BACKGROUND:It is important to produce and save a large amount of high-activity feeder cel s for the culture of human embryonic stem cel s.
OBJECTIVE:To establish the optimal method for isolation and culture of Kunming mouse embryonic fibroblasts, and to evaluate the feasibility of preparing feeder layers for culture of human embryonic stem cel s.
METHODS:Embryonic fibroblasts were isolated and cultured by different concentrations of trypsin from Kunming mouse fetuses in vitro. The biological characteristics and growth rule of mouse embryonic fibroblasts were investigated, and then the feeder layers for human embryonic stem cel s culture were produced. The growth of human embryonic stem cel s on the prepared feeder layer was tested.
RESULTS AND CONCLUSION:The optimal fetal age for preparing Kunming mouse embryonic fibroblast feeder layer was 13.5 days. Kunming mouse embryonic fibroblasts at different concentrations grew wel with high purity and active proliferation by trypsin digestion method. There was no significant difference in the survival rate of cel s after cryopreservation for 2 weeks, 1 month, 3 months and 6 months. The cel s were proliferative from the second to fourth passage and declined sharply after the fifth passage. Human embryonic stem cel s which grew on Kunming mouse embryonic fibroblasts feeder layers were stil to remain the typical undifferentiated morphology and were strongly positive for alkaline phosphatase and periodic acid-Schiff after long-term subculture. The
mouse embryonic fibroblasts can be used as the stable and high-quality feeder cel s for human embryonic stem cel s.

Objective To study the effects of rehabilitation training on angiogenesis and its ultrastructure and expression of CD32 in the peri-infarction region of rats with focal cerebral infarction. Methods Sixty-six Spra-gue-Dawley rats with experimental left middle cerebral artery occlusion (MCAO) were used as subjects in this study. All the rats were randomly divided into three groups: a rehabilitation training group (n=30), which was given bar rotating, balancing and rolling exercises everyday after 48 hours post MCAO; a control group (n = 30) and a sham-operated group (n=6), which were fed in cages with no special training exercises. Then brain tissues were fixed on the 3rd d, 7th d, 14th d after MCAO, for observing the morphological alterations of microvessels in peri-infarction re-gion using transmission electron microscope, immunohistochemistry and Western blotting were used to measure the ex-pression of CD31, which acted as the marker of the neogenetic microvessels. Results (1) It showed that the capil-lary endothelial cells were less edematous in the rehabilitation training group, and there were less pinocytosis bullae in basal membrane more integral nucleus of endothelial cells in rehabilitation training group when compared with those in the control group. (2) Expression of CD31 can be observed in peri-infarction region in both groups from the 3rd d on-wards, and peaked on the 7th d, and then gradually went down after the 14th d. Comparison between the 2 groups showed that the expression of CD31 in rehabilitation training group was higher than that in the control group at every time point, but statistical difference between the 2 groups in this regard could be revealed only on the 7th d (P< 0.05), Conclusion Rehabilitation training could promote ultrastruetural recovery of microvessels and induce an-giogenesis in peri-infaretion region, and it might be one of the mechanisms of neural functional recovery in rats after MCAO.

Objective To study the effects of rehabilitative training on motor function and expression of GAP-43 and SYN in rats with local cerebral infarction. Methods A total of 76 male adult Sprague-Dawley rats were randomly divided into a rehabilitative training group(n=32),a control group(n=32),and a sham-operated group(n=1 2).All the rats were subjected to left middle cerebral artery occlusion(MCAO)with the suture occlu sion.Motor training programs including balancing,grasping,rotating and walking exercises were administered to the rats of the rehabilitative training group at 48 hours post-operation,while those of other two groups were reared in their original living status without any special training.The animals were given behavioral tests with Bederson test,balancing wood test,net screen test to assess the functional outcome,and immunohistochemistry staining was employed to evaluate the exDression of GAP-43 and SYN in peri-infarction cortex at the 3rd,7th,21 st,35th days after MACO,respectively. Results The scores of behavioral tests in the rehabilitative training group was better than those in the control group(P＜0.05)at the 7th,21 st,35th day after MCAO,and the immunostaining showed that expression of GAP-43 was higher in the rehabilitative training group than that in the control group(P＜0.05)and the sham operated group(P＜0.01)at the 7th and 21 st days post-operation,respectively,and that the expression of SYN was higher in the rehabilitative training group than that in the control(P＜0.05)and the sham operated groups(P＜0.05)at the 21 st and the 35th days post-operation,respectively. Conclusion Rehabilitative training can improve func tional recovery in rats with local cerebral infarction,and the function enhancement may be partially attributed to the up-regulation of expression of GAP-43 and SYN in peri-infarction cortex.

Objective To investigate the changes in serum level of microparticles (EMPs) in mice with ventilator-induced lung injury (VILI), and explore its significance in VILI. Methods Forty-eight grade SPF male C57BL/6J mice were randomly divided into two groups, with 24 mice in each group: the mice in mechanical ventilation (MV) group were given high tidal volume (VT 30 mL/kg) MV for 4 hours after tracheal intubation, and those in spontaneous breathing group were spontaneously breathed for 4 hours. The apical blood of 12 mice in each group were collected, and serum levels of interleukins (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA), and serum EMPs levels were determined by flow cytometer. The correlations between EMPs and IL-1β, IL-6, and TNF-α were analyzed by linear regression analysis. The lung tissues of other 12 mice in each group were harvested, and wet/dry weight (W/D) ratio was assessed. After hematoxylin-eosin (HE) staining, the morphological changes in lung tissue were observed under light microscope. After double staining of uranium acetate and lead citrate, the ultrastructural changes in lung tissue were observed with electron microscope. Results Compared with spontaneous breathing group, the levels of lung W/D ratio in MV group was significantly increased (5.47±0.14 vs. 4.34±0.11), the levels of IL-1β, IL-6, TNF-α and EMPs were also significantly increased [IL-1β (ng/L): 42.4±4.4 vs. 7.7±3.6, IL-6 (ng/L): 1 239.5±66.3 vs. 21.7±4.6, TNF-α (ng/L):237.6±25.8 vs. 37.1±19.1, EMPs (cells/μL): 28.6±1.8 vs. 5.9±1.8, all P < 0.01]. It was shown by correlation analysis that EMPs were positively related with IL-1β, IL-6, and TNF-α (r value was 0.968, 0.932, 0.945, respectively, all P = 0.000). It was shown by fitting linear regression analysis that when EMPs increased by 1 cell/μL, IL-1β increased by 2.4 ng/L [95% confidence interval (95%CI) = 1.9-2.8, P < 0.001], IL-6 increased by 34.5 ng/L (95%CI = 25.1-44.0, P < 0.001), and TNF-α increased by 13.6 ng/L (95%CI = 10.3-16.9,P < 0.001). It was shown by light microscope that the structure of lung tissue and alveolar of mice in spontaneous breathing group appeared normal, while the shrinks of alveolar and disappearance of alveolar architecture were found in MV group. It was shown by electron microscopy that alveolar wall edema and thickening and broken alveolar septa were found in MV group, by contrast, the structure of alveolar was normal in spontaneous breathing group. Conclusion 30 mL/kg VT ventilation for 4 hours could induce VILI with increase in EMPs, suggesting EMPs closely related to VILI, and EMPs level may be putative biomarker of VILI.