Arsenite-loaded nanoparticles inhibit the invasion and metastasis of a hepatocellular carcinoma: in vitro and in vivo study.

Postoperative recurrence and metastasis are the major problems for the current treatment of hepatocellular carcinomas (HCC) in the clinic, including hepatectomy and liver transplantation. Here, we report that arsentic-loaded nanoparticles (ALNPs) are able to reduce the invasion of HCC cells in vitro, and, more importantly, can strongly suppress the invasion and metastasis of HCC in vivo without adverse side effects. Compared to free drug arsenic trioxide , ALNPs can deliver the drug into cancer cells more efficiently, destroy the structure of microtubules and reduce the aggregation of microfilaments in cell membranes more significantly. Furthermore, our results also reveal that tumor cells in murine blood were reduced remarkably after intravenous injection of ALNPs, indicating that this nano-drug may efficiently kill circulating tumor cells in vivo. In conclusion, our nano-drug ALNPs have great potential for the suppression of metastasis of HCC, which may open up a new avenue for the effective treatment of HCC without metastasis and recurrence.

Cationic niosomes an effective gene carrier composed of novel spermine-derivative cationic lipids: effect of central core structures.

CONTEXT: Cationic niosomes formulated from Span 20, cholesterol (Chol) and novel spermine-based cationic lipids of multiple central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) were successfully prepared for improving transfection efficiency in vitro. The niosomes composed of spermine cationic lipid with central core structure of di(oxyethyl)amino revealed the highest gene transfection efficiency. OBJECTIVES: To investigate the factors affecting gene transfection and cell viability including differences in the central core structures of cationic lipids, the composition of vesicles, molar ratio of cationic lipids in formulations and the weight ratio of niosomes to DNA. METHODS: Cationic niosomes composed of nonionic surfactants (Span20), cholesterol and spermine-based cationic lipids of multiple central core structures were formulated. Gene transfection and cell viability were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge were also characterized. RESULTS AND DISCUSSION: High transfection efficiency was obtained from cationic niosomes composed of Span20:Chol:cationic lipid at the molar ratio of 2.5:2.5:0.5 mM. Cationic lipids with di(oxyethyl)amino as a central core structure exhibited highest transfection efficiency. In addition, there was also no serum effect on transfection efficiency. CONCLUSIONS: These novel cationic niosomes may constitute a good alternative carrier for gene transfection.

Interleukin-6 primarily produced by non-hematopoietic cells mediates the lipopolysaccharide-induced febrile response.

Interleukin-6 (IL-6) is critical for the lipopolysaccharide (LPS)-induced febrile response. However, the exact source(s) of IL-6 involved in regulating the LPS-elicited fever is still to be identified. One known source of IL-6 is hematopoietic cells, such as monocytes. To clarify the contribution of hematopoietically derived IL-6 to fever, we created chimeric mice expressing IL-6 selectively either in cells of hematopoietic or, conversely, in cells of non-hematopoietic origin. This was performed by extinguishing hematopoietic cells in wild-type (WT) or IL-6 knockout (IL-6 KO) mice by whole-body irradiation and transplanting them with new stem cells. Mice on a WT background but lacking IL-6 in hematopoietic cells displayed normal fever to LPS and were found to have similar levels of IL-6 protein in the cerebrospinal fluid (CSF) and in plasma and of IL-6 mRNA in the brain as WT mice. In contrast, mice on an IL-6 KO background, but with intact IL-6 production in cells of hematopoietic origin, only showed a minor elevation of the body temperature after peripheral LPS injection. While they displayed significantly elevated levels of IL-6 both in plasma and CSF compared with control mice, the increase was modest compared with that seen in LPS injected mice on a WT background, the latter being approximately 20 times larger in magnitude. These results suggest that IL-6 of non-hematopoietic origin is the main source of IL-6 in LPS-induced fever, and that IL-6 produced by hematopoietic cells only plays a minor role.

Intrarenal transfer of an intracellular fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats and mice.

