BACKGROUND:Our previous studies have shown that adipose-derived stem cels under vascular endothelial growth factor C (VEGF-C) can be induced to differentiate into lymphatic endothelial cels that are confirmed by lymphatic vascular endothelial hyaluronan receptor-1 staining. However, its optimal induction program is not clear.
OBJECTIVE:To investigate the best condition for the differentiation of adipose-derived stem cels into lymphatic endothelial cels under induction of VEGF-C156s.
METHODS: Adipose tissues from healthy adults were colected to isolate adipose-derived mesenchymal stem cels using trypsin digestion method. Flow cytometry was employed to detect cel surface markers, andin vitro differentiation capacity was identified by adipogenic and osteogenic induction. Passage 3 cels at good growth state were selected and divided into six groups: cels in control group were cultured in low-glucose DMEM, and those in the rest five groups were treated with 25, 50, 100, 200, 300 μg/L VEGF-C156s, respectively.
RESULTS AND CONCLUSION:Adipose-derived stem cels were successfuly obtained by trypsin digestion and purification, and then differentiated into lymphatic endothelial cels under the induction of VEGF-C156s, basic fibroblast growth factor and other growth factors. No cels were positive for lymphatic vascular endothelial hyaluronan receptor-1 in the control group. After 8 days of induction, few cels were positive in the 25 μg/L VEGF-C156s group; a great amount of positive cels were visible in the 50 and 100 μg/L VEGF-C156s groups; 200 and 300 μg/L VEGF-C156s resulted in a large number of deaths in the cels. These findings indicate that it is optimal for adipose-derived stem cels to differentiate into lymphatic endothelial cels under 8-day induction of 50 μg/L VEGF-C156s.

Objective To explore the clinical features and treatment of children with acute severe viral myocarditis.Methods The clinical data of presentation,diagnosis,therapy and prognosis of children who were admitted in our hospital from Jan 2005 to Jan 2012 with acute severe viral myocarditis(severe myocarditis group) were analyzed retrospectively.Twenty-three cases of normal healthy children in the same period were selected as control group.The levels of serum cardiac troponin(CTn)-Ⅰ and N-terminal pro-brain natriuretic peptide(NT-proBNP) were detected by ELISA method,the changes of left ventricular ejection fraction and left ventricular fraction shortening were understood by color doppler echocardiography.Results The level of CTn-Ⅰin severe myocarditis group was significantly higher than that of control group,the difference was statistically significant [(18.67 ± 12.31) ng/ml vs (0.02 ±0.01) ng/ml,P ＜0.05].Compared with the acute phase,the level of CTn-Ⅰshowed a trend of gradual decline in 7 d [(0.55 ±0.24) ng/ml],basic close to normal in 14 d [(0.06 ±0.03) ng/ml] (P ＜0.05).The level of NT-proBNP increased significantly in severe myocarditis group compared with control group [(3 067.26 ± 902.79) pg/ml vs (80.04 ± 17.79) pg/ml,P ＜0.05].Compared with acute phase,the levels of NT-proBNP were closed to normal in 7 d [(648.63 ±342.37) pg/ml] and 14 d [(213.58 ± 129.51) pg/ml] (P ＜ 0.05).The left ventricular ejection fraction [(52.63 ± 6.98) ％ vs (71.39 ± 2.41) ％] and left ventricular fraction shortening [(32.1 ± 2.97) ％ vs (40.04 ± 2.31) ％] in severe myocarditis group were significantly lower than those in control group (P ＜ 0.05).Conclusion Acute severe viral myocarditis of children was characterized by rapid onset,severe illness and high mortality.Early use of adrenal cortical hormone and gamma globulin under the comprehensive treatment and application temporary pacemaker can help patients to recover from the disease.

OBJECTIVE: To assess the safety and efficacy of transcutaneous electrical acupoint stimulation (TEAS) for depression in late pregnancy and impacts on inflammatory cytokines. METHODS: A total of 150 pregnant women with depression in late pregnancy were randomized into a high intensity group (n＝52), a low intensity group (n＝49) and a control group (n＝49). TEAS was applied at bilateral Neiguan (PC 6) and Shenmen (HT 7) for 6 weeks. The intensities of TEAS in the high intensity group, the low intensity group and the control group were 10, 5 and 0 mA, respectively. During the process of TEAS, the blood pressure, pulse, uterine contraction and the fetal heart rate were recorded. Depression was evaluated by 24-item Hamilton depression scale (HAMD) before TEAS and after 2-week, 4-week, 6-week treatment. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1 β) and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA) before and after 6-week treatment. Delivery outcomes were observed. The correlation was analyzed between HAMD difference value and the diffe-rence values of TNF-α， IL-1 β and IL-6, respectively. RESULTS: The blood pressure, pulse and fetal heart rate had no statistical significance before and after treatment in the three groups (P>0.05). The HAMD scores at all the time points were lower than that before treatment in the high intensity group (P<0.05), which were lower compared with those in the low intensity group and the control group (P<0.05). The HAMD score in the low intensity group decreased after 6-week treatment compared with that before treatment and was lower than that in the control group (P<0.05). The serum levels of IL-1 β and IL-6 in the high intensity group decreased compared with those before treatment and were lower compared with those in the low intensity group and the control group after 6-week treatment (P<0.05). There was no significant difference in the deliver outcomes among the three groups (P>0.05). The variation of HAMD score did not have significant correlation with those of TNF-α， IL-1 β and IL-6 (P>0.05). CONCLUSION: TEAS is safe and effective for depression in late pregnancy and can regulate the serum levels of IL-1 β and IL-6 without influencing on delivery outcome.

