ObjectiveTo establish an animal model of anaplastic thyroid cancer with high metastatic activity as in human body. MethodsHuman anaplastic thyroid cancer cell line TAK was injected into one of the lateral lobes of the thyroid gland, as well as in the subcuitis in a series of nude mice. Mice were sacrificed when found moribund, and autopsy and histology were performed subsequently.ResultsThe implantation of human anaplastic thyroid cancer cells in an ectopic enviroment did not permit expression of metastasis potential. In contrast, intrathyroid implantation did. Lymph node (5/10), lung (3/10) and one metastasis (1/10) were noted upon histological examination. ConclusionAn animal model with high metastatic activity is established when human anaplastic thyroid cancer cell line TAK is implanted orthotopically into nude 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 To explore whether epithelial to mesenchymal transition ( EMT) occurs in bleomycin( BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells( BECs) in the EMT. Methods BLM-induced peribronchial fibrosis in an α-smooth muscle actin-Cre transgenic mouse( α-SMACre /R26R) was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. Results BLMtreated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some alveolar epithelial cells( AECs) in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. Conclusions EMT occurs in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.
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.
【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.
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.
Objective To provide the seed cells for bone tissue engineering, to establ ish immortal ized human bone marrow mesenchymal stem cells (MSCxj) and to investigate the ectopic osteogenesis of MSCxj. Methods MSCxjs of the 35thand 128th generations were maintained and harvested when the cell density reached 2 109. Then, these cells were co-cultured with heterogeneous bone scaffold in groups A (the 35th generation, n=12) and group B (the 128th generation, n=12); heterogeneous bone alone was used in group C (n=12). The cell prol iferation was observed by scanning electron microscopy (SEM) after 48 hours and 18 days of osteogenic induction culture. The complex was implanted subcutaneouly through a 3-mm-incision at both sides of the back in 18 nude mice. Tetracycl ine label ing was performed before the animals were sacrificed. Tetracycl ine fluorescence staining, HE staining, ponceau staining, and immunohistochemistry staining for osteocalcin were performed at 4, 8, and 12 weeks after transplantation; the morphologic quantitative analysis was made. Results After 48 hours, SEM showed that MSCxjs adhered to heterogeneous bone and grew well; after 18 days, a large number of new filamentous extracellular matrix and small granules were found to cover the cells. The results of tetracycl ine fluorescence staining, HE staining, and ponceau staining in groups A and B showed that the osteogenesis was not obvious at 4 weeks after transplantation; osteoid matrix deposition was noted around and in theheterogeneous bone at 8 weeks; and osteogenesis was increased at 12 weeks. There was no significant difference in bone formation between groups A and B. Osteogenesis was not observed in group C. The osteocalcin expressions were positive in groups A and B. The bone ingrow percentages of groups A and B were 5.64% ± 2.68% and 4.92% ± 2.95% at 8 weeks, and 13.94% ± 2.21% and 14.34% ± 3.46% at 12 weeks, showing significant differences between 8 weeks and 12 weeks at the same group (P lt; 0.05) and no significant difference between groups A and B at the same time (P gt; 0.05). Conclusion MSCxj has favorable abil ities of ectopic osteogenesis and can be appl ied as seeded cells in bone tissue engineering.
Objective To investigate the effect of myoblast transplantation on duchenne muscular dystrophy (DMD) and to explore the method and feasibil ity of applying gene therapy to DMD. Methods Myoblast of C57/BL10 mice were cultured using multiple-step enzyme digestion method and differential velocity adherent technique. The morphology of the cells was observed with inverted phase contrast microscope. The cells at passage 4 were labeled with 5-BrdU. Twenty-four DMDmodel mice (mdx mice: aged 4-6 weeks, male, 13.8-24.6 g) were randomly divided into two groups (n=12 per group): group A, 1 × 106/mL labeled myoblast were injected via ven caudal is twice at an interval of 2 weeks; group B: 1 mL DMEM/F12 was injected in the same manner serving as a control group. The mice were killed 4 weeks after operation and the motor abil ity of the mice was detected by one-time exhaustive swimming before their death. HE staining and immunohistochemistry staining observation for 5-BrdU, desmin, and dystrophin (Dys) were preformed, and the imaging analysis was conducted. Results The primary myoblast could be sub-cultured 5-7 days after culture, providing stable passage and sufficient cells. The time of onetime exhaustive swimming was (60.72 ± 5.76) minutes in group A and (47.77 ± 5.40) minutes in group B, there was significant significance between two groups (P lt; 0.01). At 4 weeks after injection, HE staining showed that in group A, there were round and transparent-stained myocytes and the percentage of centrally nucleated fibers (CNF) was 67%; while in group B, there were uneven muscle fiber with such pathological changes as hypertrophia, atrophia, degeneration, and necrosis, and the percentage of CNF was above 80%. Immunohistochemistry staining revealed that the expression of 5-BrdU, desmin, and Dys was positive in group A; while in group B, those expressions were l ittle or negative. Image analysis result displayed that integral absorbency (IA) value of desmin was 489.70 ± 451.83 in group A and 71.15 ± 61.14 in group B (P lt; 0.05) and the ratio of positive area to thetotal vision area was 0.314 3 ± 0.197 3 in group A and 0.102 8 ± 0.062 8 in group B (P lt; 0.05); the Dys IA value was 5 424.64 ± 2 658.01 in group A and 902.12 ± 593.51 in group B (P gt; 0.05) and the ratio of positive area to the total vision area was 0.323 7 ± 0.117 7 in group A and 0.035 2 ± 0.032 9 in group B (P lt; 0.05). Conclusion Myoblast transplantation has certain therapeutic effect on DMD of mice.
Objective To investigate the effect of hepatocyte-l ike cells induced by CD34+ cells in vitro on the repair of the injured hepatic tissues of mice in vivo. Methods Mononuclear cells were isolated from umbil ical blood by density gradient centrifugation and enriched CD34+ cells were obtained. The cells were (1 × 105 cells/mL) cultured in serumfreemedium containing stem cell factor (SCF), hepatocyte growth factor (HGF), EGF, oncostatin M (OSM), bFGF (the concentration were 50, 20, 20, 10, 10 ng/mL respectively) in vitro for 10 days. Forty-eight 6-week-old female ICR mice werechosen to prepare l iver injury model by injecting carbon tetrachloride and 2-acetylamionoflu-orene. The mice were randomly divided into two groups (n=24 per group): the experimental group, the cultured cells were injected into the mice through the tail vein; the control group, the equivalent serum-free medium was injected. Six mice from each group were killed at 7, 14, 21, and 28 days after operation to receive HE staining, PCR gel electrophoresis, immunohistochemistry staining, and hepatic function detection. Results HE staining: the morphology of injured hepatic tissues in the control group recovered to normal 28 days after operation, while in the experimental group, it recovered to normal 14 days after operation. PCR gel electrophoresis and immunohistochemistry staining: the cells expressing human serum albumin were detected in the hepatic tissue of the experimental group at each time point after operation; while in the control group, no such cells were detected within 28 days after operation. Hepatic function detection: the activity of alanine aminitransperase in the control group recovered to normal 14 days after operation; the mean activity of aspartate aminotransferase of two groups failed to recover within 28 days. Conclusion The hepatocyte-l ike cells induced by CD34+ cells in vitro can promote the morphological and functional recovery of the injured hepatic tissue in mice. Moreover, it can be transformed into human-derived hepatic cells in l iver-injured mice.