The present study tested the hypothesis that intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of angiotensin II (ECFP/ANG II) selectively in proximal tubules of the kidney increases blood pressure by activating AT(1) (AT(1a)) receptors. Intrarenal transfer of ECFP/ANG II was induced in the superficial cortex of rat and mouse kidneys, and the sodium and glucose cotransporter 2 (sglt2) promoter was used to drive ECFP/ANG II expression selectively in proximal tubules. Intrarenal transfer of ECFP/ANG II induced a time-dependent, proximal tubule-selective expression of ECFP/ANG II in the cortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANG II expression was low in the glomeruli and the entire medulla and was absent in the contralateral kidney or extrarenal tissues. At its peak of expression in proximal tubules at day 14, ANG II was increased by twofold in the kidney (P < 0.01) and more than threefold in proximal tubules (P < 0.01), but remained unchanged in plasma or urine. Systolic blood pressure was increased in ECFP/ANG II-transferred rats by 28 ± 6 mmHg (P < 0.01), whereas fractional sodium excretion was decreased by 20% (P < 0.01) and fractional lithium excretion was reduced by 24% (P < 0.01). These effects were blocked by losartan and prevented in AT(1a) knockout mice. Transfer of a scrambled ECFP/ANG IIc had no effects on blood pressure, kidney, and proximal tubule ANG II, or sodium excretion. These results provide evidence that proximal tubule-selective transfer of an intracellular ANG II fusion protein increases blood pressure by activating AT(1a) receptors and increasing sodium reabsorption in proximal tubules.

Fetally derived CCL3 is not essential for the migration of maternal cells across the blood-placental barrier in the mouse.

In mammals with a hemochorial placenta (e.g., primates and rodents), the maternal and fetal bloodstreams are separated by the blood-placenta barrier. However, a few maternal cells in the general circulation pass through the barrier during normal pregnancy. So far, the transfer mechanism has not been investigated. In this study, we established a chemokine (C-C motif) ligand 3 (CCL3)-deficient mouse model to examine the effect of fetus-derived chemokine(s) on the migration of maternal cells through the blood-placenta barrier. Using this model, we obtained CCL3-positive and -negative littermates from a mother expressing both CCL3 and green fluorescent protein (GFP). The numbers of GFP positive maternal cells in the lung, liver, spleen and heart of CCL3-positive and -negative fetuses were compared. A few GFP-positive cells were detected in the lung and liver of both types of fetus. These results indicate that maternal cells can migrate through the blood-placenta barrier even in the absence of fetal CCL3.

Focused ultrasound for safe and effective release of brain tumor biomarkers into the peripheral circulation.

The development of noninvasive approaches for brain tumor diagnosis and monitoring continues to be a major medical challenge. Although blood-based liquid biopsy has received considerable attention in various cancers, limited progress has been made for brain tumors, at least partly due to the hindrance of tumor biomarker release into the peripheral circulation by the blood-brain barrier. Focused ultrasound (FUS) combined with microbubbles induced BBB disruption has been established as a promising technique for noninvasive and localized brain drug delivery. Building on this established technique, we propose to develop FUS-enabled liquid biopsy technique (FUS-LBx) to enhance the release of brain tumor biomarkers (e.g., DNA, RNA, and proteins) into the circulation. The objective of this study was to demonstrate that FUS-LBx could sufficiently increase plasma levels of brain tumor biomarkers without causing hemorrhage in the brain. Mice with orthotopic implantation of enhanced green fluorescent protein (eGFP)-transfected murine glioma cells were treated using magnetic resonance (MR)-guided FUS system in the presence of systemically injected microbubbles at three peak negative pressure levels (0.59, 1.29, and 1.58 MPa). Plasma eGFP mRNA levels were quantified with the quantitative polymerase chain reaction (qPCR). Contrast-enhanced MR images were acquired before and after the FUS sonication. FUS at 0.59 MPa resulted in an increased plasma eGFP mRNA level, comparable to those at higher acoustic pressures (1.29 MPa and 1.58 MPa). Microhemorrhage density associated with FUS at 0.59 MPa was significantly lower than that at higher acoustic pressures and not significantly different from the control group. MRI analysis revealed that post-sonication intratumoral and peritumoral hyperenhancement had strong correlations with the level of FUS-induced biomarker release and the extent of hemorrhage. This study suggests that FUS-LBx could be a safe and effective brain-tumor biomarker release technique, and MRI could be used to develop image-guided FUS-LBx.

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model.

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55+/-1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.

[Green fluorescent protein absorption and accumulation in the renal proximal tubule cells after its increased entry into blood].