Directed evolution is a cyclic process that alternates between constructing different genes and screening functional gene variants. It has been widely used in optimization and analysis of DNA sequence, gene function and protein structure. It includes random gene libraries construction, gene expression in suitable hosts and mutant libraries screening. The key to construct gene library is the storage capacity and mutation diversity, to screen is high sensitivity and high throughput. This review discusses the latest advances in directed evolution. These new technologies greatly accelerate and simplify the traditional directional evolution process and promote the development of directed evolution.
Base Sequence ; Directed Molecular Evolution ; Gene Library ; Mutation ; Proteins/genetics*

ObjectiveTo discuss the relationship between objective sleep quality and cognitive function in remission depression patients with insomnia. MethodsA total of 47 patients with depression in remission in Beijing Huilongguan Hospital were enrolled. Insomnia Severity Index （ISI） and the sleep efficiency calculated by the first four items of Pittsburgh Sleep Quality Index （PSQI） were defined as insomnia. Among the selected patients， 22 subjects were classified into insomnia group， and 25 subjects in non-insomnia group. The objective sleep status of all subjects was evaluated by polysomnography （PSG）， and the cognitive function was evaluated with the Repeatable Battery for Assessment of Neuropsychological Status （RBANS）. A regression model was established to explore the relationship between objective sleep quality and cognitive function in remission depression patients with insomnia. ResultsThere was statistically significant difference in immediate memory factor scores of RBANS between depression patients in remission with and without insomnia （t=-2.102， P<0.05）. The sleep efficiency （Z=-2.113， P<0.05）， the proportion of N3 sleep stage （Z=-3.175， P<0.01） and the duration of N3 sleep stage （Z=-3.196， P<0.01） in insomnia group were all lower than those in non-insomnia group， and the nocturnal awakening （Z=-2.058， P<0.01）， the proportion of N1 sleep stage （Z=-2.399， P<0.05） and the duration of N1 sleep stage （Z=-2.463， P<0.05） were higher than those of the insomnia group. Within insomnia group， the increased proportion of REM sleep was correlated with the improvement of attention function （β=0.953， 95% CI： 0.123~2.038， P<0.05）， and the increase of nocturnal awakening was correlated with the decline of delayed memory （β=-0.173， 95% CI： -0.325~-0.024， P<0.05）. ConclusionObjective sleep is associated with cognitive impairment in remission depression patients with insomnia， the proportion of REM sleep has a positive impact on attention function， and frequent nocturnal awakening at night has a negative impact on delayed memory.

OBJECTIVE：To study the repa ir，anti-inflammatory and analgesic effects of Compound crocodile oil burn ointment on superficial second-degree burned skin. METHODS ：The heated weight was attached to the right depilated skin of guinea pigs for 4 s to induce the model of superficial second-degree burn. After modeling ，guinea pigs were randomly divided into model group ， Jingwanhong ointment group （positive control ），formula Ⅰ，Ⅱ and Ⅲ groups of Compound crocodile oil burn ointment （volume fraction 1.5％，3％，4.5％，hereinafter），with 8 guinea pigs in each group. Except for model group ，other groups were smeared with 0.7 g/guinea pigs twice a day for 14 consecutive days. The wound healing was recorded every day ，the healing rate of wound was calculated. HE staining was used to observe the histopathological changes of the wound. The serum levels of EGF ，VEGF， SOD，MDA，TNF-α and IL-1 were detected by ELISA. Eighty Kunming mice were divided into 2 groups，and then sub-grouped into model group ，Jingwanhong ointment group （positive control ），formula Ⅰ and Ⅲ groups of Compound crocodile oil burn ointment，with 10 mice in each group. Then xylene auricle swelling method and acetic acid writhing method were used to investigate the anti-inflammatory and analgesic effects of Compound crocodile oil burn ointment. RESULTS ：In the burn repair experiment，after intervention of Compound crocodile oil burn ointment ，the wound area of guinea pigs gradually decreased ，and on the 14th day ，the wound had healed greatly ，and the wound healing rate increased significantly （P＜0.01）；serum levels of EGF and SOD were increased significantly （P＜0.01），while the levels of VEGF ，MDA，TNF-α and IL-1 were decreased significantly（P＜0.05 or P＜0.01）. The thick new epidermal layer was found in wound tissue ，and the connective tissue and neovascularization in the dermis increased significantly. In the anti-inflammatory and analgesic experiment ，after intervention of Compound crocodile oil burn ointment ，the degree of ear swelling and the times of writhing decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Compound crocodile oil burn ointment shows good skin repair ，anti-inflammatory and analgesic efficacy；the mechanism may be associated with increasing the serum levels of EGF and SOD and reducing the levels of VEGF ， MDA，TNF-α，IL-1.