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Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
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Animals , Mice , Adipose Tissue/cytology , Cytokines , Encephalomyelitis, Autoimmune, Experimental/therapy , Interleukin-17 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mice, Inbred C57BL , Phosphotransferases (Alcohol Group Acceptor)/genetics , T-Lymphocytes, Regulatory/cytology , Th17 Cells/cytology , TransfectionABSTRACT
Objective: To observe and compare the cytological and biological differences between human normal and degenerated nucleus pulposus (NP), and to investigate the repair effect of insulin-like growth factor 1 (IFG-1) and platelet derived growth factor (PDGF) on human degenerated NP. Methods: Human degenerative and normal NP tissues were obtained from operative patients, a portion of which were processed into tissue sections and HE staining was performed to observe the morphological changes of nucleus pulposus cells (NPCs) before and after degeneration of NP. Immunohistochemistry staining was used to determine the expression levels of collagen type Ⅰ, collagen type Ⅱ, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) proteins. Another portion of tissues were isolated and cultured and NPCs morphology was observed under inverted microscope. Western blot analysis was used to detect collagen type Ⅱ protein expression. Then, the gene transfection experiments were launched, including 4 groups, with group A designed as degenerated NPCs only, and groups B, C, and D of degenerated NPCs transfected with IGF-1 gene lentiviral particles, PDGF gene lentiviral particles, and lentiviral particles carrying IGF-1 and PDGF double genes, respectively. At 21 days after transfection, the cell morphology of each group was observed under inverted microscope, the positive rates of IGF-1 and PDGF of each group were measured by flow cytometry, and the expression of collagen type Ⅱ protein was detected by using immunohistochemistry staining and Western blot. Results: HE staining showed that there were a large number of notochordal cells and a small number of chondrocytes in the central NP tissue of normal group, while the NPCs in degeneration group were significantly reduced, and a large proportion of fibrocartilage tissues were found in NP tissue. Immunohistochemistry staining showed that the percentages of collagen type Ⅰ and Bax protein-positive cells in degeneration group were significantly higher than those of normal group, while the percentages of collagen type Ⅱ and Bcl-2 protein-positive cells were significantly lower than those of normal group ( P<0.05). Western blot showed that the relative expression level of collagen type Ⅱ protein in degeneration group was significantly lower than that in normal group ( t=65.493, P=0.000). At 21 days after gene transfection, compared with group A, the cell viability of groups B, C, and D increased and the morphology became more regular. Flow cytometry showed that the percentages of IGF-1-positive cells in groups B and D were significantly higher than that in group A, and the percentages of PDGF-positive cells in groups C and D were significantly higher than that in group A ( P<0.05). Immunohistochemistry staining showed that the positive stainings of collagen type Ⅱ in groups A, B, C, and D was (±), (+), (+), and (++), respectively. Western blot showed that the relative expression of collagen type Ⅱ protein in groups A, B, C, and D increased by degrees, and the differences between groups were significant ( P<0.05). Conclusion: Both IGF-1 and PDGF can reverse the degeneration of intervertebral discs NPCs and they have synergistic effects, providing experimental basis for its application in clinical treatment approaches for degenerative disc disease.
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BACKGROUND: Studies have shown that the loss of sex combing protein 1 (Asxl1) can lead to the occurrence of bone dysplasia and bone defects, but the relationship between this factor and bone destruction in the microenvironment of apical periodontitis has not been reported. OBJECTIVE: To study the effect of Asxl1 on proliferation and differentiation of osteoblasts in an inflammatory microenvironment. METHODS: MC3T3-E1 cells were excited by lipopolysaccharide to establish an in vitro inflammatory microenvironment. The best concentration and optimal action time of lipopolysaccharide were screened by cell counting kit-8 test. MC3T3-E1 cells were then stimulated with 20 mg/L lipopolysaccharide for 24 hours. The expression levels of Asxl1 protein and mRNA were detected by immunofluorescence and real-time PCR, respectively. After lipopolysaccharide stimulated the formation of inflammatory microenvironment, Asxl1-Si RNA was transfected for 24 hours, cell counting kit-8 was applied to detect the activity of cell proliferation, and real-time PCR was used to detect the expression levels of Asxl1 and osteogenic related genes ALP and RUNX2 mRNA. RESULTS AND CONCLUSION: After lipopolysaccharides stimulation of MC3T3-E1 cells, the expression levels of Asxl1 protein and mRNA were decreased. Under the inflammatory microenvironment, the proliferation activity of MC3T3-E1 cells transfected with Asxl1-Si RNA for 24 hours was significantly decreased, and the expression levels of Asxl1, ALP and RUNX2 mRNA were markedly decreased. These findings indicate that Asxl1 may influence the proliferation and differentiation of osteoblasts by involvement in the process of inflammatory reaction, thereby participating in bone destruction.