The uptake of green fluorescent protein (GFP) by the proximal renal tubules was studied in the anaesthetized rats using laser confocal microscopy after GFP intravenous injection or administration into the small intestine lumen. The specific green fluorescence revealed in the proximal tubule cells after intravenous injection correlated with the logarithm of GFP dose injected intravenously (r = 0.96, p < 0.05). GFP fluorescence after its intravenous injection was higher than that one after GFP infusion into the small intestine (p < 0.05). Following the increase of injected GFP dose, the epitheliocyte cytoplasm, in addition to diffuse fluorescence, demonstrated large intensely fluorescent vesicles, that was confirmed by a graphical analysis. The reported changes in the intensity and pattern of specific fluorescence indicate the enhancement of GFP absorption by the cells of proximal tubules and GFP accumulation in the intracellular compartments during its increased entry into circulation.

Viral labeling of neurons synaptically connected to nucleus accumbens somatostatin interneurons.

The nucleus accumbens, a key brain reward region, receives synaptic inputs from a range of forebrain and brainstem regions. Many of these projections have been established using electrophysiology or fluorescent tract tracing. However, more recently developed viral tracing techniques have allowed for fluorescent labeling of synaptic afferents in a cell type-specific manner. Since the NAc is comprised of multiple cell types, these methods have enabled the delineation of the cell type-specific connectivity of principal medium spiny neurons in the region. The synaptic connectivity of somatostatin interneurons, which account for <5% of the neurons in the region, has been inferred from electrophysiological and immunohistochemical data, but has not yet been visualized using modern viral tracing techniques. Here, we use the pseudorabies virus (PRV)-Introvert-GFP virus, an alphaherpes virus previously shown to label synaptic afferents in a cell type-specific manner, to label first order afferents to NAc somatostatin interneurons. While we find GFP(+) labeling in several well established projections to the NAc, we also observe that several known projections to NAc did not contain GFP(+) cells, suggesting they do not innervate somatostatin interneurons in the region. A subset of the GFP(+) afferents are c-FOS(+) following acute administration of cocaine, showing that NAc somatostatin interneurons are innervated by some cells that respond to rewarding stimuli. These results provide a foundation for future studies aimed toward elucidating the cell type-specific connectivity of the NAc, and identify specific circuits that warrant future functional characterization.

Expression of functional human (pro)renin receptor in silkworm (Bombyx mori) larvae using BmMNPV bacmid.

The circulating RA (renin-angiotensin) system is essential for the regulation of blood pressure and electrolyte balance. Recently, plasma prorenin has been reported to significantly increase its level in diabetes and to be possibly non-proteolytically activated by binding to the PRR [(pro)renin receptor] on the cell membrane reported in several tissues during circulation. Although many pathological aspects have been researched, there is a lack of sufficient information on the biochemical structure and biological function of this hPRR (human PRR) because of the difficulty in increasing hPRR expression. In the present study, GFP(uv)-hPRR (hPRR fused with green fluorescence protein when excited with long-wave UV light) was successfully expressed by using BmMNPV (Bombyx mori multiple nucleopolyhedrovirus) bacmid DNA in silkworm (Bombyx mori) larvae. Some of the hPRR was expressed in the haemolymph of silkworm larvae and some of the hPRR was located in the fat body of silkworm larvae. The binding ability of hPRR expressed in the haemolymph and fat body with renin or prorenin was analysed by ELISA and surface plasmon resonance using a biosensor respectively. These binding assays suggest that the expressed hPRR has a functional bioactivity. hPRR preparation in silkworm larvae would, therefore, be useful for biochemical and biomedical researches related to PRR.

Transfer and Integration of Breast Milk Stem Cells to the Brain of Suckling Pups.

Beside its unique nutritional content breast milk also contains live cells from the mother. Fate of these cells in the offspring has not been adequately described. In this study, we aimed to detect and identify maternal cells in the suckling's blood and the brain. Green fluorescent protein expressing transgenic female mice (GFP+) were used as foster mothers to breastfeed wildtype newborn pups. One week and two months after the birth, blood samples and brains of the sucklings were analyzed to detect presence of GFP+ cells by fluorescence activated cell sorting, polymerase chain reaction and immunohistochemistry on the brain sections and optically cleared brains. The tests confirmed that maternal cells were detectable in the blood and the brain of the pups and that they differentiated into both neuronal and glial cell types in the brain. This phenomenon represents breastfeeding - induced microchimerism in the brain with functional implications remain to be understood.

Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression in mammalian cells and mice.

BACKGROUND: Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. RESULTS: We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. CONCLUSION: Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.

Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein.

