ObjectiveTo investigate the effectiveness of human placental decidua basalis derived mesenchymal stem cells (PDB-MSCs) in repairing full-thickness skin defect of nude mice. MethodsHuman placenta samples were obtained from healthy donor mothers with written informed consent. PDB-MSCs were isolated through enzymic digestion and density gradient centrifugation; the 4th passage cells were identified by cellular morphology, cell adipogenic and osteogenic differentiation, and phenotype evaluation. Forty-two 4-5-week-old BALB/c female nude mice were randomly divided into experimental group (n=21) and control group (n=21). The 4th passage PDB-MSCs solution (200 μL, 5×106/mL) was injected into the mice of experimental group via caudal vein; the mice of control group were given equal volume of PBS. The full-thickness skin defect model of 1.5 cm×1.5 cm in size was made after 3 days. The wound healing was observed generally at 1, 2, 4, 7, 14, 18, 21, 25, and 30 days after operation, and the wound healing rate was calculated after wound decrustation. HE staining was used to observe the wound repair at 1, 7, 14, 21, and 31 days; immunofluorescent staining was used for cellular localization at 7, 14, and 31 days after operation. ResultsCells isolated from human placenta were MSCs which had multipotential differentiation ability and expressed MSCs phenotype. Animals survived to the end of the experiment. The general observation showed that the experimental group had a faster skin repairing speed than the control group; the time for decrustation was 12-14 days in experimental group and was 14-17 days after operation in the control group. The wound healing rate of experimental group was significantly higher than that of control group at 14, 18, and 21 days (t=4.001, P=0.016; t=3.380, P=0.028; t=3.888, P=0.018), but no significance was found at 25 and 30 days (t=1.565, P=0.193; t=1.000, P=0.423). HE staining showed lower inflammatory reaction, and better regeneration of the whole skin and glands with time in the experimental group. The immunofluorescent staining was positive in skin defect area of experimental group at different time points which displayed that human PDB-MSCs existed. ConclusionThrough enzymic digestion and density gradient centrifugation, PDB-MSCs can be obtained. Pre-stored PDB-MSCs can mobilize to the defect area and participate in repair of nude mice skin.
ObjectiveTo investigate the inhibitory effect of short hairpin RNA (shRNA) mediated contactin-1 (CNTN1) gene silencing on growth of human breast cancer cell line MDA-MB-468 transplanted tumors in nude mice.MethodsEighteen nude mice (4-week-old male BALB/c) were randomly equally divided into three groups: blank control group, empty vector group, and silencing group. The MDA-MB-468 cells (blank control group), MDA-MB-468 cells transfected by nonsense shRNA (empty vector group), and MDA-MB-468 cells transfected by shRNA (silencing group) were collected in the logarithmic growth period, respectively. The subcutaneous tumor models of nude mice were prepared by the subcutaneous injection of the different group cells. The tumor growth was observed and the expressions of CNTN1 and Ki-67 proteins in the transplanted tumor were detected by the immunohistochemistry.ResultsThe xenograft models of human breast cancer cells were established successfully. The tumor growth in the silencing group was significantly slower than that of the other two groups at every 3 d point (P<0.05). The tumor volume and the tumor weight in the silencing group were significantly smaller or slighter than those of the other two groups at day 18 (P<0.05). The positive rates of CNTN1 and Ki-67 protein expressions in the tumor tissues of the silencing group were lower than those of the other two groups (P<0.05), respectively.ConclusionSilencing expression of CNTN1 gene might inhibit growth of breast cancer cell line MDA-MB-468 transplanted tumors in mude mice.