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BACKGROUND: Human amniotic mesenchymal stem cells have a wide variety of sources, low immunogenicity, and multilineage differentiation potential. Studies have confirmed that Scleraxis gene can induce human amniotic mesenchymal stem cells to differentiate into ligaments and accelerate tendon-bone healing. OBJECTIVE: To explore whether Scleraxis induces human amniotic mesenchymal stem cells to promote tendon-bone healing in vivo in rabbits, providing new options for clinical treatment of tendon-bone healing. METHODS: The study protocol was approved by the Ethic Committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The healthy full-term maternal placenta was taken and cultured, and human amniotic mesenchymal stem cells were isolated and cultured by trypsin digestion twice. Then the morphology of the cells was observed under an inverted microscope, and the cells were further cultured until the third generation for subsequent experiments. The lentivirus carrying the Scleraxis gene was transfected into human amniotic mesenchymal stem cells in vitro. Expression levels of ligament-related genes were detected by real-time fluorescent quantitative PCR, and the expression levels of related proteins were detected by immunofluorescence. Human amniotic mesenchymal stem cells transfected with Scleraxis gene were injected into the extraarticular tendon-bone model of rats. After 3 months, specimens were taken to observe the tendon-bone healing. RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells from passage to third generation showed long fusiform and vortex-like adherent growth under the inverted phase contrast microscope. (2) The third-generation human amniotic mesenchymal stem cells expressed green fluorescence after 24 hours of infection with the Scleraxis gene lentivirus, and the fluorescence expression was strong and stable. (3) Cell counting kit-8 findings indicated that lentivirus transfection of Scleraxis gene showed no influence on the cell growth rate. (4) Real-time fluorescent quantitative PCR findings showed that the mRNA expression of Scleraxis and ligament-related genes type I collagen, type III collagen, Fibronectin and Tenascin-C was significantly increased after lentivirus transfection of Scleraxis gene. (5) The results of immunofluorescence showed that the expression levels of ligament-related proteins type I collagen, type III collagen, Fibronectin and Tenascin-C were increased after lentivirus transfection of Scleraxis gene. To conclude, in vivo animal experiments have confirmed that the lentivirus transfection of Scleraxis gene can accelerate the tendon-bone healing of the rabbit extraarticular tendon-bone model.
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Objective To investigate the regulation effects of Ngn2 gene transfection on retinal neuron differnetion in three-dimentional optic vesicle (OV) of mice.Methods OV was cultured in vitro using mouse induced pluripotent stem cells (iPSC) under specific conditions.During OV culture,it was transfected multiple times by lentivirus-mediated Ngn2 gene and then it was induced after maturation.The cells were specificly differentiated toward retinal nerve cells in OV.Using the green fluorescent protein (EGFP) gene as control,the differentiation of retinal nerve cells in OV was detected by immunohistochemistry.Reverse transcription PCR and Western blot were used to quantitatively detect the expressions of retinal neuron-specific proteins Pax6,Islet1 and Brn3b.Results The mouse iPS-derived OV was successfully cultured.The number of neural cells in the OV transfected with the Ngn2 gene was increased by the lentiviral-mediated lentivirus.The expressions of PAX6,Islet1 and Brn3b in the Ngn2 transfection group were significantly higher at the gene and protein levels than those in the control group,with significant differences between the two groups (P＜0.05).Conclusions The Ngn2 gene can effectively increase the number of retinal neuron differentiation in OV and make in vitro cultured OV more mature and form a more perfect retinal cell neural circuit.
