Lentivirus mediated CCN1 gene on growth and migration of rat bone marrow mesenchymal stem cells

AIM：To investigate the role of cysteine-rich 61 (Cyr61/CNN1) in proliferation and migration of bone marrow mesenchymal stem cells (BMSCs). METHODS：The lentiviral vector carrying CCN1 (Lenti-GFP-CCN1) was constructed and then transfected into the rat BMSCs. The cells were divided into non-transfection group, transfection group (transfected with Lenti-GFP-CCN1) and negative control group (Lenti-GFP). The fluorescence intensity of the transfected BMSCs was observed under inverted fluorescence microscope. The effects of CCN1 on the proliferation and migration of BMSCs were detected by MTT assay and scratch wound healing assay. RESULTS：The proliferation of BMSCs transfected with Lenti-GFP CCN1 had no significant difference compared with negative control group and control group. The width/thickness ratio of migrated BMSCs in wound healing was significantly higher in Lenti-GFP-CCN1 group than that in negative control group and control group (P<0.05). CONCLUSION：Exogenous CCN1 promotes the migration of BMSCs.     

Effects of angiotensin II type 2 receptor blocker on wound healing in a mouse skin full-thickness injury model

AIM：To observe the effect of angiotensin II (Ang II) and its type 2 receptor (AT2R) on re-epithelialization, granulation tissue formation and growth factor production during wound healing, and to explore the possible mechanism by which Ang II and AT2R influence wound healing. METHODS：Two full-thickness skin wounds were created on the dorsum of C57BL/6J mice. The animals were treated with or without AT2R blocker PD123319 at a dose of 10 mg/kg daily after wounding. Specimens were taken from the wound of each mouse on days 3, 5, 7, 9, 11, 13 and 15 after wounding. Re-epithelialization and granulation tissue formation in the wounded skin tissues were evaluated by HE staining. The production of growth factors,epidermal growth factor (EGF),vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounded tissues during the healing process was detected by ELISA. RESULTS：Treatment with PD123319 significantly increased the rate of re-epithelialization and the area of granulation tissue compared with control group at 5 d and 7 d after wounding. Moreover, peritoneal application of PD123319 increased the production of EGF, VEGF and bFGF in the wounded tissues at the indicated time points after wounding. CONCLUSION：AT2R blocker PD123319 accelerates wound healing via promoting re-epithelialization,granulation tissue formation and growth factor production.     

Curcumin inhibits HGF induced vascular endothelial cell migration and VEGF expression in vitro and in vivo

ATM: To explore the molecular mechanism that curcumin inhibits hepatocyte growth factor (HGF) induced angiogenesis. METHODS: The effects of curcumin, c-Met inhibitor SU11274, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and mTOR inhibitor rapamycin on HGF-induced endothelial cell migration, tubule formation ability, vascular endothelial growth factor (VEGF) expression, related signaling pathways and the density of blood vessels in tumors were observed by the methods of capillary forming experiments, wound healing assay, Western blot and animal study. RESULTS: SU11274, LY294002, rapamycin and curcumin significantly inhibited HGF induced endothelial cell migration, tubule formation and VEGF expression, suppressed the phosphorylation of c-Met/AKT/mTOR/S6 pathway related molecules, reduced VEGF expression and microvascular density in the tumors. CONCLUSION: Curcumin inhibits HGF induced angiogenesis by inhibiting c-Met/AKT/mTOR/S6 pathway activation.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.     

Attenuated Salmonella carrying pGRIM-19-si-survivin co-expression plasmid inhibits growth of prostate cancer subcutaneous xenografts in vivo

