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Objective To explore the interaction between follicle stimulating hormone (FSH) and transforming growth factor Beta (TGF-β) signaling pathway in rat ovarian granulosa cells. Methods The granulosa cells isolated from the follicles of 21 days SD rats. The experiments were divided into three groups: control group, FSH treated group and transforming growth factor beta receptor II (TGF-β R II) neutralizated group. Immunocytochemistry (ICC) and Western blotting were then used to locate and detect the expression level of TGF-β R II and proliferating cell nuclear antigen (PCNA). The proliferation index (PI), cell cycle and percentage of apoptotic cells were assessed by flow cytometry (FCM), and the level of estradiol (E2) was determined by ELISA. Results FSH increased the expression of PCNA and PI, changed the cell cycle and inhibited apoptosis of GCs, and these actions were reduced significantly when TGF- βsignaling pathway was inhibited (P0.05). Conclusion The effects of FSH on ovarian granulosa cells are partly affected by the TGF-β signaling pathway.
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BACKGROUND:Platelet lysate has been known as a kind of lysate of autologous or alogeneic platelet-rich products. It not only removes the residual cel structure, reduces immunogenicity, but also retains many growth factors. Platelet lysate has been suggested as a substitute for fetal bovine serum to expand mesenchymal stem cels in vitro. OBJECTIVE:To observe the effects of platelet lysate on biological characteristics of human bone marrow mesenchymal stem cels and adipose mesenchymal stem cels, and provide some experimental data for clinical cel therapy and regenerative medicine. METHODS:Platelet lysate was prepared by repeated freezing and thawing from fresh blood. Healthy adult bone marrow and adipose tissue were colected. Human bone marrow mesenchymal stem cels and adipose mesenchymal stem cels were obtained by density gradient centrifugation and type I colagenase digestion. We tested the morphology, cel phenotype, differentiation characteristics, proliferation capacity, colony forming ability and the level of cytokine secretion of bone marrow mesenchymal stem cels and adipose mesenchymal stem cels after cultured with platelet lysis. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cels and adipose mesenchymal stem cels were successfuly cultured in vitro using platelet lysate. There were no significant differences in morphology, cel phenotype, colony forming ability and the level of cytokine secretion, and chondrogenic, osteogenic and adipogenic capacities between bone marrow mesenchymal stem cels and adipose mesenchymal stem cels. Adipose mesenchymal stem cels had a high cumulative population doublings than bone marrow mesenchymal stem cels (P < 0.05). These findings suggest adipose mesenchymal stem cels had a stronger proliferative ability, and are more suitable for large-scale expansionin vitro cultivation system of platelet lysate compared with bone marrow mesenchymal stem cels.
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Cytokine induced killer (CIK) cells are a new kind of cytotoxic cells,which are generated by stimulation of peripheral blood mononuclear cells (PBMC) with IFN-γ,IL-2,IL-1 and anti-CD3 in vitro.These cells represent phenotype of both T cell and NK cell and have advantages of the non MHC restriction,so they have a strong ability of antitumor activity in vivo and vitro.Now,CIK cells as the main effector cells of adoptive immunotherapy have been extensively used in a variety of solid tumors such as liver cancer,lung cancer and hematological malignancies including leukemia,lymphoma,myeloma.In this article,the optimized cultivation of CIK cells as well as their most prominent clinical application in hematologic malignancies were summarized.
