals a well-ordered orientation of collagen fibers in the stromal layer. The results demonstrate the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue.     

Surface modification with fibronectin or collagen to improve the cell adhesion

Objective

The surface of biomaterials plays a critical role in determining bioactivity. The aim of this study was to evaluate the cell adhesion and proliferation of ADSCs on the surface of biomaterial which is modified with fibronectin or collagen.

Materials and methods

Adipose-derived stromal cells (ADSCs) were obtained from SD rats, expanded in culture, and seeded onto scaffold surface-modified with fibronectin or collagen. To characterize cellular attachment, cells were incubated on scaffold for 1 and 2 h and then counted the cells attached onto the scaffold. The MTT assay was chosen to evaluate the proliferation at days 1, 4, 7 and 14. After 7 d of culture, scanning electron microscope was chosen to observe cell morphology and attachment of ADSCs on the scaffolds.

Results

Attachment at 1 and 2 h of cells on scaffold modified with fibronectin was significantly greater than in control, but not with collagen. The MTT assay revealed that ADSCs proliferation tendency was nearly parallel to that in control. The scanning electron microscope (SEM) showed that ADSCs in experiment expanded thoroughly and excreted much extracellular materials.

Conclusions

Surface modification with fibronectin or collagen can enhance the attachment of cultured ADSCs on the scaffold, but it had not evident effect to proliferation.     

Preparation of cross-linked copolymer microspheres 4VP/St and cobalt tetraphenylporphyrins supported on it

The cross-linked microspheres 4VP/St made of 4-vinylpyridine (4VP) and styrene(St) were prepared with suspension copolymerization method using ethyl glycol dimethacylate (EGDMA) as cross-linker and polyvinyl alcohol (PVA) as disperser. The cobalt tetraphenylporphyrins (CoPs) were immobilized on 4VP/St microspheres via the axial coordination reaction between CoPs and the pyridine groups of 4VP/St microspheres, resulting in the functional microspheres CoP-4VP/St. The chemical structure of 4VP/St and CoP-4VP/St were characterized with infrared spectrum and their morphologies were observed with the scanning electron microscope. The experimental results show that via controlling the various reaction conditions of the suspension copolymerization, the 4VP/St microspheres with excellent sphericity and narrow particle diameter distribution can be gained. In addition, CoPs are successfully immobilized on 4VP/St microspheres by means of Co-N bonds, on which the immobilized content of CoPs goes up to 10.7-17.5 μmol/g.     

Microscopic evaluation of proliferative disorders in the gerbil female prostate: evidence of aging and the influence of multiple pregnancies

The gerbil female prostate is located paraurethrally and has all the histological components of the male prostate, like secretor epithelium and fibromuscular stroma. This gland, like the prostate in males, is targeted by testosterone action, which promotes morphofunctional development. Furthermore, estrogens are required to maintain the male and female prostate and this gland presents both estrogen receptors (ER-α and ER-β). In the present work the structural and morphometric-stereological and serological aspects, as well as the quantification of the incidence, multiplicity and percentage of acini affected by different lesions were analyzed. Animals were divided into four groups: five adult nuliparous (AN) gerbils; five adult multiparous (AM) gerbils; five senescent nulliparous (SN) gerbils; five senescent multiparous (SM) gerbils, and were weighed and sacrificed by CO(2) inhalation. The ventral prostate was dissected out, weighed and fixed to perform histological and morphometric-stereological analysis and quantification of prostate disorders. A high rate of lesions, mainly dysplasia, was identified in tissue from senescent multiparous and adult multiparous animals. Prostatitis was found mainly in SN animals, while dysplasia, hyperplasia, neoplasia, PIA and adenocarcinoma were common in SM ones. Although the proliferative lesion incidence was high in AN group, it was highest in the SM group. The hormonal events which occur due to the estrous cycle in female gerbils (after and before each pregnancy) may be responsible for the high number of lesions observed in our study and all the data presented herein lead us to assume that pregnancy promotes augmentations in both the incidence and the multiplicity of proliferative disorders in the gerbil female prostate since progesterone levels remain high during pregnancy.     