AIM：To explore the effects of pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella on prostate cancer subcutaneous xenograft growth in nude mice. METHODS：Prostate cancer xenograft model was established in nude mice. Co-expression plasmids carried by attenuated Salmonella were introduced by intraperitoneal injection. The xenograft volumes were monitored timely. Immunohistochemical staining, RT-PCR and TUNEL assay were applied to investigate the related mechanisms that pGRIM-19-si-survivin inhibited tumor growth in vivo. RESULTS：Compared with psi-survivin and pGRIM-19 carried by attenuated Salmonella (control groups), the tumor volumes were reduced markedly in pGRIM-19-si-survivin plasmid group. The mean shrinkage rates were 2.36 and 3.02 times. pGRIM-19-si-survivin co-expression plasmid carried by attenuated Salmonella inhibited survivin expression but strengthened GRIM-19 expression obviously (P<0.05). The mRNA expression of apoptosis-related proteins such as Bcl-xL, Stat3, cyclin D1 and c-Myc was inhibited, and the vascular endothelial growth factor (VEGF) mRNA and Ki67 protein were also inhibited, but the caspase-3 mRNA expression was up-regulated (P<0.05) with significant cell apoptosis. CONCLUSION： pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella inhibits the growth of prostate cancer subcutaneous xenografts in nude mice by promoting cell apoptosis and inhibiting prostatic cancer proliferation.     

Effect of CD151 on biological characteristics of human umbilical cord mesenchymal stem cells

AIM：To study the effect of CD151 on the biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS：CD151 expression on hUC-MSCs was interfered by siRNA. The cells were divided into siRNA-CD151 group and negative control group (treated with siRNA-NC). The efficiency of interference after 72 h and the changes of other surface markers were detected by flow cytometry. The ability of differentiation was assessed by oil red O and von Kossa staining. The cell cycle was analyzed by flow cytometry. The mRNA expression of CD151, hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), cyclooxygenase 2 (COX-2) and indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs was detected by real-time PCR. The secretion of HGF by hUC-MSCs was measured by ELISA. RESULTS：The results of flow cytometry showed that the expression of CD151 (11.97±2.63 vs 95.66±1.56, P<0.01) and CD105 (93.66±0.21 vs 83.37±0.71, P<0.05) on hUC-MSCs in siRNA-CD151 group was lower than that in negative control group. The consistent results were also achieved by using the method of real-time PCR. Treatment with siRNA-CD151 down-regulated the progress of the cell cycle as the G1 phase increased and the S phase decreased. The mRNA expression levels of HGF and TGF-β1 in hUC-MSCs in siRNA-CD151 group were lower than those in negative control group, and opposite result of COX-2 mRNA expression was observed. The IDO mRNA in hUC-MSCs was unchanged with IFN-γ stimulation for 24 h. HGF concentration in siRNA-CD151 group was decreased as compared with negative control group. CONCLUSION：Interfering CD151 expression on hUC-MSCs doesn’t change other surface markers except CD105, and maintains the capacity of adipogenic differentiation. However, it changes the osteogenic differentiation, proliferation and the expression of immunomodulatory cytokines.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Human umbilical cord mesenchymal stem cells migrate to Raji cells and promote their proliferation

AIM：To investigate the interaction between human Burkitt lymphoma cell line Raji and human umbilical cord mesenchymal stem cells (hUC-MSC). METHODS：The direct and indirect effects of MSC on Raji cells were investigated. The viability of Raji cells was tested by CCK-8 assay, and the cell cycle was determined by flow cytometry. The importance of vascular endothelial growth factor (VEGF) in the migration of MSC to Raji cells was analyzed by blocking VEGF expression in Raji cells with small interfering RNA (siRNA). VEGF level in the supernatant was detected by ELISA, and the mRNA expression of VEGF was measured by quantitative real-time PCR (qRT-PCR). RESULTS：Both MSC and MSC-conditioned medium (MSC-CM) promoted the growth of Raji cells. The viability of Raji cells co-cultured with MSC-CM was 0.99±0.05 at 72 h, and that in control group was 0.71±0.07. Both direct co-culture with MSC and MSC-CM turned the Raji cells from G0/G1 phase to S phase. The number of Raji cells co-cultured with MSC-CM in S phase was increased from 16.33±1.37 to 28.50±1.41, and the number in G0/G1 phase was decreased from 77.70±1.57 to 54.40±1.57. The expression of VEGF was down-regulated either at mRNA or protein level after transfection with siRNA. The ability of MSC migrated to Raji cells was significantly declined (96.00±5.28 vs 143.00±7.20). CONCLUSION：Raji cells recruit MSC by secreting VEGF, and MSC promote the proliferation of Raji cells by turning the cells from G0/G1 phase to S phase.     