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Objective To explore biological characteristics of chondrogenic differentiation of human periosteum-derived cells and the role of BMP4 in chondrogenic differentiation of these cells.Methods From October 2009 to September 2012,periosteum was obtained from tibia of patients undergoing leg amputation surgery,and isolated periosteum-derived cells by tissue culture method.Cells were cultured in DMEM/F12 containing 10％ fetal bovine serum,and morphology of cells were observed under inverted microscope.Periosteum-derived cells growth and the effect of BMP4 on cells growth examined by cell count using trypan blue,and cells growth curve was made.Experiment was divided into control group,chondrogenic differentiation group and BMP4 group,cells were expanded and differentiated in the presence or absence of BMP4 and complete medium.Then toluidine and immunohistochemical staining analyzed proteoglycan and collagenⅡ expression of these cells after 14 and 21 days.The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of these cells using real-time PCR.Results (1) Periosteumderived cells adhered to growth in vitro,the shape of cell presented fibroblast-like morphology changing into polygonal after 1 week and round cell formation after 2 weeks chondrrogenic differemtiation.Growth curve showed that the passage 3 and 9 cells had similar reproductive activity.The passage 3 cells were positive for CD90 (21.07％) and CD105 (25.84％).(2)Toluidine bule staining and type Ⅱ collagen immunohistochemical staining showed BMP4 group (40.29 ± 4.29,56.74 ± 5.12) and chondrogenic differentiated group (19.27 ± 3.71,38.31 ± 4.25) ccould secrete proteoglycan and collagen Ⅱ,control group were negative (10.24 ± 1.21,15.28 ± 2.23),BMP4 group were significantly than chondrogenic differentiated group.(3) The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of BMP4 group(25.76 ±0.57,6.48 ±0.48,2.91 ±0.18)were significantly higher than that of control group(2.37 ±0.24,1.12 ± 0.31,1.07 ± 0.22)and chondrogenic differentiated group(11.12 ± 0.38,2.24 ± 0.41,1.54 ± 0.35)using real-time PCR.Conclusion Periosteum-derived cells have strong proliferative,and have good potentials of differentiating into chondroblasts like mesenchymal stem cells.BMP4 can promote chondrogenic differentiation of periosteum-derived cells in vitro cultures.
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Objective To evaluate the biomechanical properties of fibroblasts for rabbit experimental myopia after Posterior Scleral Reinforcement (PSR) treatment, and discuss the mechanism of PSR in myopia treatment as viewed from biomechanics. Method 45 rabbits of three week old were randomly monocular treated by eyelid suture to prepare experimental myopia eye. After 60 days, the experimental myopia eyes were divided into two groups randomly. Group A was treated by PSR. Group B was treated by similar operation without placing reinforce strap. After three months and six month, the fibroblasts from each group were isolated and cultured in vitro respectively. The cultured cells were then determined to be fibroblasts by using immunocyte chemistry method. Micropipette aspiration technique was used to investigate the viscoelastic properties of the fibroblasts from each group with mechanical model of semi infinite somatic cells. ResultsThree months after operation, the viscoelastic properties of the scleral fibroblasts in Group A and Group B exhibit no significant difference (P>0.05) three months and six months as well (P>0.05) after operation with the equilibrium modulus, E∞, and apparent viscosity, μ of the scleral fibroblasts in Group A (E = (361.2± 121.1)Pa、μ=(2928.2±669.4)Pa·s) compared with that in Group B (E =(347.6± 82.1)Pa、μ=(2820.6± 593.5)Pa·s). Neither in Group A nor Group B, the E∞ and μ at different stages after operation have significant difference (P>0.05). The E∞ and μ in transition zone tissues at different stages after operation have no significant difference(P>0.05) either. Conclusions The enhancement of PSR is caused by transition zone tissues and the strip itself.
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Objective: To observe the effects of angiopoietin-like 4 (ANGPTL4) gene on the growth of different hepatocellular carcinoma (HCC) cell lines. Methods: The expression of ANGPTL4 was tested by reverse transcriptase-polymerase chain reaction and Western blotting in 9 HCC cell lines and immortal liver cells. Huh-7 cells and MHCC-LM3 cells with low ANGPTL4 expression and MHCC-97L cells with moderate ANGPTL4 expression were transfected with fused ANGPTL4-GFP and empty pEGFP-N1 plasmids via Lipofetamine 2000, respectively. GFP-positive cells were screened with G418 and sorted by flow cytometry (FCM) to establish stable cell lines with over-expression of ANGPTL4, Cell array was prepared and Ki-67 fluorescent immunocytochemical techniques were used to detect in vitro proliferation of the three HCC cell lines. Xenografted tumor formation in nude mice was used to observe the effect of ANGPTL4 on the growth of the three HCC cells in vivo. Results: ANGPTL4 mRNA had low expression in Huh-7 and MHCC-LM3 cell lines, moderate expression in MHCC-97L cells, and high expression in Hep3B cells. The results of cell array showed that cell proliferation rate was lower in Huh-7 cells (P = 0.000 74), but higher in MHCC-LM3 (P = 0.073 08) and MHCC-97L (P = 0.011 52) cells with over-expression of ANGPTL4 compared with the cells transfected with empty plasmid. Over-expression of ANGPTL4 significantly inhibited xenografted tumor formation of Huh-7 cells (P = 0.001 10), but markedly promoted the xenografted tumor formation of MHCC-LM3 (P=0.057 50) and MHCC-97L (P = 0.018 91) cells in nude mice. Conclusion: ANGPTL4 plays different roles in the growth of different HCC cell lines, but the effects of ANGPTL4 on the growth of the same cell line was consistent in vitro and in vivo.
