Objective To explore the protective effect of HGF on primary cultured spinal neurons in vitro.Methods Cultured neurons were transfected with Ad-GFP or Ad-HGF in different multiplicity of infection(MOI),then flow cytometer and PI-Hoechst double stains were used to assay transfer rate or to determine the status of cells respectively.ELISA was used to detect the expression of HGF in Ad-HGF transfected neurons,and the activity of neurons was judged by neutral red stain,MTT and NSE-ELISA methods.Results After transfection with Ad-GFP in MOI of 50,the transfer rate was high as detected by flow cytometer,and the status of cells was well as judged by PI-Hoechst double stain.The results of ELISA showed that HGF was expressed in medium.After transfection with Ad-HGF,the activity of neurons was better than neurons without treatment(P

Objective To establish an aging model of mesenchymal stem cells (MSCs) and to investigate aging related biological mechanism for the purpose of studying the senesence of MSCs .Methods MSCs were separated and purified from human placenta, and the cells of the third passage(P3-MSCs) were cultured in the medium for 2 hours, then 100,200 and 300 μmol/L hydrogen peroxide ( H2 O2 ) was added to the cells for 2 hours to establish the MSCs aging model in vitro. Biological characteristics of aging MSCs were evaluated by cell cycle assay and senescence associated β-galactosidase staining.The expression of p16,p21 and p53 genes was further measured using quantitative real-time PCR (RT-PCR).Re-sults Compared with the control , the number of MSCs treated with 200μmol/L H2 O2 for 2 hours was significantly decreased and the cells displayed less adipogenic ,osteogenic and chondrogenic differentiation .Moreover ,after exposure to 200 μmol/L H2 O2 , the majority of the cells were in the G 0/G1 phase as showed by cell cycle analysis .The percentage of senescence-associated β-galactosidase-positive cells was increased , and the expression of p 16 , p21 and p53 mRNA and protein was significantly increased.Conclusion The results of this study has demonstrated that the H 2 O2 (200 μmol/L) can be used to establish the aging model of MSCs in vitro, and the cellular phenotypic alteration may attribute to the cell cycle associated gene expression (p16, p21, and p53).

Objective To compare two sources of mesenchymal stem cells ( MSCs) from human placenta and umbilical cord, and to optimize a technical solution for bench or clinical studies of MSCs.Methods MSCs were isolated from human placenta and umbilical cord and expanded for analysis.The cell morphology was observed under invert microscope, the immunophenotypic feature of MSCs was analyzed with flow cytometer, the cell proliferation ability was determined by cell cycle assay and cell doubling time, the cell differentiation potential was evaluated by osteogenic and adipogenic induction in vitro as well.Results Both sources of MSCs were adherent cells and exhibited fusiform and fibrous morphology. Furthermore, both MSCs high expressed CD90 and CD105, and were negative for the markers of CD34, CD45 and HLA-DR.The population doubling time of MSCs form human placenta and umbilical cord was 39.5 h and 40.8 h separately, and the results of cell cycle analysis showed that the percent of the two sources of MSCs in G0/G1 phase was 52.12%and 57.50% respectively. The above results demonstrated that both sources of MSCs possessed the similar biological characteristics in morphology, phenotype and as well as proliferation ability.In addition, both of them could be induced into osteoblasts and adipocytes in vitro.Conclusion MSCs from human placenta have the similar biological characteristics to these from human umbilical cord, and both of them are better candidates for bench and clinical research.