Effect of advanced glycation end products on inflammation in cultured cardiomyocytes

AIM: To investigate the effect of advanced glycation end products on inflammation in cultured cardiomyocytes.METHODS: Primary cardiomyocytes were isolated from Sprague-Dawley neonatal (1 to 2 days old) rats ventricles.The insulin resistant cardiomyocyte model was established.Neonatal rat ventricular myocytes were exposed to AGEs for 24 hours.TNF-α mRNA and PPAR-γ mRNA expressions were determined by RT-PCR.Activation of NF-κB in the cells was examined by using immunocytochemistry.The ultrastructure of the cells was detected by transmission electron microscope.RESULTS: The exprssion of TNF-α mRNA and the activation of NF-κB increased,the expression of PPAR-γ mRNA decreased compared with control group (P<0.05).The differences among different AGE-BSA groups were significant (P<0.05).The numbers of chondriosome and smooth endoplasmic reticulum increased.CONCLUSION: AGEs significantly increase TNF-α mRNA expression and NF-κB activation,and restrain the expression of PPAR-γ mRNA.These data suggest that AGEs play an important role in the onset of diabetic cardiomyopathy.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Effect of HGF gene transfection on human lymphoma xenografts in nude mice

AIM：To explore the promotion effect of hepatocyte growth factor (HGF) gene transfection on human lymphoma xenografts in nude mice. METHODS：The model of human lymphoma xenograft in nude mice was established by transplantation of Raji cells, which were transfected with recombinant plasmid pVITRO2-HGF harboring the HGF gene. The body weight of the nude mice and the tumor size were dynamically monitored and the tumor tissues were obtained after 8 weeks. Additionally, the methods of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and immunohistochemistry were used to detect the apoptotic index (AI) and microvessel density (MVD). RESULTS：The success rate of the human lymphoma xenografts in nude mice was 96.7%. The tumor volume in HGF transfection group was significantly greater than that in HGF transfection+VP-16 group and control groups (non-transfection group and empty vector group). The tumor volume in HGF transfection+VP-16 group was also bigger than that in control groups. No difference of the tumor volume between non-transfection group and empty vector group was observed. AI in HGF transfection group was substantially lower than that in control groups. AI in HGF transfection+VP-16 group showed a little higher than that in HGF transfection group, yet was still lower than that in control groups. MVD in HGF transfection group was extraordinary higher than that in control groups, but decreased after VP-16 induction (P<0.01), which was still higher than that in control groups. CONCLUSION：HGF gene transfection significantly promotes the growth of human lymphoma xenografts in nude mice and substantially inhibits the apoptosis presumably owing to promoting tumor angiogenesis and inhibiting tumor cell apoptosis.     

Coriaria sinica Maxim extract promotes burn wound healing in rats

AIM：To investigate the effects of Coriaria sinica Maxim extract (CSME) on burn wound healing in rats. METHODS：Forty male SD rats were randomly divided into normal saline (NS) group, white petrolatum jelly (WPJ) group, silver sulfadiazine (SSD) group and CSME group. After the animals were anesthetized, the skin of their backs was burnt to induce deep Ⅱ degree burn wounds. These wounds were treated respectively for 21 d by covering dressings with NS, WPJ, SSD and CSME, respectively. On the 1st, 3rd, 7th, 14th and 21st days, after the clinical symptoms of the animals and conditions of the wounds such as the epithelization rate, crusting and hair growth were observed, the wound tissues were also taken for histological examination. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD), and the expression of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and collagen were detected. RESULTS：On the 21st day after burn, the new epithelial tissues in CSME group extraordinarily　developed and a great deal of hair also grew along the margin of the wounds. The epithelization rate of the wound tissues in CSME group was higher than that in other groups. There was rare new hair in the center of the wound area in SSD group, but intensive new hair was observed in CSME group. On the 14th day and 21st day after burn, multilayer of epithelial cells was entirely covered with the wound area in CSME group. Some of the healing signs, such as sufficient differentiation and a line up in order of collagen fibers, clear tissue structure, exceedingly active hyperplasia of sebaceous glands and hair follicles, and so on, were also observed in the wound area in CSME group. From the 1st day to 21st day after burn, the increase in protein expression of EGF and bFGF in the wound tissues was significantly higher than that in other groups in the early stage, which was quickly decreased and was obviously lower than other groups in the latter stage. The mRNA expression ratio of type I and III collagens in SSD group was significantly higher than that in other groups, while that in CSME group was significantly lower than that in other groups. CONCLUSION：CSME promotes burn wound healing without scarring. The effects of CSME are likely to associate with the expression reinforcement of EGF and bFGF at mRNA and protein levels in the early stage, and associate with the expression inhibition of them later. Moreover, CSME may also inhibit the mRNA expression of type I collagen and promote the synthesis of type III collagen in the wound tissues of burn.     