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Objective: To investigate the changes of gene expression in CD34+ hematopoietic stem and progenitor cells (HSPCs) under different growth environments. Methods: Umbilical cord blood mononuclear cells (UCB MNCs) were cultured in static and stirred systems. After 7 days of culture, CD34+ cells were isolated and total RNA was extracted. Gene expression patterns of CD34+ cells from fresh, static and stirred cultures were compared using differential display (DD). Results: 30 gene fragments displayed differential expression levels based on the conditions of DD. One of differentially expressed genes was identified as RAN, which is a member of oncogene RAS family. This gene may be associated with proliferation of hematopoietic cells. Conclusion: Different growth environments induced differential gene expression patterns of CD34+ HSPCs. These differentially expressed genes would give new insights into optimizing in vitro environments for expanding hematopoietic cells.
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@#Objective To screen the optimum conditions carrying murine melanoma B16 cells in spaceflights without cares.MethodsMurine melanoma cells were cultured on micocarriers and grouped depending on cells concentration, serum concentration, microcarrier number and temperature.After 33 days, B16 cells were stained by Giemsa, observed with phase-contrast microscope and counted for surviving percentage.ResultsThe optimum conditions,in which the surviving percentages were 8% and 10%, were obtained in the experiments.B16 cells were carried in the 20th recoverable satellite orbiting 18 days under the optimum conditions. After recovering, 110 strain monocloned cells were survived and the surviving percentage was 1.1%.ConclusionThe optimum conditions carrying murine melanoma B16 cells in spaceflights without cares seems to be obtained,and it did improve the surviving time and percentage of cells in spaceflights without cares.
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PURPOSE: To treat limbal stem cell deficiency, we investigated the epithelial characteristics in rabbits with total limbal stem cell deficiency (LSCD) after reconstruction with autologous limbal corneal epithelial cells in vivo expanded on amniotic membrane. METHODS: The right eyes of 10 rabbits were rendered total LSCD, as verified by impression cytology. In the left eyes of 10 rabbits, in vivo limbal corneal epithelial cells culture was achieved. In group A (n=4), we evaluated the characteristics of the cultured limbal corneal epithelial cells and in group B (n=6) we evaluated the characteristics of the transplanted limbal corneal epithelial cells at postoperative 1, 2, and 4 weeks. RESULTS: Successful epithelial growth was observed on amniotic membrane in all eyes of group A. Transplanted epithelial cells were well remained in five eyes of group B. The epithelial cells of group A and B were confirmed with corneal specificity by immunohistochemical staining (AK-2, AE-5). CONCLUSIONS: The corneal surface with unilateral total LSCD can be successfully reconstructed by transplantation with autologous limbal corneal epithelial cells in vivo expanded on amniotic membrane.
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Conejos , Amnios , Células Epiteliales , Sensibilidad y Especificidad , Células MadreRESUMEN
BACKGROUND: It is evident that inhalational anesthetics, such as nitrous oxide, possess a certain degree of myelodepressive effect in humans. However, unlike nitrous oxide, propofol is frequently recommended for the anesthesia of oncologic patients or for harvesting bone marrow from donors. To evaluate the possible toxicity of propofol on hematopoietic stem cells, the in vitro sensitivity of colony growth to propofol was assessed using murine clonogenic hematopoietic progenitor cells. METHODS: Femoral and tibial marrow cells were obtained from 4- to 6-week-old male BALB/c mice. Propofol was diluted in culture medium (30microM, 300microM and 1 mM) and added into methylcellulose semi-solid culture media. After 14 days of culturing, the numbers of colony-forming unit granulocyte/monocyte (CFU-GM) colonies were counted. An advance liquid culture (RPMI 1640) of 5 hours duration was also applied prior to culturing in semisolid media to assess the short term exposure toxicity. RESULTS: The colony counts were significantly decreased compared to the control at higher concentrations than 1 mM (P<0.05). The pre-exposure to propofol did not affect the number CFU-GM colony count at the concentrations of 30microM and 300microM or under conditions of co-culture. CONCLUSIONS: No myelodepressive effect was observed in mouse bone marrow cells with exposure of propofol at concentrations under 300microM.