BACKGROUND:How to obtain sufficient autogeneic chondrocytes is a problem which must be answered as soon as possible in both the transplantation of chondrocytes and the development of cartilage engineering.Bone marrow-derived mesenchymal stem cells have the potential of multidirectional differentiation. Under different induced conditions, they can differentiate into multiple tissue cells, such as chondrocyte, osteoblasts , sarcoblast, nerve cells and so on. Insulin-like growth factor-I plays an important role in regulating the formation and development of limb and cartilage.OBJECTIVE:To observe the effect of the insulin-like growth factor-I and culture solution of chondrocyte on inducing bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes in vitro.DESIGN:Open experiment.MATERIALS:This experiment was carried out at the Medical Study Center, Second Hospital Affiliated to Sun Yat-sen University from March to November 2003.Human bone marrow-derived mesenchymal stem cells were harvested from 4 to 6-month old embryos, all of which were from pregnant women who needed to terminate of pregnancy by induction delivery with water bag for health.METHODS:Human embryonic mesenchymal stem cells were isolated with Percoll separating medium. Subsequently, the cells were amplified in vitro,and the expression of surface makers of bone marrow-derived mesenchymal stem cells, such as CD44, CD71, CD34 and CD45 were measured with flow cytometer to identify the cells in our experiment. 100 μg/L insulin-like growth factors and culture solution of chondrocytes were added in the culture medium of bone marrow-derived mesenchymal stem cells of the fourth generation. The morphological changes of induced cells were observed with an inverted microscope. The expression of type Ⅱ cartilage matrix was observed by collogen immunohistochemistry. The proteoglycan level in the cells was detected, too.MAIN OUTCOME MEASURES:Phenotype of bone marrow-derived mesenchymal stem cells was identified through detecting the expressions of CD34, CD44 and CD4.Type Ⅱ collagen immunohistochemistry and the change of cellular ability to secrete proteoglycan after induction were observed to determine whether bone marrow-derived mesenchymal stem cells can differentiate into chondrocytes.RESULTS: ① Being observed under an inverted microscope,the bone marrow-derived mesenchymal stem cells presented a morphology like fibroblasts when they were cultured in vitro. ②Identification of surface antigen of bone marrow-derived mesenchymal stem cells: These cells were detected to have good homogenicity with flow cytometer. The fourth generation of bone marrow-derived stem cells positively expressed CD44, negatively expressed CD34 and CD45, suggesting these cells had the characteristics of bone marrow-derived mesenchymal stem cells. ③Observation of the morphological change of chondrocytes induced by bone marrow-derived mesenchymal stem cells under optical microscope: chondrocyte condition culture solution and insulin-like growth factors-Ⅰ were added to co-culture.During the process of culture, bone marrow-derived mesenchymal stem cells were seen to have a shape of round gradually. Fifteen days later, some cells presented a shape of short-shuttle or polygon with short mutations,which were the shape characteristics of chondrocytes. ④Immunohistochemical staining of Type Ⅱ collagen: In the insulin-like growth factor group,72.5% cells had many brown granules in cytoplasm, which were weakly positive or strongly positive expression of Type Ⅱ collagen. In the control group, bone marrow-derived mesenchymal stem cells was negative expression of type Ⅱ collagen on the 15th day. ⑤ Measurement of proteoglycan level: After co-culture with insulin-like growth factor-Ⅰ and chondrocyte culture solution for 15 days, proteoglycan was higher in the cells of co-culture group [ (8.92±0.91) μg/L ] than in culture group [(2.56±0.26) μg/L,P ＜ 0.05], but lower than in the chondrocyte group[(13.69±1.51) μg/L, P＜ 0.05].CONCLUSION:Insulin-like growth factor-Ⅰ and chondrocyte culture solution can induce bone marrow-derived mesenchymal stem cells to differentiate into chondrocytes.

BACKGROUND:Human umbilical cord mesenchymal stem cells with capabilities for self-renewal and multi-differentiation have attracted widespread attention. OBJECTIVE:To develop an efficient method for isolation and culture of human umbilical cord mesenchymal stem cells, and to analyze the cellbiological features. METHODS:Mesenchymal stem cells were isolated and cultured from human umbilical cord by improved tissue cultivation. Immunophenotype and cellcycle were analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated by in vitro osteogenic and adipogenic induction as wel . RESULTS AND CONCLUSION:Some fusiform cells crawled out from human umbilical cord tissues after cultivation for 5 days and formed colonies about 10 days later. When the removed tissues were further cultured, more cells appeared again within 2 days and formed colonies after 5 days. The isolated cells exhibited similar morphology of fibroblast-like shape after passage. Furthermore, the cells expressed CD90, CD105, but were negative for the markers of CD34, CD45, HLA-DR. Population doubling time of the cells calculated from the result of MTT was about 50 hours and cellcycle analysis showed that 41.24%cells were in the G 2/S phrase. Therefore, the isolated cells had a high prolification ability. In addition, the isolated cells could be induced into osteoblasts and adipocytes in vitro. In a word, the results of this study demonstrated that the cells from the second tissues culture possessed the biological characteristics of mesenchymal stem cells and more primary umbilical cord mesenchymal stem cells were acquired through the improved method.

