Effect of EGFP gene transfection on the cell cycle distribution of primary cultured human chondrocytes

AIM: To investigate the effect of enhanced green fluorescence protein (EGFP) gene transfection on the cell cycle distribution of primary cultured human chondrocytes in order to establish a tracking method of cultured human nasoseptal chondrocytes. METHODS: pEGFP-N1 plasmid was amplified in E.coli, and purified by high purity kit. Primary cultured human chondrocytes,which were initially obtained from the nasoseptal cartilage, were cultured in vitro and transferred with pEGFP-N1 by means of electroporation with Amaxa nucleofector device. Transfering process and transient expression were evaluated by laser scanning confocal microscope (LSCM), the transfer efficiency and the cell cycle distribution were evaluated by flow cytometry. RESULTS: There was significant expression of EGFP at 24 h after transferring. The transfection efficiency of pEGFP-N1 into primary cultured human chondrocytes reached 35.37％ at 48 h. It didn't affect the process of cell adherance and had no effect on the cell cycle distribution. CONCLUSION: Primary cultured human chondrocytes, which were transfected with pEGFP, are alive in vitro, and the transferring process doesn't affect the cell cycle distribution. These results suggest that pEGFP-N1 is an ideal transient expression vector for primary cultured human chondrocytes and it might be a well tracer in construction tissue engineered cartilage.     

Effect of shock lymph on apoptosis relative gene expression in pulmonary micro-vascular endothelial cells

AIM:To observe the effects of shock lymph on apoptosis relative gene expressions of pulmonary micro-vascular endothelial cells (PMVECs), and explore its mechanism.METHODS:The model of severe hemorrhagic shock was established by maintaining the blood pressure of rats in the condition of sepsis, mesentery lymph and shock portal vein blood was taken out. As control, mesentery lymph, portal vein blood of normal rats was taken out. The primary PMVECs of passages 3 were treated by different treatment factors, respectively. The apoptosis rate was analyzed by flow cytometry, and the expressions of relative genes of apoptosis such as fas, fas L, bcl-2 and bax were detected by RT-PCR. RESULTS:The apoptosis rate of PMVECs was 9.86%±3.24% after exposed to shock lymph at the final concentration of 4% for 4 hours and significantly higher than that in control (P<0.01). The expression levels of fas, fas L and bax mRNA were higher and bcl-2 mRNA was lower in shock lymph group than those in control group.CONCLUSION:The results demonstrated that the apoptosis of PMVECs of rats was induced by shock lymph, and its mechanism relate to high expression of apoptosis accelerative genes such as fas, fas L, bax mRNA and low expression of apoptosis inhibitory gene bcl-2.     

Study on adherent precursors from human cord blood

AIM: To confirm the existence of the endothelial progenitor cells in human cord blood and to study its differentiation and development process. METHODS: The mononuclear cells in human cord blood were isolated using lymphocyte separation solution. Then the mononuclear cells were cltured in MCDB131 containing 20% fetal bovine serum. The effects of 5 μmol/L dexamethasone, the extract from bovine brain, insulin and hypoxanine on the proliferation and differentiation of the adherent cells were observed. The morphology of the adherent cells were examined twice daily by inverted phase contrast microscope. CD34 and CD14 expression were determined by FACS. Immunohistochemistry was used to confirm the expression of factor Ⅷ. RESULTS: The proliferative endothelial progenitor cells existed within the CD34^-adherent mononuclear cells of human cord blood. Dexamethasone and hypoxanine decreased the number of spindle-shaped cells and caudated cells. Bovine brain extract, insulin and FCS enhanced the number of spindle-shaped cells and caudated cells. CONCLUSION: The existence of endothelial progenitor cells within the CD34 - adherent monouclear cells of the human cord blood was observed and these cells were able to differentiate into endothelial-like cells in vitro.     

Experimental study on biocompatibility between canine bone marrow stem cells and copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate

AIM: To evaluate the biocompatibility between copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and bone marrow stem cells (BMSCs). METHODS: Canine BMSCs were isolated and cultured. The cells in passage 3-4 were seeded onto the PHBV films and three-dimensional foams. The seeded cells were observed under inverted microscope for morphology and cell attachment onto the PHBV films at 1, 2 or 3 weeks after seeding. With 4% paraformaldehyde formalin and staining, the protein content in seeded cells was determined by bicinchoninic acid assay (BCA). The content of DNA was quantified using the Hoechst 33258 assay. RESULTS: Observation under inverted microscope showed that the PHBV fabric was fairly thickness, lucency is weak. Unser contrast phase microscope, PHBV fabric was uneasy to be observed. Most cells attached onto the PHBV films 2 h after seeding, and extended well and acquired a spindle fibrecyte-like morphology 3 d later. Moreover, on the three-dimensional foams, the seeded cells lay in micropores and grew tri-dimensionally. The conjunction of cells appeared about 1 week, and extended at 3 weeks, with a large amount of extracellular matrix around cells. The content of DNA and protein has no significant difference with control group. CONCLUSION: As a kind of tissue engineering material for BMSCs seeding, PHBV has an excellent biocompatibility.     

