Objective To explore the electrophysiological characteristics and the expression of mRNA and protein of L-type calcium channel in rat bone mesenchymal stem cells(MSCs). Methods MSCs were isolated,cultured and purified.RT-PCR was used to detect the mRNA expression of ?1C,?1D,?1H,?1S.The protein expression of L-type calcium channel(?1C) was testified by immunohistochamical.Ion currents were recorded in MSCs using whole-cell patch clamp technques.Results CD29,CD44,CD106 expressed in about 93% MSCs and CD14,CD34,CD45 expressed negatively.A high expression of mRNA in ?1C was detected by RT-PCR but no expressions were observed in ?1D,?1G,?1H,?1S.Immunofluorescent double labeling showed an expression of ?1C subunits in MSCs.Moreover,inward currents were recorded in 16 of 36 cells using whole-cell patch clamp techniques.The currents were activated around-30?mV and peaked at 0 to 10?mV and were blocked by nifedipine(10??mol/L).These cells had larger currents with Ba~(2+)(10?mmol/L) in bath solution than with Ca~(2+)(2?mmol/L).Conclusion The results indicated that adult rat MSCs expresse functional L-type calcium channels.It is possible that this channel plays a role on the proliferation and differentiation of MSCs.

Objective To isolate,culture and identify epidermal stem cells(ESCs).The gene of enhanced green fluorescent protein(eGFP) was introduced via recombinant adeno-associated virus(rAAV) infection into the epidermal stem cells in vitro and the transfection efficiency under various tite of culture was determined. Methods 1.Getting fetal Wistar rats'dissociated single epidermal cells.2.Making laminin and CollagenⅣ the substitution of basal memrane,ESCs were isolated by adhering to murine laminin and CollagenⅣ.3.Epidermal stem cells were cultured in twenty-four-well plate,and cells number was controled 5?10~4 or so.After epidermal stem cells adhered to plate,diluted rAAV_2/eGFP virus fluid with serum-free medium.The diluted rAAV_2/eGFP virus fluid was added to the twenty-four-well plate according to the different MOI(viru gene/cells).The number of green fluorescence cells were counted under fluorescence microscope.The transfection efficiency was determined. Results 1.ESCs had better adhesive ability to laminin and higher colony formation efficiency(CFE) than that of keratinocytes.2.ESCs wer strongly positive with immunocytochemical staining of integrin ?1 and keration 19(K19).3.After rAAV_2/eGFP genetic transfection of ESCs,the positive cloning expressed highly eGFP gene along with time.Conclusion 1.These results suggest that rats'epidermal stem cells could been successfully isolated and enriched in vitro by means of rapid adherence to laminin and CollagenⅣ.After rAAV_2/eGFP genetic transfection of epidermal stem cells,the positive cloning expressed highly eGFP gene stably along with time.

Objective We compare the effects of fibrinogen gel and chitin on BMSCs to chondrocytes differentiation in order to explore the relationship between three-dimensional scaffold and cartilage tissue engineering seed cells BMSCs differentiation. Methods BMSCs together with chitin and fibrin gel complexes were cultured in vitro and implanted into rat's articular cartilage defect location. After 14 days in vitro culture, HE staining, toluidine blue staining and type II collagen immunohistochemical staining were performed;After transplantation in vivo, for 2 weeks, 4 weeks and 6 weeks, morphology observations, expression of cartilage-specific protein analysis and BMSCs in vivo tracer method were performed. We analyzed the differentiation of BMSCs into chondrocytes by statistical methods. Results The comparison of positive cell rate of type II collagen immunohistochemical staining in BMSCs-fibrin gel group and BMSCs-chitin group which were cultured in vitro, showed no significant difference with control group. Integrated absorbance(IA) change rate of toluidine blue staining and type II collagen immunohistochemical staining in BMSCs-fibrin gel group which was cultured in vivo, showed significantly different with other groups and control group.Conclusion The results showed that in vitro fibrin gel or chitin has very weak induction of BMSCs to cartilage differentiation, while in vivo BMSCs-fibrin gel can facilitrate BMSCs to differentiate into chondrocyte-like cells.

This investigation was made on 30 adult female rats weighing approximately 150～200g. during estrus as judged from vaginal smear examination. Ovaries which investigated with electron microscope, were fixed in 3％ glutaraldehyde. Other ovaries were embeded in paraffin and examined under light microscope.Theca folliculi of large antral follicles were examined with electron microscope. Three kinds of theca cells were distinguished. They were secretory cell, fibroblast and smooth muscle cell. The secretory cell showed the characterastics of the steroidproducing cell, having smooth endoplasmic reticulum, mitochondria and lipid droplets. The fibroblast contained rough endoplasmic reticulum, free ribosomes and mitochondria in the cytoplasm. The smooth muscle cell was located in the outer layer of theca folliculi, and its structural features were typical according to the criteria of Somlyo and Somlyo: (1) pinocytotic vesicles of the cytoplasmic membranes; (2) myofilaments in the cytoplasm; (3) dense bodies intimately related to the myofilaments; and (4) location of mitochondria and endoplasmic reticulum at the nuclear poles.The ultrastructure of smooth muscle cell, its differentiation and other problems were discussed.

