Objective To optimize and establish the methodology for culturing and inducing differentiation of mouse preadipocytes 3T3-L1. Methods The mouse cells 3T3-L1 were incubated in DMEM medium contained with 10%FBS, during which the incubation medium was refreshed every 2 to 3 days. Two methods were used to introduce differentiation, including three-drug combination group and four-drug combination group. The protocol of mediumⅠin three-drug combination group including insulin 10 mg/L, IBMX 0.5 mmol/L and DEX 1.0μmol/L. The protocol of mediumⅠin four-drug combination group including indometacin 0.1 mmol/L based on those of three-drug combination group. Both of them were incubated for 2 days and continuous for 2 times. And medium Ⅱ included insulin 10 mg/L for 2-day culturing and continuous for 2 times. Oil red O staining was used to observe the morphological changes of two groups of cells before and after treatment under inverted microscope. Results Mouse preadipocytes 3T3-L1 appeared in good conditions and grew in a paving stone fashion. These cells covered homogeneously the bottom of incubators, the culture medium refreshed every 2 days. The results of four-drug combination group were better than those of three-drug combination group. After three-drug combination induced differentiation, there was no significant change in cell morphology. Comparing with three-drug combination induced differentiation, four-drug combination was successfully achieved in over 90% of the cell inducing, which were round-shaped, with jacinth ester droplets by oil-red O staining. Conclusion We have optimized the method for culturing and inducing differentiation of mouse preadipocytes 3T3-L1 by adding indometacin on the basis of the three-drug combination induced differentiation.

The authors summarize Professor SHI Qi's clinical experience in diagnosing and treating chronic tendon and bone disease.The specific diagnosing and treating thinking and methods could be summarized as follows:1)Three stages,which means chronic tendon and bone disease could be treated according to early,medium and late stages.2) Three differentiations,which include differentiating disease,type and syndrome.3) Three examining,which include seeing patient clearly,reading the disease and getting the key point.In addition,Prof.SHI emphasizes threepoint syndrome differentiation which means the combination of the lesion's target,peri-target and whole syndrome characteristics differentiation.In the process of treatment,Prof.SHI emphasizes three methods combination of herb,technique and breathing technique.Both internal and external treatments should be used.Prof.SHI advocates that the control strategy should be the prevention,treatment and recuperation integration concept,including preventing disease,early treatment to prevent deterioration and preventing reoccurrence after cure.

Objective To explore the mechanism of cell apoptosis of immortalized human keratinocytes (HaCaT cells) and protein expression related to this process after long term exposure to sodium arsenite (NaAsO2,1.0 μmol/L).Methods Malignant transformation model was set up through long-term exposure of HaCaT cells to 1.0 μmol/L NaAsO2.Cell passage for 0,1,7,14,21,28 and 35 generations in the process of malignant transformation were collected for measurement of cell apoptosis rate by flow cytometry,and apoptosis related proteins by Western blotting,including activation of cysteine protease 3,8 (cleaved-caspase-3,8),C/EBP homologous protein (CHOP),B-cell leukemia/lymphoma 2 (Bcl-2),and Bcl-2 associated X protein (Bax).Results Along with the arsenite treatment,the apoptosis levels were significantly decreased (F =26.770,all P ＜ 0.05),the apoptosis levels (0.307 ± 0.049,0.213 ± 0.055,0.163 ± 0.057,0.147 ± 0.035,0.053 ± 0.012) of the 7th,14th,21st,28th and 35thgenerations of cells after arsenite treatment were lower than that of control group of the 0th generation (0.393 ±0.021,all P ＜ 0.05).Compared between generations,there were statistical differences of the protein expression levels of cleaved-caspase-3,Chop,Bax and Bcl-2 in arsenite group (cleaved-caspase-3:1.000 ± 0.000,1.030 ± 0.027,1.104 ± 0.069,1.016 ± 0.087,0.838 ± 0.075,0.753 ± 0.082,0.677 ± 0.073;Chop:1.000 ± 0.000,1.059 ± 0.018,0.934 ± 0.095,0.976 ± 0.216,0.793 ± 0.136,0.651 ± 0.042,0.564 ± 0.056;Bax:1.000 ± 0.000,1.069 ± 0.037,1.028 ± 0.042,0.954 ± 0.118,0.641 ± 0.135,0.531 ± 0.132,0.429 ± 0.085;Bcl-2:1.000 ± 0.000,1.072 ± 0.023,1.249 ± 0.134,1.334 ± 0.143,1.633 ± 0.221,1.507 ± 0.152,1.461 ± 0.145,F =7.730,7.355,27.802,12.438,all P ＜ 0.05),compared with control group of the 0th generation (1.000 ± 0.000) and the same generation control group (1.000 ± 0.000),after the 21st generation,the differences were statistically significant (all P ＜ 0.05),while there was no difference of the protein expression levels of cleaved-caspase-8 (F =0.832,P ＞ 0.05).Conclusion In the process of malignant transformation,the apoptosis levels of HaCaT cells are inhibited after long term sodium arsenite exposure through mitochondria and endoplasmic reticulum (ER) stress signaling pathways.