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Objective To modify polyethyleneimine (PEI) by using Poloxamer 188 (P188) , and evaluate its related feature as carriers of genes in vitro. Methods PEI was modified through conjugating one hydroxyl group of P188 to the amino group of PEI by carbonate method. Structural analysis of synthesized polymer was performed by using 1H-NMR. The particle size and Zeta potential of synthesized polymer /DNA complexes were measured. The cytotoxicity of the complexes was evaluated using MTT method in MCF-7 cells, HeLa cells and HepG2 cells. The pGL3-lus was used as a reporter gene, and the transfection efficiency of complexesat HeLa cells was evalutated by measuring activity of luciferase. Results The result of 1H-NMR showed the purity of these synthesized polymers was high. The particle size of complexes were decreased with the increment of N /P ratios. The Zeta potential of complexes increased with the increment of N /P ratios. The cytotoxicity of the complexes increased with the increment of N /P ratios. The synthesized polymers showed lower cytotoxicity than unmodified PEI. The new synthesized polymers had maintained the high transfection efficiency at high N /P ratios. In particular, the optimal transfection efficiency of (P188) 1- PEI (N /P = 24) was significantly higher than that of PEI (N /P = 6) . Conclusion The P188 modifed PEI can serve as a effective non-viral gene carriers to transfect Hela cells.
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Objective: To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods: mESCs were cultured in vitro, and the recombinant vector pEFla-Pax6-IRES-AcGFP and the empty vector pEFlot-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1 (SSEA1) and octamer binding transcription factor 4(0CT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results: Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and 0CT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion: Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.
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Objective To evaluate the relationship between the expression of microRNA-146a (miR-146a) in liver tissue and the inflammatory hepatic injury induced by ischemia/reperfusion (I/R) in rats. Methods One hundred and forty-four Sprague-Dawley (SD) rats were randomly divided into three groups: control (group N), sham operation (group S) and group I/R. Each group was subdivided into four subgroups (n = 12), and different substances were respectively injected intravenously to rats in different subgroups at 1 hour before the experiment: 220 μL physiological saline (group A), 20 μL miR-146a mimic + 200 μL physiological saline (group B), 20 μL miR-146a mimic + 200 μL ultrasound microbubble contrast agent (group C) and 20 μL miR-146a inhibitor + 200 μL ultrasound microbubble contrast agent (group D). Before the experiment and after experiment for 24 hours, the plasma concentrations of alanine aminotransferase (ALT), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected, the reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression of miR-146a in liver tissue, and Western Blot was applied to detect protein expressions of Toll-like receptor 4 (TLR4), IL-1 receptor associated kinase 1 (IRAK-1), IL-6 and TNF-α, and the pathological hepatic cell injury was observed. Results Before the experiment and 24 hours after experiment in various subgroups of N and S groups, there were no statistical significant differences in the plasma concentrations of ALT, IL-6 and TNF-α, and the expression of miR-146a level and the protein expressions of TLR4, IRAK-1, IL-6 and TNF-α in liver tissues; the pathological examination also did not show any obvious