BACKGROUND:Placenta is a valuable source of mesenchymal stem cels for stem cel therapy and future application in the field of regenerative medicine. However, conventional methods cannot acquire a large amount of purified human placenta-derived mesenchymal stem cels. Here, we present a new method for isolating human placenta-derived mesenchymal stem cels suitable for banking strategies and for future clinical applications. OBJECTIVE:To analyze the biological characteristics of human placenta-derived mesenchymal stem cels cultured by tissue dissociating and colagenase digestion. METHODS: Human placenta-derived mesenchymal stem cels were obtained from human placenta by tissue dissociating and colagenase digestion method. Immunophenotype was analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated byin vitro adipogenic, osteogenic and chondrogenic induction as wel. RESULTS AND CONCLUSION:Human placenta-derived mesenchymal stem cels could be passaged stablyin vitro. Furthermore, the cels expressed CD73, CD90, CD105, but were negative for the markers of CD11b, CD19, CD34, CD45, and HLA-DR. Human placenta-derived mesenchymal stem cels proliferated actively and began to grow logarithmicaly at days 3-5 folowed by a plateau period at day 6. In addition, the isolated cels could be induced into adipocytes, osteocytes, chondrocytesin vitro. In a word, the results of this study demonstrated that the tissue dissociating and colagenase digestion method is an efficient method for obtaining a large amount of human placenta-derived mesenchymal stem cels that can be stably cultured in vitro and have strong proliferative ability.

OBJECTIVE: To establish a New Zealand rabbit model of acute kidney injury induced by cisplatin. METHODS: A total of 24 male New Zealand rabbits were randomly divided into control group,low-,medium-and high-dose cisplatin group according to the body mass. Rabbits were injected with cisplatin at 0. 0,1. 0,2. 0,4. 0 mg/kg body weight by auricular vein. Rabbits in low-dose group was continuously injected for 5 days,medium-dose group was continuously injected for 3 days,and the high-dose group was injected for once per day. Rabbits in the control group did not receive any treatment. Blood was collected from the middle ear artery and 24 h urine was taken before exposure and on day 1,day 3,day 5 and day 7 of injection. The serum creatinine( Scr) and urea nitrogen( BUN) were detected by colorimetric method,and 24 h urine kidney injury molecule 1( KIM-1) was measured by enzyme-linked immunosorbent assay. Plasma platinum,24 h urinary platinum and renal platinum level were detected by inductively coupled plasma mass spectrometry.At the end of the experiment,rabbits were sacrificed and the left kidney was taken for histopathological examination.RESULTS: The body mass of rabbits of the low-,medium-and high-dose groups on day 7 after cisplatin exposure was lower than that of the control group( P < 0. 05),and lower than that of the same group before exposure( P < 0. 05). After 3 days of exposure,the Scr level in each dose group was higher than that of the control group( P < 0. 05),the Scr level on day 3and day 5 in medium-and high-dose groups were higher than that of the low-dose group( P < 0. 05). The BUN levels on day 3 and day 5 in medium-and high-dose group were higher than that of the control group and low-dose group( P <0. 05),the BUN levels on day 7 in three dose groups were higher than that of the control group( P < 0. 05). The levels of plasma platinum and 24 h urinary platinum in the three doses groups of New Zealand rabbits on day 1,day 3,day 5 and day 7 after exposure were higher than that of the control group( P < 0. 05),and were higher than the pre-treatment levels of the same group( P < 0. 05). The level of 24 h urinary KIM-1 in the meclium-dose group of New Zealand rabbits was higher than that of the control group on day 3 of exposure( P < 0. 05). The level of 24 h urinary KIM-1 in the mediumdose group of New Zealand rabbits on the 5th day after exposure was higher than that of the control group( P < 0. 05). The renal platinum levels in the three groups of New Zealand rabbits were higher than that in the control group( P < 0. 05).The pathological changes of rabbit kidney caused by cisplatin are mainly tubular dilatation,protein cast,alkalophilic and interstitial nephritis. CONCLUSION: Cisplatin can induce acute kidney injury in rabbits,and the degree of injury is dosedependent. The dose of 1. 0 mg/kg body weight continuous injection for 5 days is closely related to clinical use of cisplatin,which is recommended for model establishment.