Gene silencing of indoleamine 2,3-dioxygenase 2 influences proliferation,migration and invasion of B16-BL6 melanoma cells

AIM: To investigate the effects of indoleamine 2, 3-dioxygenase 2 (IDO2) silencing on proliferation, migration and invasion of B16-BL6 melanoma cells.METHODS: IDO2-siRNA was transfected into the B16-BL6 melanoma cells in vitro. The expression of IDO2 or IDO1 at mRNA and protein levels was detected by real-time PCR and Western blot. Colony formation assay was performed to analyze the proliferation of IDO2-silencing tumor cells. The migration ability of B16-BL6 cells after silencing of IDO2 was measured by wound healing assay and Transwell cell migration assay. The invasion ability of the tumor cells was detected by Transwell cell invasion assay.RESULTS: IDO2-siRNA signi-ficantly down-regulated IDO2 expression in B16-BL6 melanoma cells, and did not affect IDO1 expression. Compared with control group, the colony formation ability, the migratory distance measured by wound healing assay, and the migration and the invasion cell numbers detected by Transwell assay all remarkably decreased in the IDO2-silencing cells.CONCLUSION: IDO2 silencing affects the proliferation, migration and invasion abilities of the B16-BL6 melanoma cells.     

Basic fibroblast growth factor promoting the healing of palatal perforation in rats

AIM: To study the influence of basic fibroblast growth factor(bFGF) on treating ed palatal perforation in rats. METHODS: bFGF was given to the early palatal perforation in rat. The granulation tissues in perforations were grossly and pathologically obserVed. RESULTS: The the of wound healing was significantly in- crease in the bFGF group (P<0.01 ). bFGF could obviously promote the proliferation of the granulation tissues and fibroblasts. The number of AgNORs granules in fibrablasts were 3.73 ±0.52 in the buy group and 2.11 ±0.31 in control group (P < 0.05). CONCLUSION: bFGF could promote the growth of granulation tissues and had a strong promotion on the wound healing in palatal perforation. It was helpful in repairing palatal perforation.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Effect of annexin A2 on proliferation,migration and apoptosis of human cervical cancer HeLa cells

AIM：To explore the effect of annexin A2 (ANXA2) on the proliferation, migration and apoptosis abilities of human cervical cancer HeLa cells. METHODS：Overexpression vectors and siRNA of ANXA2 were constructed, and then transfected into HeLa cells. The HeLa cells were divided into 4 groups:control group, scramble group, ANXA2 overexpression group and ANXA2-siRNA group. The expression of ANXA2 at mRNA and protein levels was examined by real-time PCR and Western blotting. MTT assay, Boyden chamber and flow cytometry were used to determine the effect of ANXA2 on the proliferation, migration and apoptosis abilities of the HeLa cells. RESULTS：The proliferation and migration of HeLa cells were obviously promoted by ANXA2 overexpression. The proliferation and migration of HeLa cells were remarkably inhibited by the transfection of ANXA2-siRNA. ANXA2 had no effect on apoptosis of HeLa cells. CONCLUSION:Silencing of ANXA2 effectively inhibits the proliferation and migration of cervical cancer cells, but has little effect on apoptosis. ANXA2 may play a pivotal role in the occurrence and development of cervical cancer, and may be used as a molecular target for the treatment of cervical cancer.