hepatic cell injury. After the experiment for 24 hours: compared to the group S, the liver tissue miR-146a expression was significantly decreased in the subgroups A and D of group I/R (miR-146a/U6nsRNA: 0.51±0.13, 0.22±0.09 vs. 1.01±0.02, both P < 0.01), and the plasma concentrations of ALT, IL-6 and TNF-α and the protein expressions of TLR4, IRAK-1, IL-6 and TNF-α in liver tissues were significantly increased [ALT (U/L): 103.23±26.64 vs. 44.16±18.55, 176.46±7.26 vs. 49.74±6.83, IL-6 (μg/L): 64.28±16.19 vs. 17.68±7.54, 88.49±3.23 vs. 15.58±2.38; TNF-α (μg/L): 31.28±2.57 vs. 5.58±3.35, 59.12±8.74 vs. 5.27±1.37; TLR4/GAPDH: 2.43±0.36, 3.23±0.71 vs. 0.96±0.24, IRAK-1/GAPDH: 2.34±0.52, 3.14±0.63 vs. 0.76±0.21, IL-6/GAPDH: 1.01±0.22, 1.11±0.16 vs. 0.98±0.37, TNF-α/GAPDH: 2.05±0.48, 2.86±0.27 vs. 0.59±0.16, all P < 0.01], moreover, the hepatic pathological lesions were obvious; the liver tissue expression of miR-146a was significantly increased after being transfected with miR-146a mimic in subgroups B and C of group I/R (miR-146a/U6nsRNA: 1.56±0.31, 2.40±0.53 vs. 1.01±0.02, both P < 0.01), especially in group C combined with ultrasound microbubble (P < 0.01). However, the protein expressions of TLR4, IRAK-1, IL-6 and TNF-α in liver tissues were significantly decreased (TLR4/GAPDH:0.77±0.18, 0.65±0.27 vs. 0.96±0.24, IRAK-1/GAPDH: 0.61±0.14, 0.47±0.20 vs. 0.76±0.21, IL-6/GAPDH:0.80±0.13, 0.54±0.22 vs. 0.98±0.37, TNF-α/GAPDH: 0.41±0.14, 0.16±0.03 vs. 0.59±0.16; all P < 0.01), and the expressions were more significant in the group C combined with ultrasound microbubbles (P < 0.01), and the hepatic pathological damage was mild, however, the plasma concentrations of ALT, IL-6 and TNF-α were of no statistical significant differences. Conclusion Ultrasound microbubble can efficiently transfect miR-146a mimic and inhibitor into the liver tissue, and miR-146a may negatively regulate the I/R inflammatory liver injury mediated by TLR signaling pathway.
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Objective To achieve the purpose of promoting movement function of the injury nerve by using the joint therapy of NT- 3- HUMSCs and SOCS3 gene silencing on SD rats'spinal cord injury. Methods (1) We used adherence method in vitro human umbilical cord-derived mesenchymal cells (HUMSC) during separation, purification and identification. (2) Then constructed NT-3 gene eukaryotic expression vector, which was transfected into its HUMSC, and constructed NT-3- HUMSC cell survival in vitro assay conditions and NT-3 expression. (3) We selected specific targets for SOCS3 screening and for sequence homology analysis. A negative control group was established. siRNA was designed and synthesized in vitro detection. (4) SD rats with spinal cord injury model were divided into two categories: (1) sham group with 10 rats; (2) T12 whole spinal cord injury model with 40 rats. The 40 rats were randomly divided into four groups with 10 rats in each group (saline treatment group,siRNA +NT-3-HUMSCs treatment group,NT-3-HUMSCs treatment group and siRNA treated group) . Motor function of the rats were evaluated respectively in 1, 2 and 3 months after the modeling was established successfully.Results(1) siRNA + NT-3-HUMSCs treatment group's BBB scores was significantly higher than NT-3-HUMSCs, SOCS3-siRNA and physiological saline groups ( P＜0.05) . (2) The grid climbing experiments showed that the neural functional recovery performed better in siRNA+the NT- 3- HUMSCs treatment group compared to the NT - 3 - HUMSCs, SOCS3 - siRNA and physiological saline groups (P＜0.05) . Conclusion The NT- 3- HUMSCs joint SOCS3 gene silencing in the treatment of SD rat spinal cord injury can improve the motor function of SD rat spinal cord injury.