The Wistar Kyoto (WKY) rat and the spontaneously hypertensive (SHR) rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN) and metabolic syndrome, respectively. Novel transgenic (Tg) strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP) under the rat elongation factor 1 alpha (EF1a) promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminaryin vitroandin vivoimaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats.

Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 µM ADP and 10 µg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl3, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl3 caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl3, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, P = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.

Quantification of plasma exosome is a potential prognostic marker for esophageal squamous cell carcinoma.

Exosomes play important roles in cancer progression. Although its contents (e.g., proteins and microRNAs) have been focused on in cancer research, particularly as potential diagnostic markers, the exosome behavior and methods for exosome quantification remain unclear. In the present study, we analyzed the tumor-derived exosome behavior and assessed the quantification of exosomes in patient plasma as a biomarker for esophageal squamous cell carcinoma (ESCC). A CD63-GFP expressing human ESCC cell line (TE2-CD63-GFP) was made by transfection, and mouse subcutaneous tumor models were established. Fluorescence imaging was performed on tumors and plasma exosomes harvested from mice. GFP-positive small vesicles were confirmed in the plasma obtained from TE2-CD63-GFP tumor-bearing mice. Patient plasma was collected in Chiba University Hospital (n=86). Exosomes were extracted from 100 µl of the plasma and quantified by acetylcholinesterase (AChE) activity. The relationship between exosome quantification and the patient clinical characteristics was assessed. The quantification of exosomes isolated from the patient plasma revealed that esophageal cancer patients (n=66) expressed higher exosome levels than non-malignant patients (n=20) (P=0.0002). Although there was no correlation between the tumor progression and the exosome levels, exosome number was the independent prognostic marker and low levels of exosome predicted a poor prognosis (P=0.03). In conclusion, exosome levels may be useful as an independent prognostic factor for ESCC patients.

Expression Analysis of CB2-GFP BAC Transgenic Mice.

The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells.

A Laser Scanning Cytometry-based method was developed for identification of transgenic mice expressing green fluorescent protein (GFP) using minute amounts of peripheral blood. The difference between the autofluorescence of cells not expressing GFP and the fluorescence of GFP expressing cells after excitation with Ar-ion laser (wavelength 488 nm) and detection of emitted fluorescent light in the green channel was high enough for unambiguous identification of the GFP expressing mice. The sensitivity of this method was estimated 1:10(4) for detection of rare GFP expressing cells under the conditions used. This sensitivity should be sufficient for many studies on microchimerism. Because of the possibility for relocation of the cells, this method will be particularly useful for characterizing the cells with high GFP expression using other markers of cell phenotype or conventional morphological analysis.

Role of recombinant plasmid pEGFP-N1-IGF-1 transfection in alleviating osteoporosis in ovariectomized rats.

Decreased levels of serum insulin-like growth factor-1 (IGF-1) have been proven to cause osteoporosis. Gene transfer of IGF-1 offers an attractive technology to treat skeletal metabolic disorders including osteoporosis, but the viral vectors are limited by their high antigenicity and immune response. Our purpose was to investigate the expression of a non-invasive vector, recombinant plasmid enhanced green fluorescent protein-N1 (pEGFP-N1) that transferred IGF-1 gene into ovariectomized (OVX) rats in vivo and evaluate the effect of this therapy on osteoporosis. OVX or sham operations were performed in 60 female, 7-month-old unmated SD rats. 12 weeks after OVX operation, the vectors were transfected to the 10-month-old rats and experimental data were detected from 48 h to 7 week after transfection. Our results showed that remarkable expression of fluorescence and serum IGF-1 was observed in the rats transfected by recombinant plasmids, indicating that IGF-1 gene was successfully transferred to OVX rats by injecting the vector through hydrodynamic method via the tail vein. The bone metabolism index including serum alkaline phosphatase, the histomorphometric parameters of lumbar vertebra including trabecular area percentage, trabecular thickness, trabecular number and trabecular separation, and the bone mineral density (BMD) and biomechanical parameters of lumbar vertebra including BMD, maximum condensing force, crushing strength in OVX rats transfected by pEGFP-N1-IGF-1 were improved remarkably compared with OVX+pEGFP-N1 rats, indicating that the transfection of recombinant plasmid pEGFP-N1-IGF-1 played a significant role in alleviating osteoporosis in rats induced by OVX. This encouraged a potential approach of IGF-1 gene therapy to the treatment of osteoporosis.