Morphology and protein content of hepatocytes in type I diabetic rats submitted to physical exercises

The importance of physical exercise practice in the treatment of diabetes has been reported in many studies recently, but only limited data can be found regarding its benefits on liver morphology and protein content of hepatocytes. In order to assess the changes arising from the development of type I diabetes and the benefits of a training protocol, Wistar rats were divided into four groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). The training protocol consisted of swimming for 60 min a day, 5 days/week, during 8 weeks. Liver samples were collected, processed and analyzed by histochemical and ultrastructural techniques. Biochemical tests were also conducted to examine the protein content and quantity of DNA in the liver. In morphological assessment, the presence of areas of cytoplasmic basophilia observed in control subjects was not visualized in sedentary diabetics. It was related to differences in the amount of mitochondria in the cytosol. The mitochondrial structure has not undergone relevant changes, and the number of rough endoplasmic reticulum cisterns was clearly inferior in sedentary diabetics, suggesting lower protein production. However, the biochemical analysis of protein content indicated no statistical differences between groups. The exercise, in turn, was not responsible for major changes in these characteristics. On the whole, the morphological damages arising from type I diabetes were noteworthy. Nevertheless, regular physical training was not responsible for significant improvements in some respects, making evident the need for combined application of a distinct form of treatment.     

Novel Application of Multiscale Cross-Approximate Entropy for Assessing Early Changes in the Complexity between Systolic Blood Pressure and ECG R-R Intervals in Diabetic Rats

Cardiac autonomic neuropathy (CAN) is a common complication of diabetes mellitus, and can be assessed using heart rate variability (HRV) and the correlations between systolic blood pressure (SBP) and ECG R-R intervals (RRIs), namely baroreflex sensitivity (BRS). In this study, we propose a novel parameter for the nonlinear association between SBP and RRIs based on multiscale cross-approximate entropy (MS-CXApEn). Sixteen male adult Wistar Kyoto rats were equally divided into two groups: streptozotocin-induced diabetes and age-matched controls. RRIs and SBP were acquired in control rats and the diabetic rats at the onset of hyperglycemia and insulin-treated euglycemia to determine HRV by the ratio of low-frequency to high-frequency power (LF/HF) and Poincaré plot as SSR (SD1/SD2), BRS, and MS-CXApEn. SSR and BRS were not significantly different among the three groups. The LF/HF was significantly higher in the hyperglycemic diabetics than those in the controls and euglycemic diabetic rats. MS-CXApEn was higher in the diabetic hyperglycemic rats than the control rats from scales 2 to 10, and approached the values of controls in diabetic euglycemic rats at scales 9 and 10. Conclusions: We propose MS-CXApEn as a novel parameter to quantify the dynamic nonlinear interactions between SBP and RRIs that reveals more apparent changes in early diabetic rats. Furthermore, changes in this parameter were related to correction of hyperglycemia and could be useful for detecting and assessing CAN in early diabetes.     

Volume-weighted mean volume of the submandibular gland acini in male and female diabetic rats

Mouth dryness is one of the complications of diabetes mellitus but there has been little work on morphological changes of the salivary glands. In the present study, the effects of diabetes mellitus on the serous and mucous acini of submandibular gland of male and female rats, 4 and 12 weeks after diabetes induction were studied. Male and female rats were divided into experimental and control subgroups. Diabetes was induced to experimental rats by streptozotocin. At the end of 4 and 12 weeks, the submandibular glands were removed, random sections obtained and volume-weighted mean acini volume was estimated by point-sampled intercepts method. The data revealed that volume reduction occurred only in serous acini in both male and female rats 12 weeks after diabetes induction and the others remained unchanged. The present research using stereological methods demonstrates that diabetes make some morphological changes in serous acini, the main exocrine part of the rat submandibular gland.     

Intravoxel incoherent motion and diffusion tensor imaging of early renal fibrosis induced in a murine model of streptozotocin induced diabetes