Biological characteristics of newborn rabbit tracheal chondrocytes

AIM：To investigate the biological characteristics of newborn rabbit tracheal chondrocytes in vitro. METHODS：Newborn rabbit tracheal chondrocytes were obtained by the method of enzyme digestion, and then cultured in monolayer in vitro. Morphological and growth observations were performed under inverted phase contrast microscope. The ultrastructures of the cells were observed under scanning electron microscope and transmission electron microscope. The biological characteristics of secreted extracellular matrix components were detected by real-time PCR, immunocytochemistry staining and toluidine blue staining. RESULTS：Newborn rabbit tracheal chondrocytes isolated and cultured in vitro showed short triangular or irregular shapes, and adherent growth very well. The ultrastructures of the cells showed pore and abundant cytoplasm and organelles, with a lot of protein secretions in the cells. The chondrocytes expressed the mRNA of collagen I, collagen II and proteoglycans, mainly collagen II and proteoglycans. Immunocytochemistry staining showed collagen II and SOX9 positive, and collagen I weakly positive. Toluidine blue staining was also positive. CONCLUSION：Enzyme digestion and monolayer culture are suitable method to obtain newborn rabbit tracheal chondrocytes. These cells， secreting extracellular matrix components, are able to be selected as seed cells for tissue engineering of trachea in vitro, and used to study the therapeutic method for neonatal rabbit tracheal stenosis.     

Amplification of mesenchymal stem cells from human bone marrow and orientation to induce MSCs differentiating into endothelial cells in vitro

AIM: Our purpose was to induce MSCs differentiating into endothelial cells (EC) in vitro and to provide the seed cells for study of cardiovascular tissue-engineering. METHODS: MSCs were separated by gradient centrifugation on Percoll (density 1 073 g/L) from human bone marrow (HBM), and incubated for purification and amplification in DMEM (low glucose) with 10% fetal bovine serum (FBS). Then, the MSCs were incubated for orientation differentiated into EC in DMEM (high glucose) with 20% FBS, VEGF (10 μg/L), bFGF (5 μg/L), L-glutamine (2 mmol/L), penicillin (1×105U/L) and streptomycin (100 mg/L) for about 14-21 days and their phenotypic characteristics were analyzed by flow cytometry. Afterwards, the differentiating cells were evaluated by histology and immunohistochemistry. RESULTS: The quantity of MSCs was increased from 5.0×105 in the primary culture to 8.0×1012, or to increase to 1.6×107 times after 15 generations of incubation. The purity of MSCs was above 95% and 98% homogeneous at passages 2 and 3, respectively. About 80%-90% of the differentiating cells from MSCs after 14-21 days were positively stained for Ⅷ factor (vWF) related antigen by immunohistochemistry assay, and Weible-palade corpuscle was also observed by transmission electron microscopy in the cytoplasm. CONCLUSION: MSCs from HBM have the capability of differentiation into ECs in vitro, which may be a potential source of seed cells for fabrication of tissue-engineering heart valve, particularly in children with congenital heart disease.     

Reconstructed corneal endothelial tissue on allo-stroma in vitro

AIM: In order to search carrier material and better tissue culture method, the morphology and structure of the cultured cat corneal endothelium was observed. METHODS: The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, and then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology and structure of reconstructed tissue was tested by the inverted microscope and the scanning electron microscope. RESULTS: As shown by the morphological observations, the cultured cat corneal endothelial cells formed an integrated membrane, and attached to the Descemet's membrane, which was similar to the nature tissue. The cells connected tightly to each other, and some of them arranged in hexagon approximately. CONCLUSION: The cat corneal endothelial cells could be rebuilt on the carrier of the dehydrated swine corneal stroma successfully, which is similar to the nature tissue.     