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Objective To investigate the effect of joint therapy by NT-3-HUMSCs and SOCS3 gene silencing in promoting the injury nerve regeneration repair after spinal cord injury in SD rats. Methods (1) Adherence method was used to culture human umbilical cord-derived mesenchymal cells (HUMSC) in vitro for separation, purification and identification. (2) We constructed NT-3 gene eukaryotic expression vector, and used gene transfection technology into its HUMSC, and tested the survival of NT-3-HUMSC cells and NT-3 expression in cells. (3) We screened specific targets of SOCS3, made sequence homology analysis, and set a negative control, designed and synthesized siRNA and detected the function. (4) SD rats model of spinal cordinjury were established and divided into: 1. sham group 10; 2.T12 whole spinal cord injury model 40, were randomly divided into four groups, respectively; saline treatment group 10; siRNA + NT-3-HUMSCs treatment group 10; NT-3-HUMSCs treatment group 10; siRNA treated group 10. After each group above modeling success, they received respectively the neural electrophysiological monitoring for 12 weeks survival. (5) We perfused SD rats for fixation and collect samples, and observed the local glial scar degradation situation and axon regeneration, meanwhile, used biotin glucan fluorescent (BDA) anterograde tracing. The injury transplant area-host junction spinal cord tissues were collected to observe the corticospinal tract regeneration under microscope. Results (1) In siRNA + NT-3-HUMSCs treatment group, the transection syringomyelia was significantly reduced as compared with normal saline group (P ＜ 0.05). (2) BDA anterograde tracing results showed that in the siRNA + NT-3-HUMSCs treatment group, neural axon grew significantly compared with the normal saline group. (3) Neural electrophysiological testing 12 weeks after injury: in the treatment group, the incubation period P40 was shorter as compared with control group; in siRNA + NT-3-HUMSCs treatment group, the incubation period was shorter obviously than normal saline, but the amplitude increased obviously (P ＜ 0.05). Conclusion NT-3-HUMSCs joint with SOCS3 gene silencing can promote the injury nerve regeneration repair in the treatment of SD rat spinal cord injury.
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Objective To observe the effects of pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells (PEDF-MSCs) on the expression of pigment epithelial-derived factor (PEDF) and vascular endothehal growth factor (VEGF) in a rat model of retinal ischemia-reperfusion injury (RIRI) and its protection on retinal ganglion cells.Methods Lentivirus (LV) labeled with green fluorescent protein (GFP) was used as a vector to transfect the PEDF gene into human umbilical cord mesenchymal stem cells (hUCMSCs) at a multiplicity of infection (MOI) of 1,10,20,and 50 in vitro,and then the expression of PEDF gene and protein in cells transfected with the best MOI value was detected by RT-PCR and ELISA.The healthy male SD rats were randomly divided into normal group (N group) and experimental group.The RIRI model was made by high intraocular pressure in the experimental group,and the RIRI rats with PBS treatment were allocated as the PBS group,with hUC-MSCs treatment as M group and LV-PEDF-MSCs treatment as P group,and the N group was left untreated.All rats were sacrificed on day 5,and the number of retinal ganglion cells were counted by Nissl staining,the thickness of the retina was calculated,as well as the expression of PEDF and VEGF mRNA in rat retina was detected by RT-PCR.Results The transfection efficiency was as high as 75.8％ under fluorescence microscope.The results of RT-PCR showed that the relative expression of PEDF mRNA in PEDF-MSCs supernatant (4.34 ± 0.29) was significantly higher than that in hUCMSCs (1.08 ± 0.15),and the difference was statistically significant (P ＜ 0.05);moreover,the results of ELISA showed that PEDF protein expression in PEDF-MSCs supernatant [(83.09 ± 7.58)μg · L-1] was significantly higher than that in hUCMSCs [(12.30 ±1.24) μg · L-1],and the difference was statistically significant (P ＜ 0.05).Nissl staining results showed that the number of ganglion cells in group PBS decreased after model establishment.After 5 days of treatment,the number of ganglion cells in P group and M group was higher than that in PBS group,and the difference was statistically significant (both P ＜ 0.05);and P group was higher than M group,with the significant difference (P ＜ 0.05).And this was true of the thickness of the retina.RT-PCR showed that the expression of PEDF mRNA in P group was significantly up-regulated,but VEGF mRNA expression was significantly down-regulated,and the differences were statistically significant when compared with PBS and M group (both P ＜ 0.05).Conclusion Intravitreal injection of PEDF-MSCs can up-regulate the expression of PEDF but down-regulate the expression of VEGF in the retina of RIRI rats,which can protect the retinal ganglion cells against RIRI.