Objective To use tetraploid embryo complementation combined with gene transfer to produce genetically modified embryonic stem cells (EsCs) clones. Methods In this study, EGFP was introduced into ESCs by electroporation, and transfected positive cells were selected by G418 resistance. The tetraploid embryos were obtained from diploid blastomere electrofusion which preformed at 2-cell stage. Afterwards, 19-21 EGFP-ESCs were inserted into each tetraploid blastocyst cavity by piezo drilled microinjection,then the injected blastocysts were transferred into the uterus of pseudo-pregnancy at 2.5-day or the oviduct of 0.5-day female mice. Results The transfected ESCs maintained normal karyotype even after long-term passage (2n=40). The rate of fusion was 95.07%, and the developmental rate of tetraploid blastocyst was 95%.Totally 410 injected blastocysts were obtained. Unfortunately, we have not got any vital offsprings, except 151 implantation sites (pseudo-pregnancy 2.5 days:29.41%;the oviduct of half one day:64.37%). Furthermore, scattered EGFP expressions in transgenic fetus were observed under invert fluorescent microscope. Conclusion The transfected ESCs were observed in transgenic fetus, and the implantation rate in oviduct was higher than that in uterine.

BACKGROUND: Tissue engineering method has been employed to recover cartilage defect and overcome many traditional shortages.OBJECTIVE: In vitro marrow mesenchymal stem cells (MSCs) of adult rat was induced to differentiate into chondrocyte phenotype so as to probe into the feasibility of MSCs to be cartilage seed cell in tissue engineering.DESIGN: Completely randomized design and controlled experimental study.SETTING: Department of Histology and Embryology, and Department of Neurobiology, Harbin Medical University MATERIALS: Six Wistar rats of either gender, cleanness grade, were provided by Experimental Animal Center, Affiliated Second Hospital, Harbin Medical University. Permission number of experimental animal production was SCXK(black) 20020002.METHODS: The MSCs of the second generation adult rat was taken and divided into test group and control group. The test group was induced with serum free and control group was induced with 10% fetal calf serum. Collagen type Ⅱ immunohistochemical staining, toluidine blue staining was performed to detect differentiation.MAIN OUTCOME MEASURES: Identification of chondrocyte, comparison of the positive rate of Collagen type Ⅱ immunohistochemical staining at different induction time point.RESULTS: Induced MSCs had identical characteristic to the chondrocyte.CONCLUSION: In vitro culture can induce MSCs to differentiate directly to chondrocyte-like cell. The results suggest that it is feasible to use MSCs as seed cells in the cartilage tissue engineering.

Objective To observe the influence of simulated microgravity on rat islet. Methods Isolated islet were assigned to flask-culture or bioreactor-culture. Gross structure and ultrastructure of islet were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Results Islets ultrastructure on 7th day in bioreactor closely resembled fresh islets,with well-formed secretory granules and abundant mitochondria. SEM showed under microgravity islets communicating each other with cavity-like areas. Conclusions The ultrastructure of islets cultured under microgravity closely resembled fresh islets.

Objective To reveal the pathophysiological changes of sepsis, the plasma metabonomics of septic rats was determined and differential metabolites were determined by gas chromatography-mass spectrometry (GC-MS). Methods Male Spraguee-Dawley (SD) rats about 8 weeks were randomly divided into sham group (n = 18) and sepsis group (n = 24). Cecal ligation and puncture (CLP) was used to build sepsis model, while cecum was kept intact only in the sham group. 6 hours after the operation, rats were anesthetized, and blood was harvested through heart thoracotomy. Then the plasma metabonomics was detected by GC-MS and metabolic profile analysis was performed to find the relative differential metabolites.Results Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) showed that the metabolic profiling of the sepsis group was significantly different from the sham group. 259 kinds of metabolites were got by GC-MS, and 69 kinds of differential metabolites were found between sham group and sepsis group, in which 23 differential metabolites were related to amino acid metabolism. Compared with sham group, the contents of putrescine, N-glutamic acid, hydroxynorvaline, 3-cyanuric acid, D-alanyl-D-alanine and urea in the plasma of septic rats increased significantly, which ratios of sepsis/sham group were 10.876, 6.394, 2.800, 2.226, 1.323, 1.203, respectively (allP < 0.05). On the other hand, the contents of oxygen generation of proline, citrulline, glutamine, su-beta-hydroxy aspartic acid, citric acid, N-methyl-DL-alanine, serine, lysine, threonine, N-formyl-L-methionine, methionine, alanine, nicotinuric acid, N-methyl-L-glutamic acid, trans-4-hydroxy-L-proline, proline, L-glutamic acid in the plasma of septic rats decreased significantly, which ratios of sepsis/sham group were 0.858, 0.853, 0.834, 0.816, 0.816, 0.814, 0.813, 0.801, 0.793, 0.792, 0.774, 0.766, 0.748, 0.727, 0.716, 0.674, 0.603, respectively (allP < 0.05).Conclusions Through the GC-MS analysis of plasma metabonomics of septic rats, we found abnormal energy metabolism changes. The content of amino acid in plasma might be a method to evaluate the energy metabolism status of sepsis.