Objective To study effect of carcinoembryonic antigen (CEA) positive targeted lymphocytes on gastric cancer cells in vitro and in vivo. Methods The peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers. The recombinant vector anti-CEA-scFv-CD3ζ-pcDNA3.0 was transfected into the PBMCs by lipofectamine 2000, by this means, the CEA special lymphocytes were obtained. Meanwhile, the PBMCs transfected with empty plasmid pcDNA3.0 were used as control (empty vector lymphocytes). The different lymphocytes and gastric cancer cells (CEA positive KATOⅢ gastric cancer cells and CEA negative BGC-823 gastric cancer cells) were co-cultured, then the ability to identify the gastric cancer cells and it’s effect on apoptosis of gastric cancer cells were observed at 24 h or 36 h later respectively. The CEA special lymphocytes and empty vector lymphocytes were injected by the tail vein of nude mice bearing gastric cancer cells, then it’s effect on the tumor was observed. Results ① The CEA special lymphocytes could strongly identify the KATOⅢ gastric cancer cells (identification rate was 72.3%), which could weakly identify the BGC-823 gastric cancer cells (identification rate was 7.8%). ② The apoptosis rate of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly higher than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (P=0.032), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (P=0.118). ③ The tumor volume of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly smaller than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (F=5.010, P<0.01) or the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells (F=4.982, P<0.01), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (F=1.210, P>0.05). Conclusion CEA special lymphocytes can promote cell apoptosis and inhabit tumor reproduction of CEA positive gastric cancer cells in vitro and in vivo.
Objective To investigate the effect of human placental-derived mesenchymal stem cells (PMSCs) on immunological rejection in mouse allogeneic skin transplantation. Methods The placenta fetal tissues from voluntary donors were used to isolate and culture the PMSCs, and the 3rd passage PMSCs were used in the experiment. Thirty Vr ∶ CD1 (ICR) mice at age of 1-2 days were used as skin donors for allogeneic skin transplantation. Thirty C57BL/6 mice at age of 6-8 weeks as recipients were made back skin defect of 12 mm in diameter and were randomly divided into 3 groups (n=10): group A, autograft; group B, allogeneic graft + PBS tail vein injection; and group C, allogeneic graft + human PMSCs (1 × 105 cells/mouse) tail vein injection. The flap survival was observed. At 7 days after skin transplantation, blood leukocyte counting, abdominal fluid macrophage activation, and the expression levels of interleukin 4 (IL-4), interleukin 17 (IL-17), and interferon γ (INF-γ) in blood and spleen were detected by ELISA and RT-PCR, respectively. Results The flap survival time was significantly longer in group A [(58.33 ± 4.04) days] than in groups B and C [(3.80 ± 0.92) days and (6.80 ± 0.82) days] (P lt; 0.05), and in group C than in group B (P lt; 0.05). At 7 days after transplantation, the blood leukocyte number was (6.32 ± 0.45) × 109/L in group A, (7.45 ± 0.52) × 109/L in group B, and (6.35 ± 0.39)× 109/ L in group C, and it was significantly more in group B than in groups A and C (P lt; 0.05). The macrophage activation rate of the abdominal fluid was 6.87% ± 2.40% in group A, 7.84% ± 0.44% in group B, and 15.98% ± 2.87% in group C; group C was significantly higher than groups A and B (P lt; 0.01). ELISA results showed that there was no significant difference in the concentrations of IL-4 among 3 groups (P gt; 0.05). Compared with group B, the concentrations of IL-17 and IFN-γ were significantly reduced in group C (P lt; 0.05), while the concentration of IFN-γ was significantly increased in group B when compared with group A (P lt; 0.05). RT-PCR results showed that there were significant differences in the expressions of IL-4, IL-17, and IFN-γ mRNA between groups B, C and group A (P lt; 0.05); the expressions of IL-4 and IFN-γ mRNA were significantly lower in group C than in group B (P lt; 0.05). Conclusion Human PMSCs transplantation can suppress the acute immunological rejection in allogeneic skin transplantation. The possible mechanism may be partially related to the inhibitory effect on the secretion of IL-17 and IFN-γ.