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<p><b>OBJECTIVE</b>This study aims to investigate the effect of human osteoprotegerin (hOPG) gene-modified rat bone marrow mesenchymal stem cells (rBMSCs) combined with hydroxyapatite (HA) scaffolds on the repair of mandibular defects in ovariectomized rats.</p><p><b>METHODS</b>rBMSCs were transfected with adenovirus carrying pDC316-hOPG-EGFP. The expression of hOPG and the inhibition of osteoclast function were detected by Western blot and bone-grinding experiment respectively. The model of mandibular bone defect in rats with osteoporosis was established; HA, untransfected rBMSCs-conjugated HA, and transfected rBMSCs-conjugated HA scaffolds were implanted into the mandibular bone defects. After six weeks, tartrateresistant acid phosphatase staining and hematoxylin-eosin staining were used to observe the number of osteoclasts and repair of bone defect.</p><p><b>RESULTS</b>Adenovirus carrying hOPG gene in vitro were successfully transfected into rBMSCs. The hOPG with anti-osteoclast activity was expressed by hOPG-rBMSCs, and rBMSCs expressing hOPG combined with HA scaffolds promoted mandibular defect repair.</p><p><b>CONCLUSIONS</b>rBMSCs transfected with hOPG gene inhibited the function of osteoclasts both in vitro and in vivo, and transfected rBMSCs combined with HA scaffolds promoted the repair of mandibular defects in rats with osteoporosis.</p>
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Objective To establish nasopharyngeal carcinoma cell line (CNE1) with eIF1 gene stable over-expression and to study its effects on the proliferation and migration of nasopharyngeal carcinoma cells.Methods EIF1 over-expression vector was constructed by adopting the pEGFPC1 eukaryotic expression system for transfecting nasopharyngeal carcinoma CNE1 cells.Thus the stably transfected EIF1-elF1 and its control cells were obtained.The over-expression situation of eIF1 in these cells was verified by real time fluorescence quantitative PCR(qPCR) and Western blot.The proliferation and migration activity of CNE1-eIF1 cells were tested by adopting the cell proliferation and migration tests.Results The enzyme digestion electrophoresis identification and sequencing showed that the pEGFPC1-eIF1 eukaryotic expression vector was successfully constructed.After mRNA and protein expression identification,compared with the reloading plasmid transfection group,the eIF1 gene mRNA and protein expression levels in nasopharyngeal carcinoma cell line CNE1 stably over-expressing eIF1 were up-regulated by 2.85 folds and 2.58 folds respectively (P＜ 0.05),while its proliferation and migration activities were down-regulated by 55 ％ and 36 ％ respective (P＜ 0.05).Conclusion The nasopharyngeal carcinoma cell line over-expressing elF1 is successfully constructed,the eIF 1 over-expression could significantly down-regulate the proliferation and migration activities of nasopharyngeal carcinoma cells,suggesting that eIF1 has potential anti-tumor effect.
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Aim To observate the effect of chemokine receptor(CXCR4) gene transfection on biological behavior of bone marrow mesenchymal stem cells in vitro.Methods Firstly, bone marrow mesenchymal stem cells were divided into three groups:GFP(transfected GFP into MSCs), CXCR4+(transfected CXCR4+ into MSCs) and CXCR4-(transfected CXCR4-into MSCs) group.Then, their capacity of proliferation, differentiation and migration ability (in vitro) was assessed with immunofluorescence cytochemistry method, flow cytometry assay and Transwell cell chemotaxis test.Results The high or low expression of CXCR4 had no effect on their ability of proliferation and differentiation into lung tissue.Compared with GFP group, however, CXCR4+-MSCs group significantly increased the number of migrating cells, while CXCR4——MSCs group showed no change in the number of migrating cells.Conclusions The proliferation and differentiation capacities are not affected by the high or low expression of CXCR4.The high expression of CXCR4 can significantly enhance the migration ability of MSCs to inflammatory lesions, and the low one has no effect on the migration of the cells.After the transplantation of MSCs, CXCR4′s high expression will access to the lesion area to participate in tissue repairing rapidly and largely, significantly enhancing the therapeutic efficacy.