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Animales , Humanos , Masculino , Ratones , Anestesia , Anestésicos , Células de la Médula Ósea , Médula Ósea , Técnicas de Cocultivo , Medios de Cultivo , Células Progenitoras de Granulocitos y Macrófagos , Hematopoyesis , Células Madre Hematopoyéticas , Metilcelulosa , Óxido Nitroso , Propofol , Células Madre , Donantes de TejidosRESUMEN
Objective To discuss the effect of nicotine on the growth of rabbit tendon cells.Method 1% concetration Nicotine(0 5ml/kg) was injected into rabbit's auditive vein,then femoral vein blood was extracted at 3 minutes later. Separating blood serum and add it into F-12 medium and 10% fetal bull serum,penicillin,streptomycin,vitC which was then used to culture rabbit tendon cells, observating the time of cell number growth duplication of test group and drawn up its growth curve. The control group was treated as the test group except the rabbit was not injected by nicotine.Results The time of cell number growth duplication of test group was 9?2 1 days and the control group was 7?1 1 days,there was difference significatly between the two groups.Conclusion Nicotine can restrain the growth of rabbit tendon cells and enhance tendon's adhesiveness in post-operation.
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Purpose:To evaluate the feasibility, advantages and disadvantages of chemosensitivity test of human gastric cancer using MTT assay with short term culture tumor cells contaminated by nonmalignant cells compared with purifiedly primary culture tumor cells.Methods:Fifty nine fresh samples from patients with gastric cancer were obtained from operating rooms. Chemosensitivity results were provided by the MTT assay with short term culture cells. The primary culture cells were purified by means of a series of methods such as repeated cell attachment, differential trypsinization and natural purification which removed fibroblasts and other nonmalignant cells. The same MTT assay was conducted using purified cells. Chemosensitivity results between short term method and purification method were compared for seven antitumor drugs. Results:The success rate of short term method using the MTT assay for chemosensitivity testing was 81 4% (48 of 59 patients),and for the purification method it was 50.8(30 of 59 patients). The average was 20.2?9.5 days when primary cells were cultured into purified cancer cells. The optical density ( A ) value correlated directly with the the number of tumor cells with the two methods( P
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Objective :To study a simplified method of isolation of rat hepatocytes and to observe the pro-cess of cell morphology in long-term culture. Methods :Rat hepatocytes were isolated by a single two-stepperfusion method. The yield and viability were assessed by trypan blue exclusion. [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] (MTT) was used to test the effect of serum concentration of newborn calf serum on the proliferation of hepatocytes. Hepatocytes were inoculated in the culture mediumconsisted of Williams' E supplemented with insulin,dexamethasone and 10％ new born calf serum. Themorphologic change of cultured hepatocytes was observed. Results:The average yield of hepatocytes was 2.26× 108 cells per rat, with an average viability of 95.6％. New born calf serum had strong biological activi-ty to stimulate the proliferation of hepatocytes and there was close-effect relationship followed by the in-crease of new born calf serum concentration. The rat hepatocytes can be cultured for 5～ 6 weeks withpreservation of normal morphologic appearance. Conclusion:The rat hepatocytes isolated by the abovemethod have high yields and viability and can be long-term cultured in vitro.
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Objective To study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2. Methods The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by[3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT]-test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method. Results The A value in the hypoxia group was higher than that in the normal group after 24 hours (P
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Objective To develop a new culture system in which hepatocytes can restore in-vivo like function. Methods Sandwich configuration was used for the culture of hepatocytes, and protein synthesis as well as P 450-b ,albumin mRNA expression by in situ hybridization was studied. ResultsStable and gradually increasing protein synthesis and high level of P 450-b , albumin mRNA expression were observed. In contrast, protein secretion of hepatocytes cultured in conventional single collagen culture system was low and decreased until approximately zero seven days after, and mRNA expression of P 450-b ,albumin was low. Conclusions Sandwich configuration in which cell polarity of hepatocytes is reestablished like in vivo situation.