Objective To study the expressions of human ether-a-go-go related gene (HERG) in CD1 mice gallbladder and interstitial cells of Cajal (ICC) and explore their possible implications. Methods The expression of HERG protein in gallbladder tissue slices obtained from CD1 mice was detected by immunohistochemistry method. The expression of HERG mRNA in gallbladder tissue was detected by reverse transcription (RT)-PCR. The production of HERG protein was confirmed in the CD1 mice gallbladder by Western blot. Enzymatically dispersed cells were identified as ICC using the specific ICC marker c-kit antibody, and the double positive cells of c-kit and HERG were observed by laser passing confocal microscope. Results HERG was present in the CD1 mice gallbladder tissues for the yellow or buffy positive reaction. At the same time, the expression of mRNA specific for the HERG gene and production of HERG protein in the CD1 mice gallbladder tissues were indicated by RT-PCR and Western blot analysis, respectively. Using double labeling of anti-c-kit and anti-HERG, the double positive cells of c-kit and HERG were observed in the CD1 mice ICC by laser passing confocal microscope. Conclusion The study demonstrates that HERG is present in the CD1 mice gallbladder tissues and ICC, which is likely related to the pacemaking activity of ICC.
Objective To investigate how to establish stable mice cervical heart transplantation model. Methods Totally, 40 male C57 mice with the age of 6-8 weeks and weight of 19-24 g were randomly divided into recipients and donors (n=20 in each group). Mice cervical heart transplantation model was established by connecting the ascending aorta of donors to the right cervical common artery of recipients through end to side anastmosis and the pulmonary artery of donors to the right external jugular vein of recipients through end to end anastmosis. Results More than 95% recipients survived after surgery. Cold ischemia time was 15±5 min, warm ischemia time 23±6 min, and the whole operation took about 55±15 min. The recipients survived more than 30 d with functional heart grafts. Histologically, there was no difference between the heart graft one month after the transplantion and the normal heart. Conclusion Cervical heart transplantation of mice model is reliable and feasible, which is easy to monitor the survival condition of heart graft by visual examination and palpation, which will benefit the basic research in transplantation field.
Objective To investigate the intervention effect of 3-phosphoinositede dependent protein kinase-1 (PDK1) inhibitor on prostaglandin E2 (PGE2) in smoking-induced chronic obstructive pulmonary disease (COPD) mice. Methods Fifty C57BL/6 male mice were randomly divided into normal control group, smoking group, smoking +low dose PDK1 inhibitor group, smoking + medium dose PDK1 inhibitor group and high dose PDK1 inhibitor group with 10 mice in each group. The mice in the normal control group inhaled phosphate-buffered saline twice a day for 12 weeks, and the mice in the smoking group were fumigated twice a day, 5 days per week for 12 weeks, and the other three groups were given intraperitoneal injection of low-dose PDK1 inhibitor OSU-03012 (0.25 mg/kg), medium-dose PDK1 inhibitor (0.5 mg/kg) and high-dose PDK1 inhibitor (1.0 mg/kg) respectively before smoking. After smoking, lung function was tested, the bronchoalveolar lavage fluid (BALF) of each mouse was taken for cell count, the PGE2 in serum and BALF of mice was determined by enzyme linked immunosorbent assay, and the lung tissue of mice was sectioned with paraffin and stained by hematoxylin-eosin (HE), and pathological changes were observed under microscope. Results Compared with the control group, FEV100/FVC and FEV200/FVC of the mice in each smoking group were significantly decreased (P<0.05); The number of cells in BALF of smoking group was significantly higher than that of normal control group (P<0.05). There was no significant difference in the total number of BALF cells, the proportion of neutrophils and macrophages between the smoking + low-dose PDK1 inhibitor group and the smoking group. However, the total number of BALF cells and the proportion of neutrophils in the smoking + medium dose PDK1 inhibitor group and the high dose PDK1 inhibitor group gradually decreased, while the proportion of macrophages gradually increased, compared with the normal control group, the PGE2 concentrations of serum and BALF in the smoking group and the smoking + PDK1 inhibitor group were significantly higher than those in the control group. Compared with the smoking group, the PGE2 concentrations of serum and BALF in the middle and high dose PDK1 inhibitor groups were significantly lower than those in the smoking group. HE staining of lung tissue showed that there were a large number of inflammatory cell infiltration, alveolar cavity dilatation, alveolar wall rupture and fusion, alveolar formation, significant decrease in the number of alveoli and other pathological changes in the smoking group, which were consistent with the pathological changes of COPD. The inflammatory cell infiltration, mucus obstruction and alveolar dilatation were slightly alleviated in the smoking + low-dose PDK1 inhibitor group, while the inflammatory cell infiltration, alveolar wall thinning and alveolar dilatation were improved in both the medium-dose inhibitor group and the high-dose inhibitor group, and the improvement was more obvious in the high-dose inhibitor group. Conclusion The lung function of the smoked COPD mouse decreases, the airway inflammation is obvious, and the secretion of PGE2 is also increased, while the use of PDK1 inhibitor could reduce the secretion of PGE2, reduce airway inflammation and pathological changes, and improve lung function in a dose-dependent manner.