Objective To investigate the impacts of treating stratege for non-infarct related artery on clinical prognosis in elderly patients with acute myocardial infarction (AMI) after urgent percutaneous coronary intervention (PCI). Methods From Augst 2007 to Augst 2010,a total of 75 elderly AMI patients (aged 75 years and over) were treated by urgent PCI and confirmed as multivessel coronary disease in our hospital. Among them,30 patients received medicine combined with PCI once again (PCI group) and 45 patients received only medicine treatemt (medicine group).The major adverse cardiovascular events (MACE) and results ot coronary angiography after treatment for average one year were compared between the two groups. Results There were no significant differences in the rates of in stent restenosis[1 case(2.2 ％)vs.0 case],late loss of in-segment lumen [5 cases(11.1％)vs.3 cases(10.0％)],stent thrombosis[1 case(2.2％)vs.1 case(3.3％)] and larget vessel revascularization [2 cases (4.4 ％ ) vs.1 case ( 3.3 ％) ] between medicine group and PCI group (x2=0.00,0.00.2.03 and 0.00,all P＞0.05).The propertions of angina recurrence and second hospital admission for heart diseases were lower in PCI group than in medicine group [36 cases (80.0％)vs.14 cases(46.7％),18 cases(40.0％)vs.5 cases(16.7％),x2=9.00,4.61,P＜0.01and P＜0.05].However,no differences were found in the secondary heart failure,recurrent nonfatal myocardial infarction,severe arrhythmia,all- cause death and mortality rate of cardiovascular disease between the two groups (x2 =0.09,0.00,0.00,0.00 and 0.00,all P＞ 0.05). Conclusions Compared with single medicine therapy,the medicine combined with PCI for non- infarct-related artery may decrease the rates of angina recurrence and second hospital admission for heart diseases in elderly patients with AMI.

Objective The objective of this study is to examine the expression level of the S100A7A protein in both gastric cancer tissues and cells,as well as to evaluate its impact on the malignant phenotype of gastric cancer(GC)cells.Methods Immunohistochemical assay was used to detect the expression characteristics of S100A7A in 21 gastric cancer tissues and their corresponding paracancerous tissues,as well as to investigate its correlation with gastric cancer clinicopathological factors.Gastric cancer cells were genetically modified to overex-press S100A7A through plasmid transfection.Subsequently,the impact of S100A7A on the proliferation,migra-tion,and invasion capacities of gastric cancer cells was assessed using cell proliferation assays(EdU assay and plate cloning assay)as well as cell migration and invasion assays(Transwell assay and scratch assay).Results The expression of S100A7A protein was higher in GC tissues than in paracancerous tissues;Overexpression of S100A7A may increase gastric cancer cell proliferation,migration,and invasion.Conclusion S100A7A is a possible oncogene in GC and is predicted to serve as a new diagnostic and therapeutic target for the disease.

BACKGROUND:Skin damage caused by radiation therapy and nuclear accidents is still a serious medical problem.It is difficult to achieve effective treatment results with single prevention and treatment methods.It is an important research direction to find new comprehensive treatment methods. OBJECTIVE:To observe the protective effect and the underlying mechanism of 1,2-propanediol combined with hepatocyte growth factor-modified exosomes derived from dental pulp stem cells on human epidermal radiation damage cell models. METHODS:(1)After infection of human dental pulp stem cells using recombinant adenovirus of human hepatocyte growth factor gene,exosomes,i.e.,Ad.HGF DPSC-Exo,were isolated with ultracentrifugation.(2)HaCat cells were irradiated with X-ray.The cells were treated with 1,2-propanediol before irradiation and Ad.HGF DPSC-Exo after irradiation.Cell proliferative activity was determined by CCK-8 assay.Cell apoptosis was detected by flow cytometry.Cell migration was detected by cell scratch assay.The expression levels of P21 and P53 were detected by PCR. RESULTS AND CONCLUSION:1,2-Propanediol,Ad.HGF.DPSC-Exo,Ad.HGF.DPSC-Exo + 1,2-propanediol could significantly improve the growth inhibition of HaCaT cells,reduce cell apoptosis,elevate cell proliferation and migration,and exhibit a good radiation protection effect.Moreover,the combined effect of Ad.HGF.DPSC-Exo + 1,2-propanediol was better.Furthermore,Ad.HGF.DPSC-Exo + 1,2-propanediol alleviated the cellular G2/M phase block and decreased the expression of cell cycle genes P53 and P21.In conclusion,1,2-propanediol pretreatment combined with Ad.HGF.DPSC-Exo had significant protective effects on radiation-induced HaCaT cell injury and it provided novel ideas and potential methods for the prevention and treatment of radiation-induced skin damage.