Changes of pulmonary apoptosis and NOS mRNA in the lipopolysaccharide-induced acute lung injury

AIM: To observe the chronological changes of pulmonary apoptosis and the expression of iNOS mRNA,nNOS mRNA and eNOS mRNA in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to investigate the mechanisms of ALI.METHODS: Rats were randomly divided into 2 groups: control group and LPS treated group.The rats were injected with either saline or LPS and killed at 1,3,6,9 and 12 h after LPS injection.The expressions of iNOS mRNA,nNOS mRNA and eNOS mRNA in the lung tissue were respectively measured with RT-PCR methods.Apoptosis and expressions of Bcl-2 and Bax were respectively determined by flow cytometry (FCM) and immunohistochemistry (IHC).The pathological changes of lung tissue were observed under light and electron microscope.RESULTS: Compared with that in control group,the expression of iNOS mRNA was significantly increased at 3,6,9 and 12 h after administration of LPS (P<0.05).The eNOS mRNA was significantly decreased at 3,6,9 and 12 h after administration of LPS (P<0.05).The nNOS mRNA had no significant change during the 12 h in LPS group.Degree of ALI was gradually worsened after administration of LPS.Apoptosis of pulmonary cells was significantly increased,and reached the top level at 9 h after administration of LPS (P<0.01).The expression of Bcl-2 was markedly decreased and the expression of Bax was significantly enhanced in alveolar and airway epithelial cells in LPS treated group.CONCLUSION: The expressions of iNOS mRNA,eNOS mRNA and nNOS mRNA are not identical in LPS-induced acute lung injury.NOS regulates the apoptosis of pulmonary cells through affecting the balance of Bcl-2 and Bax.     

Functional improvement of infarct heart by implantation of human adipose-derived mesenchymal stem cells delivered by matrigel

AIM: To investigate the implantation of matrigel carrying human adipose-derived mesenchymal stem cells to enhance the cell survival and the improvement of the ventricular functions in infarct heart.METHODS: Human adipose-derived mesenchymal stem cells (ADMSCs) were isolated and cultured from adult adipose tissue. SD rats with one-week-old myocardial infarction were randomly received the following 4 treatments: injection of PBS, matrigel, PBS+ADMSCs or matrigel+ADMSCs, respectively. Labeled ADMSCs either in matrigel or in saline were injected into the border area of ischemia. The controls received the injection of matrigel or saline only. Four weeks after injection, the heart functions were determined by echocardiography. The densities of the micro-vessels within the infarct area were also measured.RESULTS: Four weeks after implantation of ADMSCs, the cell graft size, the heart functions and the micro-vessel densities within the infarct area improved in matrigel+ADMSCs group as compared to other groups.CONCLUSION: The co-injection of ADMSCs with matrigel enhances the graft survival, increases the density of the micro-vessels in the myocardium and improves the cardiac functions.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Preliminary study of endothelial progenitor cells formed vessel-like structure in vitro

AIM: Endothelial progenitor cells (EPCs) are a group of stem cells/progenitor cells, which exist in postnatal body and can be of specially homing to the foci of angiogenesis and then differentiate into endothelial cells.This investigation was to study the method for culturing endothelia progenitor cells (EPCs) in vitro, and to observe its feasibility and condition formed vessel-like structure.METHODS: The cells were isolated from born marrow, peripheral blood, umbilical cord blood or spleen in different laboratories.The EPCs derived from human umbilical cord blood and rabbit peripheral blood were cultured in vitro through adhesion selection and were differentiated into endothelial cells under the induction of special cytokines.The expression of CD34, VEGFR-2, AC133 and VE-cadherin were detected by fluorescence-activated cell sorting.The endothelial cell lineage was confirmed by DiI-ac-LDL up-taking and Ⅷ factor immunocy tochemistry.RESULTS: The EPCs derived from human umbilical cord blood and rabbit peripheral blood were cultured in vitro successfully, forming cord-like and tube-like structure.The EPCs derived from rabbit peripheral blood differentiated more mature and formed vessel-like structure.CONCLUSION: The EPCs derived from human umbilical cord blood and rabbit peripheral blood formed vessel-like structure in vitro.EPCs may be a potential resource of vessel tissue engineering.     