Objective To determine the effect of insulin-like growth factor-1 (IGF-1) on angiogenesis in mouse breast cancer model of lower and normal serum IGF-1 levels after using angiogenesis inhibitor ginsenoside Rg3 （GS Rg3）. Methods The breast cancer models were established in control mice and liver specific IGF-1 deficient (LID) mice by feeding DMBA and were treated with GS Rg3. Vascular endothelial growth factor (VEGF) and F8-RAg were detected by immunohistochemical method in breast cancer tissues. IGF-1 gene and angiogenesis relating genes were detected by gene chip in breast cancer and normal breast tissue. Results The incidence rate of breast cancer in LID mice was lower than that in control mice (P＜0.05). VEGF expression and microvessel density of LID mice were lower than those in control mice (P＜0.05). Compared to the control mice, IGF-1, FGF-1, TGF-β1 and HGF genes were increased, and FGFR-2, PDGF-A and PDGF-B genes were decreased in breast cancer of LID mice. After GS Rg3 treatment, VEGFa, EGF, EGFR, PDGF-A and FGFR-2 genes were increased, IGF-1 and TGF-β1 genes were decreased in breast cancer of LID mice compared with the control mice. Conclusion IGF-1 may be involved in mouse breast cancer progression and associated with the growth of blood vessels. Angiogenesis inhibitor may play an antitumor role by IGF-1 and TGF-β1.
On the basis of established JF305 cell line from human pancreatic cancer at this university, cell clone technique, cell electrophoresis, flower cytometer, and cancer orthotopically implanted nude mice technique were used to establish the sublines with different metastatic potential from human pancreatic cancer line-JF305 and the nude mice model implanted orthotopically with human pancreatic cancer monoclonal sublines with different metastatic potential. The results showed that the monoclonal cell sublines with different metastatic potential from human pancreatic caner-JF305 and the nude mice model implanted orthotopically with the sublines, would provided a useful method to study the metastatic mechanism of human pancreatic cancer.
Objective The human amniotic epithel ial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes. The hAECs may be a candidate seed cell for l iver regeneration. To research the survival and migrationin vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and toexplore the expression of hepatocyte-l ike function. Methods Thirty nude mice (aging 6-8 weeks, half males and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10). The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy. The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal sal ine was injected in group C. At 4 hours, the nude mice were sacrificed and the samples of l iver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes. Results No tumor tissue was found in l iver and spleen of 3 groups, and HE staining showed no tumor cells. There were a lot of roundl ike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the l iver of group A; no abnormal cells were found in l iver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C. The GFP+ cells were detected in the spleen and l iver of group A with expressing human albumin, but no GFP+ cells was found in l iver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C. Conclusion AAV-GFP infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-l ike function. It will be beneficial to further identify their biological characteristics.