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Objective To study an effective method for culturing of human vascular smooth muscle cells (hVSMCs) in tissue engineered blood vessels (TEBVs) in vitro.Methods A 3-cm segment of human long saphenous vein was harvested under sterile conditions.The primary culture and subculture were done by modified tissue-piece inoculation and trypsin digestion respectively.The cells were purified by mechanical treatment and differential attachment.PDGF was combined with high concentration of glucose DMEM as the culture medium.The cultured cells were identified by morphology and immunohistochemistry with contrast microscope and SABC kit,and RT-PCR method detected the expression of ?-SMA and calponin 1.Results The cultured cells possessed"peak and valley"characteristics for VSMCs under microscope.Immunohistochemical staining showed positive expression of intracytoplasmic ?-actin,and RT-PCR detected positve expression of ?-SMA and calponin 1.Conclusions Culture medium of PDGF combined with high concentration of glucose DMEM,and with differential attachment method can provide highly purified hVSMCs with good structure and function.
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Vital circulating endothelial cells (CEC) from peripheral blood of traumatic patients were successfuly isolated and cultured in vitro with a centrifugation method on density gradient of percoll. After culturing for 72 hr in vitro, the cells formed several dense groups. The technique of alkaline phosphatase monoclonal antioalkaline phosphatase (APAAP) found that the cells originated from vaslular endothelial cells (VEC). The results indicated that trauma could cause the detachment of VEC, and the culture of vital CEC from peripheral blood of traumatic patients may enlighten an attracting perspective.
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Objective To investigate the therapeutic mechanisms for differentiated thyroid cancer by using CIK and IL-2, to find out the better adjunctive clinical therapies for thyroid cancer patients after operation, and to evaluate the effects of cytokine-induced kill cell (CIK) and IL-2 on the secretion of thyroxine by thyroid papillary carcinoma. Methods The samples of thyroid papillary carcinoma were taken from the excised tissues of patient with thyroid cancer, and then dispersed with collagenase and trypsin for culturing. The carcinoma cells were then seeded in 24-well cell culture plates at 37℃, 5% CO2 and 95% humidity for 3 days. At the fourth day, the cells in the wells were separately stimulated four times with different dosage of CIK and IL-2, and the stimulation lasted for 72 hours each time. 12 days later, the solution of T3, T4 was detected with radio-immunity kits, TSH was detected with immuno-radiation kits, and the cell proliferation was detected with mono-nuclear cell direct cytotoxicity assay. Results The thyroid cancer cells did not respond to IL-2 in median and low concentrations, but responded to IL-2 in a higher concentration which may depress the secretary function of thyroid cancer cells. IL-2 of high concentrations can obviously decrease the hormone secretion, such as Thyroxine and Thyrotropin, of papillary carcinoma, and improve the CIK's ability of killing cancer. CIK can kill the cancer cells only when companied with IL-2. Conclusion IL-2 of high concentrations can't inhibit the proliferation of thyroid cancer cells, but can depress the secretary function of thyroid cancer cells, which is different from the killing mechanism of CIK.
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Objective To study isolation and identification and culture of rat type A spermatogonial cells in vitro.Methods Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish was used to purify the type A spermatogonial cells.The c-kit and TERT special antibodies were used to identify the type A spermatogonial cells.The purified cells were cultured in vitro. Results 0.614?10~6 cells per testis finally were obtained and the percentage of viable cells was 92.1% by trypan blue dye exclusion test.The percentage of type A spermatogonial cells expressing c-kit and TERT were 91.7?1.2% and 90.8?1.0% respectively.Type A spermatogonial cells could proliferate and self-renew in the DMEM containing 10% NBS.Conclusion Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish is an efficiency method for isolation of rat type A spermatogonial cells.The purified cells are type A spermatogonial cells by identification of the immunohistochemistry of c-kit and TERT antibodies.Type A spermatogonial cells can proliferate and self-renew in vitro.