Inhibitory effect of cytotoxin 1 from Naja atra Cantor venom on K562 cells

AIM: To investigate the inhibitory effect of cytotoxin 1 (CTX1) from Naja atra Cantor venom on human chronic myeloid leukemia cell line K562.METHODS: The MTS and cell counting methods were used to detect cell relative viability and cell numbers of K562 cells treated with CTX1 at different concentrations. In the living culture system, the dead and dying cells stained by PI were observed by inverted fluorescence microscope. After treated with CTX1, the apoptotic cells were detected by flow cytometry with annexinV-FITC and PI double staining.RESULTS: The relative viabilities of K562 cells were (90.50±3.07)%, (58.33±3.08)% and (27.43±1.99)% when the cells were treated with CTX1 for 24 h at the concentrations of 2, 5 and 10 mg/L,respectively. The median inhibitory concentration of CTX1 on K562 cells was 5.77 mg/L after 24-hour treatment. Comparment with control group, the percentages of K562 cells by cell counting were (85.01±3.54)%, (56.65±3.59)% and (43.24±4.15)% after treatment with 8 mg/L of CTX1 for 6 h, 12 h, 24 h,respectively. As treatment concentration of CTX1 was elevated and treatment time was prolonged, the cells stained by PI were remarkably observed under inverted fluorescence microscope. After treatment with CTX1 at 8 mg/L for 6 h, 12 h and 24 h, the incidences of cell necrosis were (0.73±0.06)%, (13.90±0.46)% and (23.33±0.86)%, respectively, and the incidences of late apoptosis were (16.27±0.21)%, (26.90±1.23)% and (18.77±0.81)%, respectively.CONCLUSION: CTX1 possesses obvious inhibitory effect on K562 cells and it mainly causes the late phase of apoptosis and necrosis.     

Three dimensional tissue culture in culture medium with rhbFGF

AIM:To investigate the behaviour of 3T3 fibroblast and macrophage co-culture on blood fibrin clot or adipose tissue with recombinant basic fibroblast growth factor (rhbFGF).METHODS:MTT method, inverted contrast microscopy, Giemsa staining as well as scanning electron microscope were used in the present study.RESULTS:The effect of rhbFGF on co-culture of 3T3 fibroblast and mouse macrophage on blood fibrin clot in low-serum DMEM with rhbFGF were monitored, and 3T3 fibroblast and macrophage growed well on the blood fibrin blot in low-serum DMEM with rhbFGF. CONCLUSION:The blood fibrin clot, with its low immunogenicity, could be used as a bionic support for three-dimensional tissue culture, and also a physiological carrier to distribute the growth factor rhbFGF for the cells.     

Characterizations of human retinal microvascular endothelial cells at various ages

AIM: To investigate the properties of human retinal microvascular endothelial cells (RMECs) at two different age groups. METHODS: Human RMECs with high affinity were isolated from donors at two age groups: 30 d(group A) after birth and 40-60 years of age (group B). The RMECs were characterized for expression and localization of endothelial cell markers by immunofluorescence staining of CD31, von Willebrand factor(vWF) and uptake of acetylated low-density lipoprotein. The ability of tube formation was assessed on Matrigel, and vWF distribution in cells was observed by confocal immunofluorescence microscope and Western blotting analysis, respectively. RESULTS: High purity RMECs can be obtained readily from each group with modified methods. At 6 hours, cells from both groups formed tube structures successfully, but there was a significantly higher incidence of branching in RMECs of infant group (group A) by 27.2％±2.2％ compared with adult group (group B) at 12 h （P<0.05). Group A maintained intact structure even at 30h, but group B partially lost the basic tube structure. In addition, vWF was translocated from cytoplasm to nucleus with aging. CONCLUSION: Human RMECs at different ages have specific properties. Cell properties related with age of the donors should be considered in in vitro studies.     

Effects of NGF on collagen secretion and morphology of hepatic stallete cells

AIM: To investigate the effects of nerve growth factor (NGF) on the changes of collagen secretion and morphology of hepatic stallete cells(HSCs). METHODS: Rat HSCs were incubated with different concentrations of NGF for 24 h. Collagen I and III in the supernatants of culture medium secreted by HSCs were detected by enzyme-linked immunosorbent assay. The morphological changes of HSCs were observed under inverted microscope with acridine orange staining and under transmission electronic microscope. RESULTS: When HSCs was incubated with NGF at concentrations of 100, 200 or 400 μg/L for 24 h, the content of collagen I and collagen III in the culture supernatants were significantly reduced compared with control group (P<0.05). After stimulated with NGF at the concentration of 100 μg/L for 24 h, the growth of the HSCs was inhibited and the morphous of the cells became round or oval gradually. The morphological changes of apoptotic cells were also observed by acridine orange staining and transmission electronic microscopy. CONCLUSION: NGF inhibits HSCs to synthesize collagen I and collagen III. Inhibition of collagen production and promotion of apoptosis in HSCs may be the possible mechanisms of NGF to reverse liver fibrosis.     