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Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery(UTMD) mediated a peptide nucleic acid (PNA) binding nuclear localization signal (NLS).Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor.The SV40T antigen receptors were expressed on the membranes of 293T cells.The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA,which increased nuclear localization.Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm,respectively.The study was divided into five groups:Contrast (group A),Common microbubble + DNA (group B),Antibody-targeted microbubbles + DNA (group C),Common microbubbles + NLS-PNA-DNA (group D),Antibody-targeted microbubbles + NLS-PNA-DNA (group E).Fluorescence microscope was used to observe the fluorescent light in each group;flow cytometry to test the transfection;RT-PCR and Western blot to detect genes' mRNA and protein expression level.Results Ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability(＞80％).Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully.The transfection results of flow cytometry were 0,(9.30 ± 0.46)％,(26.46 ± 2.01)％,(29.54 ± 0.62)％,(45.72 ± 1.86)％,respectively,and the differences were statistically significant(P ＜0.05).The relative mRNA and protein expression in group E were greater than those in group C and D respectively (P ＜0.05).Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nuclear DNA import.
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Objective To investigate the effects of human leptin ( hLEP) gene transfection on rat bone marrow stro-mal cells ( rBMSCs) .Methods rBMSCs were cultured and transfected with adenoviruses encoding hLEP ( Ad5-hLEP-EGFP) in vitro as experimental group while rBMSCs transfected with Ad 5-EGFP and non-transfected were control groups.The proliferation was detected by MTT and the expression of collagen type Ⅰ(Col-Ⅰ) and alkaline phosphatase ( ALP) were assessed by real-time PCR.The ability of mineralized nodule forming was also examined by Alizarin red staining .The combination of transfected rBMSCs and β-tricalcium phosphate (β-TCP ) was con-structed and the osteogenic ability of the construction was evaluated in nude mice .Results hLEP could be trans-fected into rBMSCs successfully by adenovirous .After transfection , the proliferation was not affected while Col-Ⅰand ALP expressions were more pronounced in rBMSCs transfected with Ad 5-hLEP-EGFP ( P<0.05 ) .Alizarin red staining showed the ability of mineralized nodule forming was also up-regulated in Ad5-hLEP-EGFP group (P<0.05).In addition, the transfected rBMSCs adhered to β-TCP and survived well and the combination showed more new bone like tissue formation in nude mice compared to control groups .Conclusions rBMSCs transfected with hLEP might be potently used in bone or periodontal tissue regeneration .
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Objective To evaluate the effect of ultrasound guided via chest puncture injection and ultrasound targeted microbubble destruction(UTMD) mediated the angiogenin 1 (Ang1) gene therapy in myocardial infarction (MI) canines.Methods Thirty nine dogs were divided into three groups (each group 13 dogs):① MI group (MI dogs without treatment);② intravenous injection group (MI dogs with intravenous injection and UTMD treatment);③ myocardial injection group (MI dogs with myocardial injection and UTMD treatment).Four weeks later,the safety and incidence of complications were compared.The dimensions and systolic function of left ventricular were measured by echocardiography.The percentage of collagen fiber was assessed by Masson.CD31 was applied for quantifying capillary density.The Ang1 protein was detected by Western blotting.Results ①The survival and complications showed no significant difference among the 3 groups(allP ＞0.05).②Compared with MI group,the left ventricular end systolic dimension (LVESD)reduced and the left ventricular ejection fraction (LVEF) increased in intravenous injection group;the left ventricular end-diastolic dimension (LVEDD) and LVESD were both reduced in myocardial injection group,the LVEF was increased significantly(all P ＜0.05).③ The immunohistochemistry showed lower collagen fiber percentage and higher blood vessel density in myocardial injection group than those in the other groups(P ＜0.05).④The relative quantity of Ang1 was significantly higher in myocardial injection group than those of the other groups.The differences were statistically significant (P ＜0.05).Conclusions The combination of ultrasound guided via chest puncture injection and UTMD is a safe and effective method for gene transfection.It mediates Ang1 gene transfection that can promote angiogenesis after MI and improve left ventricular systolic function.