IntroductionTo assess if parameters in intravoxel incoherent motion (IVIM) and diffusion tensor imaging (DTI) can be used to evaluate early renal fibrosis in a mouse model of diabetic nephropathy.Materials & methodsIn a population of 38 male CD1 mice (8 weeks old, 20–30 g), streptozotocin induced diabetes was created in 20 mice via a single intraperitoneal injection of streptozotocin at 150 mg/kg, while 18 mice served as control group. IVIM parameters were acquired at 0, 12 and 24 weeks after injection of streptozotocin using a range of b values from 0 to 1200 s/mm². DTI parameters were obtained using 12 diffusion directions and lower b values of 0, 100 and 400 s/mm². DTI and IVIM parameters were obtained using region of interests drawn over the renal parenchyma. Histopathological analysis of the right kidney was performed in all mice. Results were analyzed using an unpaired t-test with P < 0.05 considered statistically significant.ResultsRenal cortex fractional anisotropy (FA) was significantly lower in the diabetes group at week 12 as compared with the control group. Renal cortex apparent diffusion coefficient and tissue diffusivity were significantly higher in the diabetes group at week 12 compared with the control group at 12 weeks. Blood flow was significantly decreased at the renal medulla at 24 weeks. Histopathological analysis confirmed fibrosis in the diabetes group at 24 weeks.ConclusionFA is significantly reduced in diabetic nephropathy. FA might serve a potential role in the detection and therapy monitoring of early diabetic nephropathy.     

Stereological study of the effects of nandrolone decanoate on the rat prostate

It has been shown that nandrolone decanoate which is one of the anabolic-androgenic steroid compounds changes the testis structure and sperm quality but quantitative studies of the prostate have received less attention. Control rats received the peanut oil and experimental group received nandrolone decanoate for 14 weeks. Then the rats were left untreated for 14 weeks. After 14 weeks of withdrawal, the prostate was studied using stereological methods. The mean prostate weight decreased approximately 39% (p<0.009) in nandrolone decanoate treated rats. The mean total prostate volume, glands, epithelia, fluids and collagen bundles reduced approximately 30% (p<0.03), approximately 31% (p<0.03), approximately 41% (p<0.02), approximately 31% (p<0.05) and approximately 59.5% (p<0.02) in the experimental group. The mean total luminal surface of the glands and total length of the vessels decreased approximately 40% (p<0.02) and approximately 46% (p<0.009), respectively, in the nandrolone decanoate treated rats. The height of epithelium did show no difference. It can be concluded that nandrolone decanoate causes atrophic changes in the components of rat prostate.     

Zinc in native tissues and cultured cell lines of human prostate studied by SR‐XRF and XANES

During longlasting attempts to understand the aetiology of prostate cancer (CaP) on molecular level, attention has been paid to a unique capability of prostate epithelial cells to accumulate zinc. The latter plays role in a wide range of cellular processes such as the function of immune system, angiogenesis or apoptosis. Zinc has a significant antioxidant function, and its presence in prostate cells is strongly connected with their metabolism. This paper reports on the analysis of zinc concentration and oxidation level in the samples of human CaP tissues and cultured human prostate cell lines such as DU‐145, LNCaP and PC‐3, aided by x‐ray bioimaging. The study was performed by means of synchrotron radiation (SR) techniques. Bioimaging on cellular level, available through the SR x‐ray fluorescence (SR‐XRF) methods, applied to the samples of native prostate tissue, revealed a complexity of structures, while cell culture samples provided areas of homogeneity required for reliable analysis. SR‐XRF enabled us to establish the dependence of zinc concentration upon histological status of tissue (healthy, hyperplastic or cancerous). In‐depth studies of local chemical environment of an x‐ray absorbing zinc atom, including determination of Zn K‐edge position, were possible through the use of x‐ray absorption near edge structure (XANES) analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced effect of microdystrophin gene transfection by HSV-VP22 mediated intercellular protein transport

Background

Duchenne musclar dystrophy (DMD) is an X-linked recessive disease caused by mutations of dystrophin gene, there is no effective treatment for this disorder at present. Plasmid-mediated gene therapy is a promising therapeutical approach for the treatment of DMD. One of the major issues with plasmid-mediated gene therapy for DMD is poor transfection efficiency and distribution. The herpes simplex virus protein VP22 has the capacity to spread from a primary transduced cell to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of plasmid-mediated gene therapy and investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, expression vectors for C-terminal versions of VP22-microdystrophin fusion protein was constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo.

Results

Our results clearly demonstrate that the VP22-microdystrophin fusion protein could transport into C2C12 cells from 3T3 cells, moreover, the VP22-microdystrophin fusion protein enhanced greatly the amount of microdystrophin that accumulated following microdystrophin gene transfer in both transfected 3T3 cells and in the muscles of dystrophin-deficient (mdx) mice.

Conclusion

These results highlight the efficiency of the VP22-mediated intercellular protein delivery for potential therapy of DMD and suggested that protein transduction may be a potential and versatile tool to enhance the effects of gene delivery for somatic gene therapy of DMD.