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 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 the feasibility and characteristic of tissue engineered testicular prosthesis with highdensity polyethylene(HDPE，trade name: Medpor) and polyglycolic acid(PGA). Methods The chondrocytes were isolated from the swine articular.The PGA scaffold was incorporated with medpor which semidiameters were 6mmand 4mm respectively.Then, the chondrocytes (5×10 7/ml) were seeded onto Medpor-PGA scaffold and cultured for 2 weeks. The ten BALB／C mice were divided into two groups randomly(n=5). In the experimental group, the cell-scaffold construct was implanted into subcutaneous pockets on the back of nude mice. In the control group, the Medpor-PGA scaffold was implanted. The mice of two groups were sacrificed to harvest the newly formed cartilage prosthesis after 8 weeks. Macroscopy, histology and immunohistochemistry observations were made. Results The gross observation showed that on changes were in shape and at size, the color and elasticity were similar to that of normal cartilage and that the cartilage integrated with Medpor in the experimental group; no cartilage formed and fiberlike tissue was found in the control group. HE staining showed that many mature cartilage lacuna formed without blood vessel and some PGA did not degradated completely. Toluidine blue staining showed extracellular matrix had metachromia. Safranin O-fast green staining showed that many proteoglycan deposited and collagen type Ⅱ expression was bly positive. In the control group, Medpor was encapsulated by fiber tissue with rich blood vessel. Conclusion The newly formed complex of Medpor-PGA and cells was very similar to testicle in gross view and to normal cartilage in histology. This pilot technique of creating testicular prosthesis by incorporating tissue-engineered cartilage with Medpor demonstrated success.
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.
ObjectiveTo evaluate therapeutic effect of the targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3 on primary hepatocellular carcinoma cells (HepG2) and subcutaneous implanted tumors of nude mice. MethodsHepG 2 cells, breast cancer MDA-MB-231 cells， and normal hepatic L-02 cells were infected with the previously constructed targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3, then morphological change was observed and apoptosis index was detected by cell morphologic assay. Tumor growth and pathological changes were observed after the implanted tumors model of nude mice were injected with the recombinant adenovirus. Results Both apoptosis and obvious apoptotic peak of cells were observed by flow cytometry (FCM) in HepG 2 cell group. The apoptosis index of MDA-MB-231 and L-02 cells was 0 and 7.3%, respectively, which were significantly lower than that of HepG2 cells (48.2%), Plt;0.01. The volume of subcutaneous tumor of nude mice was (0.26±0.31) cm 3 in HepG2 cell group, (0.25±0.15) cm 3 in MDA-MB-231 cell group， and (0.26±0.28) cm 3 in control group on two weeks after implantation of cancer cells, and no statistical difference was found among them (Pgt;0.05). The volume of subcutaneous tumor of nude mice was (0.53±0.12) cm 3 in HepG 2 cell group, (0.49±0.22) cm 3 in MDA-MB-231 cell group， and (0.54±0.13) cm 3 in control group on four weeks after implantation of cancer cells, and the difference was not significant among them (Pgt;0.05). But on eight weeks after implantation of cancer cells, the volume of tumor was (0.65±0.13) cm 3 in HepG 2 cell group, (1.27±0.32) cm 3 in MDA-MB-231 cell group， and (1.43±1.09) cm 3 in control group, which suggested that the growth of tumor in HepG 2 cell group slowed down significantly when compared with MDA-MB-231 cell group and control group (Plt;0.01), although the difference in the latter two groups was not significant (Pgt;0.05). The necrosis regions were found with lymphocytic infiltration and apoptosis cells in HepG2 cell group after injection of recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3, but no pathological change of cells was found in control group after injection. It was found that the cells in MDA-MB-231 cell group were solidly arranged with small glandular lumenlike structure, marked cellular atypia, multinucleated tumor cells and megakaryocytic tumor cells, which was not different from that before injection. ConclusionThe targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3 induces the apoptosis of primary hepatocellular carcinoma in vivo and in vitro, which may provide new ways of targeted gene therapy.
ObjectiveTo investigate MUC1 over-expression on chemotherapy of 5-fluorouracil and cisplatin for esophageal cancer cells. MethodsMUC1 over-expression and stable silencing of MUC1 expression esophageal cancer cell lines were constructed. Xenograft model of esophageal cancer was established in nude mice. Cisplatin (8 mg/kg, day 1 and day 7)and 5-fluorouracil (20 mg/kg, day 1 to 6)were injected intraperitoneally. Tumor volume and body weight of nude mice were measured. Tumor growth curve and body weight curve were drawn, and tumor inhibitory rate was calculated. ResultsBoth cisplatin and 5-fluorouracil suppressed tumor growth of MUC1 over-expression esophageal cancer nude mice. Body weight and tumor volume of nude mice of cisplatin and 5-fluorouracil groups were significantly smaller than those of the control group (P < 0.05), and the inhibitory effects of cisplatin were significantly greater than those of 5-fluorouracil (P < 0.05). There was no significant inhibitory effect in stable silencing of MUC1 expression esophageal cancer nude mice. ConclusionBoth cisplatin and paclitaxel can suppress the growth of MUC1 over-expression esophageal cancer, and cisplatin has greater inhibitory effects than 5-fluorouracil in tumor volume and body weight of nude mice.
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.
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.
Objective The immunogenicity of tissue engineered skins is still vague, though it has been appl ied cl inically for several years. To observe the evidence of immunologic rejection of tissue engineered skins transplanted to severe combined immunodeficiency (SCID) mice, which are implanted human splenic lymphocytes to construct human immunesystem. Methods Tissue engineered skins and acellular dermic matrix were constructed in vitro. Twenty SCID mice, aging4-6 weeks and weighing 16-17 g, were randomly divided into four groups equally (n=5). The tissue engineered skins, human foreskins from circumcision and acellular dermic matrix were transplanted to groups A, B, and C, respectively; group D was used as a control. After 2 weeks of transplanting, 3 × 107 human splenic lymphocytes were injected into every SCID mouse intraperitoneally. After 4 weeks, the morphology, histology, immunohistochemistry and human IgG immunofluorescence were used to observe immunologic rejection. Results Group A showed that transplanted tissue engineered skins had the bilayer structure of dermis and epidermis, which was similar to the normal human skin structure. Group B showed that the transplanted human foreskins still retained normal structure of human skin. Group C showed that acellular dermic matrix were located in situ and had no sign of degradation. After injecting human splenic lymphocytes into the SCID mice, no inflammatory cells infil itration were observed basically in groups A, C, and D; the inflammatory cells infil itration of group B were significantly higher than that of other 3 groups (P lt; 0.05). The results of anti human keratin 14 monoclonal antibody (mAb) staining and anti human type IV collagen mAb staining were positive in group A; no positive cells for CD3, CD4, and CD8 were observed in groups A, C, and D; and many positive cells for CD3, CD4, and CD8 were observed in group B. The results of IgG immunofluorescence staining was negative in group A, C, and D, and positive in the great vessel wells of group B. Conclusion The immunogenicity of tissue engineered skins is very weak, and tissue engineered skins would not be rejected by host immune system after transplantation.