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Objective To evaluate gene transfection in liver , lung and kidney by ultrasound , microbubble and recombinant adenovirus mediated exogenous stromal cell derived factor-1α ( SDF-1α) gene transfer to the heart in rats with acute myocardial infarction( AMI) . Methods Forty AMI SD rats were randomly divided into control and experimental groups:myocardial infarction + ultrasound irradiation group (M+ U/control group , n = 10) ,and 3 experimental subgroups on the basis of pAd-EGFP/SDF-1α ( The biotin recombinant adenovirus expressing enhanced green fluorescent protein and SDF-1α) . Genes transfection length of time:1 day ,2 days and 3 days of transfection ( M +S1+U ,M+S2+U and M+S3+U group) . The expression of EGFP in liver ,lung and kidney were detected by laser scanning confocal microscopy at 7 days after administration . Results There was a little expression of EGFP in the liver ,lung and kidney in the drug delivery group and no expression in the control group .The differences in the expression of EGFP between all the gene delivery groups and the control group were statistically significant ( P <0 .05) . With the increase of the number of medication days , the target gene transfection increased slightly ,but there was no significant difference among the different drug delivery groups . Conclusions When the ultrasound ,microbubble and recombinant adenovirus mediated exogenous SDF-1αgene transfer to the heart in AMI rats ,liver ,lung and kidney tissues will also be transfected . However ,with the increasing of the days of administration , the transfection of target gene in non-target tissue produces only a slight accumulation . The transfection area of target gene in non-target tissue is not linear correlated with the days of administration .
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Objective To establish nasopharyngeal carcinoma cell line (CNE1) with eIF1 gene stable over-expression and to study its effects on the proliferation and migration of nasopharyngeal carcinoma cells.Methods EIF1 over-expression vector was constructed by adopting the pEGFPC1 eukaryotic expression system for transfecting nasopharyngeal carcinoma CNE1 cells.Thus the stably transfected EIF1-elF1 and its control cells were obtained.The over-expression situation of eIF1 in these cells was verified by real time fluorescence quantitative PCR(qPCR) and Western blot.The proliferation and migration activity of CNE1-eIF1 cells were tested by adopting the cell proliferation and migration tests.Results The enzyme digestion electrophoresis identification and sequencing showed that the pEGFPC1-eIF1 eukaryotic expression vector was successfully constructed.After mRNA and protein expression identification,compared with the reloading plasmid transfection group,the eIF1 gene mRNA and protein expression levels in nasopharyngeal carcinoma cell line CNE1 stably over-expressing eIF1 were up-regulated by 2.85 folds and 2.58 folds respectively (P＜ 0.05),while its proliferation and migration activities were down-regulated by 55 ％ and 36 ％ respective (P＜ 0.05).Conclusion The nasopharyngeal carcinoma cell line over-expressing elF1 is successfully constructed,the eIF 1 over-expression could significantly down-regulate the proliferation and migration activities of nasopharyngeal carcinoma cells,suggesting that eIF1 has potential anti-tumor effect.
ABSTRACT
Objective To invstigate the effect of ultrasound microbubble mediated miRNA delivery on mi-gration,invasion and cloning ability of human hepatoma HepG2 cells. Methods The migration,invasion and col-ony forming ability of HepG2 cells were measured after transfection with antisense miRNA-21/221 and miRNA-199a mimic via the optimal ultrasound microbubble transfection method. Results Compared with the control group ,the migration ,invasion and cloning ability of cells were significantly inhibited after transfection with miRNA mimics(P < 0.05,respectively),especially for miR-199a(relative cell migration rate was 31.05%,the number of invasive cells were 38.67 ± 4.51 and the number of clones were 105.67 ± 5.86). Conclusion The pres-ent study may provide new ideas and clues for gene therapy and prognosis of hepatocell ular carcinoma through ana-lyzing the effect of miRNAs on the biological characteristics of human hepatoma HepG2 cells.