Effects of puerarin on heme oxygenase-1/carbon monoxide system in lung ischemia-reperfusion injury

AIM:To investigate the variations of heme oxygenase-1/carbon monoxide system in lung ischemia-reperfusion injury and effects of puerarin on the system.METHODS:The unilateral lung ischemia-reperfusion model was replicated in vivo. Rabbits were randomly divided into three groups (n=10 in each): control group, ischemia-reperfusion (I-R) group and puerarin group. The blood specimens collected at times before ischemia, ischemia 1 h, reperfusion 1 h, 3 h and 5 h were tested for the contents of carboxyhemoglobin (COHb) and cyclic guanosine monophosphate (cGMP). The lung tissues sampled at 5 h after reperfusion were assayed for wet/dry weight ratio (W/T), the injured alveoli rate (IAR) and the activity of HO-1. The changes of ultrastructure were observed under electron microscope. The tissue slides were also stained by immunohistochemistry (IHC) and in situ hybridization (ISH) to detect the expression of HO-1.RESULTS:The plasma content of COHb and cGMP in I-R and puerarin group increased in a time-dependent manner after I-R compared with control group, but the increment in puerarin group was higher than that in I-R group (P<0.01). The activity of HO-1 was much higher in I-R group than that in control group, and the indexes in puerarin group increased more significantly (P<0.01). Expression of HO-1 was up-regulated in I-R and puerarin group than that in control group in the pulmonary vascular endothelial cells, part of pulmonary vascular smooth muscle cells, extima of vessels and epithelial cells of airway. The highest average optical density value was observed in puerarin group (P<0.01 or P<0.05). Serious ultrastructural morphological damages were observed in I-R group under electron microscope, only slightly injury was found in puerarin group.CONCLUSION:Puerarin up-regulates the expression of HO-1, improves the activity of HO-1, provides significant protective effects on lung during ischemia-reperfusion injury.     

Direct differentiation of embryonic stem cells into neural cells without embryonic body culture period in vitro

AIM:To investigate the feasibility and effect of directly differentiation of embryonic stem cells (ESC) into neural cells induced by retinoid acid (RA) without embryonic body (EB) culture period in vitro. METHODS:ESC were digested and divided into 4 groups:group A and B were undergone EB culturing. After that, cells in group A were induced by RA, cells in group B were differentiated spontaneously, cells in group C were committedly induced by RA directly without EB culturing, and cells in group D were differentiated spontaneously without EB period. Morphologic changes were observed under inverted microscope and scanning electron microscope. MAP-2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days. RESULTS:In groups A or C, neuron-like cells increased gradually, forming neural network. At the 9th day, a large part of cells in these groups were MAP-2 positive cells, and the positive rate was higher than that in groups B or D (P<0.01). Groups B and D were almost epithelial-like cells. At the 9th day, GFAP positive cells were predominant. The rate of GFAP staining cells in groups A or C was significantly lower than that in groups B or D (P<0.01). There was no significant difference of MAP-2 or GFAP positive rates between group A and C, nor between group B and D (P>0.05). CONCLUSION:ESC was directly induced into neural cells by RA without EB culture period in vitro. This modified method has the same effect as the traditional RA 4－/4＋ assay and can replace the traditional method.      

Studies of cell sheet engineering on fibrin glue cultured with three kinds of rabbit corneal cells

AIM: To study whether three kinds of rabbit corneal cells can grow well on fibrin glue (FG) constructed in vitro, and investigate the feasibility of FG for the scaffold of cell sheet engineering. METHODS: Three kinds of corneal cells were seeded on FG which was produced in vivo. Cell growth on FG was examined as follows: (1) by inverted microscope; (2) histologically by hematoxylin and eosin; (3) by scanning electron microscopy. RESULTS: Fibrin glue prepared was smooth and transparent. With cell growth, FG degradated partly, then obtained cell sheet engineering only with a small amount of FG. Corneal cells generated well on the fibrin glue in vitro and maintained the physiological state of cells. Corneal epithelial cells formed unilaminar and stratified layers and cellular joins. Corneal endothelial cells formed round or polygon, the same size cells and lined up tightly. Corneal stroma cells formed triangle and arborization, cell-cell junction was obvious, and formed network link. CONCLUSION: Fibrin glue is well compatible with three kinds of corneal cells, which can construct tissue engineered cell sheet with fibrin glue, so as to reconstruct ocular surface.