Neural development is a complex process.Its development is affected by a variety of molecular signals such as signals from intestinal microorganisms.Recent animal research showed that microorganisms play an important role in neurogenesis,myelination,microglial cell maturation and formation of blood-brain barrier,and the regulation of animal behavior from many aspects.This article reviewed the impact of prenatal and postnatal gut microbes on the development and function of the nervous system,and the relationship between gut microbes and autism spectrum disorder as well.

Objective:To analyze the composition, the changes of expense structure and the influencing factors of hospitalization expenses, for reference in optimizing the cost control of day surgery.Methods:Collection of the first page data of patients with the top three diseases(varicose veins of lower limbs, chronic cholecystitis and varicocele)in the day surgery volume ranking in three tertiary general hospitals in a city in 2020. The confounding factors were eliminated through propensity matching. The structural change of hospitalization expenses was analyzed by structural change degree, and the influencing factors of hospitalization expenses were analyzed by grey correlation degree and multiple linear regression.Results:After 1∶1 propensity matching of the first page data of 752 patients with day surgery and non day surgery, 98 patients with lower extremity varicose veins, 356 patients with chronic cholecystitis and 38 patients with varicocele were finally included. Compared with non day hand, the total hospitalization cost of day surgical instruments decreased, and the cost structure changes of chronic cholecystitis, varicocele and varicose veins of lower limbs were 14.59%, 6.20% and 16.20% respectively. Among them, the general medical service fee, nursing fee and examination and laboratory fee showed a downward trend, and the fees of materials and drugs showed an upward trend. General medical service fee, nursing fee, examination and laboratory fee, clinical diagnosis fee, treatment fee, drug fee, material fee and other expenses presented a high correlation with the cost of day surgery(grey correlation>0.90). The payment method, wound healing type and discharge diagnosis can influence the cost of day surgery( P<0.05). Conclusions:Compared with non daytime surgery, the total hospitalization cost of day surgery has a certain cost control effect, but it can not reduce the cost of all projects. The main influencing factors are the internal composition of the cost, payment method and so on. The hospitals should focus on tapping the internal cost control potential of day surgery and further expanding the coverage of day surgery diseases.

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Objective To observe the regulating effect of electroacupuncture on the polarization level of decidual macrophages in rats with thin endometrium and the repairing effect of electroacupuncture on thin endometrium.Methods Thirty SPF female SD rats were randomly divided into control group,model group and electroacupuncture group.The electroacupuncture group was treated with acupuncture of"Guanyuan"(CV4),"Zigong"(EX-CA1)and"Sanyinjiao"(SP6)and intervened continuously for 4 estrous cycles.After the intervention,the estrous cycle of rats in each group was observed,the number of implantation embryos in the uterus was recorded,the uterine morphology and decidualization,the thickness of endometrium,the number of glands and blood vessels were observed by HE staining,the polarization level of macrophages was detected by flow cytometry,and the expression levels of IGFBP-1,CD86 and CD206 genes were detected by qRT-PCR.Results Compared with the control group,the estrous cycle of rats in the model group was disturbed;compared with the model group,the estrous cycle of rats in the electroacupuncture group recovered;compared with the control group,the number of embryos in the model group decreased significantly(P<0.01);compared with the model group,the number of embryos in the electroacupuncture group increased(P<0.05),and the morphology of endometrium in the model group was damaged and the thickness of endometrium decreased compared with the control group(P<0.01).The number of glands and blood vessels decreased(P<0.01).Compared with the model group,the morphology of endometrium in the electroacupuncture group recovered,the thickness of endometrium increased(P<0.01),and the number of glands and blood vessels increased(P<0.01).Compared with the control group,the ratio of M2 to M1 macrophages in the model group decreased significantly(P<0.01).Compared with the model group,the ratio of M2 to M1 macrophages in the endometrium of the electroacupuncture group was significantly increased(P<0.05).Compared with the control group,the expression of IGFBP-1 mRNA in the model group decreased significantly(P<0.01),the expression of CD86 mRNA increased significantly(P<0.01),and the expression of CD206 mRNA decreased significantly(P<0.01).Compared with the model group,the expression of IGFBP-1 mRNA in the endometrium of the electroacupuncture group increased significantly(P<0.01),the expression of CD86 mRNA decreased significantly(P<0.01),and the expression of CD206 mRNA increased significantly(P<0.01).Conclusion Electroacupuncture can effectively regulate the polarization level of decidual macrophages in endometrium,and effectively repair thin endometrium,which is beneficial to embryo implantation.