Objective:To investigate whether interferon-gamma inducible protein-10(IP-10) and interferon-gamma(IFN-?) participated in the human cerebral ischemia injury.Methods:Twenty-one cerebral ischemia specimens, collected from patients died of cerebral infarction, were divided into three groups: less than 7 days, 7-14 days and 15-21 days according to the lasting time of cerebral infarction. The infiltrating of inflammatory cells were observed using HE stain as non-ischemic hemisphere was for controls. Expression of IP-10 and IFN-? in sections both postmortem ischemic hemisphere and non-ischemic hemisphere were detected using immunohistochemistry.Results:In the groups of less than 7 days and 7-14 days large quantity of inflammatory cells were infiltrated in ischemia tissue. Expression of IP-10 in three groups was elevated in the ischemic hemisphere compared with non-ischemic hemisphere(1.74-folds, 1.41-folds and 1.52-folds increases respectively, P0.05).Conclusion:These results showed IP-10 and IFN-? are expressed in human cerebral ischemia. It was suggested that IP-10 and IFN-? involve in cerebral ischemia injury. Moreover, it was also suggested that IP-10 participate in the repair for the later stage of cerebral ischemia injury.

Objective:To investigate the difference of vitamin A and E levels in children with different respiratory diseases at different ages.Methods:A total of 671 children in Hunan Children's Hospital from July 2017 to October 2019 were selected as the disease group, including 197 cases of pneumonia, 152 cases of recurrent respiratory tract infection, 91 cases of asthma, 88 cases of cough variant asthma and 143 cases of Mycoplasma pneumoniae pneumonia; At the same time, 245 healthy children were selected as the normal group. The serum vitamin A and vitamin E levels of the two groups were detected by high performance liquid chromatography (HPLC).Results:⑴ The vitamin A level [(0.31±0.09)mg/L] of the disease group was lower than the normal group [(0.35±0.25)mg/L], and the vitamin E level [(8.92±2.57)mg/L] was lower than the normal group [(9.62±2.79)mg/L], with statistically significant difference ( P<0.05); ⑵ The level of vitamin A in the disease group at the age of >1-3 years [(0.32±0.09)mg/L] was lower than that in the normal group of the same age group [(0.35±0.08)mg/L]; the level of vitamin A in the disease group at the age of >3-6 years old [(0.30±0.08)mg/L] was lower than that of the same age group [(0.32±0.07)mg/L], with statistically significant difference ( P<0.05); ⑶ The vitamin E level of the disease group at >1-3 years old [(9.23±2.56)mg/L], >3-6 [(8.02±1.86)mg/L] and >6-14 years old [(8.02±1.82)mg/L] were lower than that of the same age normal group [(9.76±2.81)mg/L, (9.67±2.87)mg/L, (9.19±2.58)mg/L], with statistically significant difference ( P<0.05); ⑷ There were significant differences in vitamin A levels among different age in disease group ( P<0.05). Among them, the children with high risk of subclinical deficiency accounted for the largest proportion (45.78%) in the 6-month-1-year-old group, and the proportion of children with normal vitamin A levels in other age groups was the largest; ⑸ There are significant differences in vitamin E levels in different age groups in the disease group ( P<0.05), the levels in the normal range accounts for the largest proportion of all ages; ⑹ The levels of vitamin A and vitamin E in mycoplasma pneumoniae infection group were increased compared with in recurrent respiratory infection group , asthma group, and cough variant asthma group, and the difference was statistically significant ( P<0.05). Compared with the pneumonia group, the level of vitamin E increased in the recurrent respiratory infection group, and the difference was statistically significant ( P<0.05); The vitamin E levels in the cough variant asthma group were reduced compared with the repeated respiratory infection group, asthma group and pneumonia group ( P<0.05). Conclusions:The Vitamin A and E levels of children suffering from respiratory diseases are lower than those of normal children. The Vitamin A and E levels of different respiratory diseases and different age groups are different. Vitamin A and E supplementation may be significantly targeted according to different ages and different respiratory diseases in clinical practice.